Preface

The three different types of muscle tissue found in the animal kingdom are cardiac, skeletal, and smooth. The muscle cells are not only complex but also fascinating. In recent years there has been substantial advances in our understanding of muscle cell biology, especially in the areas of molecular anatomy, basic physiology, understanding disease mechanisms, and therapeutic targets.

Consequently, this book mainly focuses not only on the biology of myocytes, but also on all-encompassing disciplines pertaining to muscle tissue, such as fundamental physiology, molecular mechanisms of diseases, muscle regeneration, etc. for all three types of muscle, namely, skeletal, cardiac, and smooth muscle. As a result, the goal of this book is to consolidate the recent advances in the areas of muscle biology/diseases/regeneration covering a broad range of interrelated topics in a timely fashion and to disseminate that knowledge in a lucid way to a greater scientific audience.

The book consists of eight chapters, contributed by leading experts in basic science and clinical care, and is organized into four sections. The first section introduces the cellular and molecular aspects of muscle function, such as the role of vascularization during skeletal muscle regenerative and/or reparative processes, electrophysiological signature of arteriolar smooth muscle in various tissues with an emphasis on the excitability of fourth-order arterioles of skeletal muscle, the expression pattern and functional role of two orexigenic peptides (orexin system) in muscle mitochondrial dynamics, and finally the role of non-coding RNAs in exercise.

The second section of the book deals with current perspectives of inflammatory myopathies. Next, the third section explores the cancer-induced muscle-wasting disorder, focusing on the anabolic effects of branched-chain amino acids supplementation, especially leucine on skeletal muscle wasting and thereby ameliorating the host response to cancer-cachexia-induced muscle damage, as well as repercussions of adipose tissue dysfunction and remodeling and its significance in the development of cachexia. Eventually, the last section of the book contains a survey of recent advances in immunological detection assays pertaining to skeletal muscle proteins in physiological and pathological conditions.

This book will prove highly useful for students, researchers, and clinicians in muscle cell biology, exercise physiology/science, stem cell biology, developmental biology, cancer biology, pathology, oncology, as well as tissue engineering and regenerative medicine. This quick reference will benefit anyone desiring a thorough knowledge pertaining to recent advances in muscle biology in the context of health and disease.

I would like to thank the staff of IntechOpen who have produced this book so efficiently, and in particular I am indebted to Sandra Maljavac, the Author Service Manager, and Petra Svob, the Commissioning Editor, for their valuable source of

**II**

**Chapter 7 131**

Muscle Markers: Immuno-Analysis **147**

**Chapter 8 149**

Current Approaches in Immunoassay Methods Focus on Skeletal Muscle

Adipose Tissue Remodeling during Cancer Cachexia *by Miguel Luiz Batista Júnior and Felipe Henriques*

**Section 4**

Proteins *by Gisela Gaina* advice throughout the preparation of this book. Finally, this book is dedicated to the memory of my parents and to the memory of my eldest brother.

> **Mani T. Valarmathi, MD, PhD** Department of Biomedical Engineering, School of Medicine and School of Engineering, UAB | The University of Alabama at Birmingham, Birmingham, Alabama, USA

> > **1**

Section 1

Muscle Function:

Cellular-Molecular

Section 1

## Muscle Function: Cellular-Molecular

**3**

**Chapter 1**

**Abstract**

tion in skeletal muscle.

**1. Introduction**

**1.1 Vasculogenesis**

on angiogenesis [1].

Vascularisation of Skeletal Muscle

Skeletal muscle is mainly involved in physical activity and movement, which requires a large amount of glucose, fatty acids, and oxygen. These materials are supplied by blood vessels and incorporated into the muscle fiber through the cell membrane. In contrast, metabolic waste is discarded outside the cell membrane and removed by blood vessels. The formation of a functional, integrated vascular network is a fundamental process in the growth and maintenance of skeletal muscle. On the other hand, vascularization is one of the main central components in skeletal muscle regeneration. In order for regeneration to occur, blood vessels must invade the transplanted muscle. This is confirmed by the fact that muscle regeneration occurred from the outside of the muscle bundle toward the inner regions. In fact, it is likely that capillary formation is a key process to start muscle regeneration. Thus, vascularization activates muscle regeneration, and a decrease in vascularization could lead to disruption the process of muscle regeneration. Also, a better understanding of vascularization of skeletal muscle necessary for the successful formation of collateral arteries and recovery of injured skeletal muscle may lead to more successful strategies for skeletal muscle regeneration and engineering. So, in this chapter, we want to review vasculariza-

**Keywords:** vascularization, angiogenesis, arteriogenesis, skeletal muscle

esis, angiogenesis, arteriogenesis, and lymphogenesis.

Vascularization of skeletal muscle occurs by four distinct processes: vasculogen-

The fundamental biological phenomenon of new vessel formation is one enormous complexity, and has excited the interest of scientific workers for many years. Vasculogenesis describes the formation of the primitive network of blood vessel in the embryo from undifferentiated precursor cells (angioblasts), and the differentiation of angioblasts to endothelial cell. This is the initial step to blood vessel formation de novo. To form a new vessel, angioblasts proliferate and join up with primary capillary plexus. The endothelial cell grid manufactured by vasculogenesis then serves as an angiogenesis framework [1]. After primary capillary plexus formation, it is altered by the sprouting and branching of new vessels from pre-existing ones. The majority of work on skeletal muscle capillaries has focused

*Kamal Ranjbar and Bayan Fayazi*

### **Chapter 1**

## Vascularisation of Skeletal Muscle

*Kamal Ranjbar and Bayan Fayazi*

### **Abstract**

Skeletal muscle is mainly involved in physical activity and movement, which requires a large amount of glucose, fatty acids, and oxygen. These materials are supplied by blood vessels and incorporated into the muscle fiber through the cell membrane. In contrast, metabolic waste is discarded outside the cell membrane and removed by blood vessels. The formation of a functional, integrated vascular network is a fundamental process in the growth and maintenance of skeletal muscle. On the other hand, vascularization is one of the main central components in skeletal muscle regeneration. In order for regeneration to occur, blood vessels must invade the transplanted muscle. This is confirmed by the fact that muscle regeneration occurred from the outside of the muscle bundle toward the inner regions. In fact, it is likely that capillary formation is a key process to start muscle regeneration. Thus, vascularization activates muscle regeneration, and a decrease in vascularization could lead to disruption the process of muscle regeneration. Also, a better understanding of vascularization of skeletal muscle necessary for the successful formation of collateral arteries and recovery of injured skeletal muscle may lead to more successful strategies for skeletal muscle regeneration and engineering. So, in this chapter, we want to review vascularization in skeletal muscle.

**Keywords:** vascularization, angiogenesis, arteriogenesis, skeletal muscle

### **1. Introduction**

Vascularization of skeletal muscle occurs by four distinct processes: vasculogenesis, angiogenesis, arteriogenesis, and lymphogenesis.

### **1.1 Vasculogenesis**

The fundamental biological phenomenon of new vessel formation is one enormous complexity, and has excited the interest of scientific workers for many years. Vasculogenesis describes the formation of the primitive network of blood vessel in the embryo from undifferentiated precursor cells (angioblasts), and the differentiation of angioblasts to endothelial cell. This is the initial step to blood vessel formation de novo. To form a new vessel, angioblasts proliferate and join up with primary capillary plexus. The endothelial cell grid manufactured by vasculogenesis then serves as an angiogenesis framework [1]. After primary capillary plexus formation, it is altered by the sprouting and branching of new vessels from pre-existing ones. The majority of work on skeletal muscle capillaries has focused on angiogenesis [1].

### **1.2 Angiogenesis**

Angiogenesis and inflammation are critical to the process of muscle regeneration, but the complex interactions between these multiple cell types are poorly understood [2]. Most normal angiogenesis occurs in the embryo where it establishes the primary vascular tree as well as an adequate vasculature for growing and developing skeletal muscles. Angiogenesis involves the growth of new capillaries from existing blood vessels within the skeletal muscle and occurs in the adult during the ovarian cycle and in physiological repair processes such as wound healing. However, very little turnover of endothelial cells occurs in the adult vasculature. In order for new blood vessel sprouts to form, as previously described by Papetti et al. [1], mural cells (pericytes) must first be removed from the branching vessel. Endothelial cell basement membrane and extracellular matrix is then degraded and remodeled by specific proteases such as matrix metalloproteinase (MMPs), and the new matrix synthesized by stromal cells is then laid down. This new matrix, coupled with soluble growth factors, fosters the migration and proliferation of endothelial cells. After sufficient endothelial cell division has occurred, endothelial cells arrest in a monolayer and form a tube-like structure. Mural cells (pericytes in the microvasculature and smooth muscle cells in larger vessels) are recruited to the abluminal surface of the endothelium, and vessels uncovered by pericytes regress. Blood flow is then established in the new vessel [1]. Angiogenesis in the skeletal muscle can occur by two primary mechanisms: sprouting and intussusception.

### *1.2.1 Sprouting angiogenesis*

Sprouting in angiogenesis refers to activated endothelial cells diverging from the existing vasculature, continuing through the encompassing matrix to form a cord-like structure (see **Figure 1**). The endothelial cell cord is changed into a tube and sticks to the extracellular matrix. It should be noted that the newly formed tube must reenter the capillary network via joining with another capillary or venule to become a functional capillary. Newly formed capillary in the beginning is leaky, but it blossoms to that of the original capillary when pericytes surround completely the endothelial cells. Like intussusception, sprouting needs to activate endothelial cells [3].

### *1.2.2 Capillary intussusceptions*

Intussusceptions point to the process by which a mature capillary is divided into two separate capillaries from within, by the formation of a pillar-like structure or longitudinal divide on the luminal side of the capillary (**Figure 2**). Activated endothelial cells extend intraluminally, effectively forming two tubes through which blood can pass. Previous studies confirmed that intussusception is a main method of capillary formation during development. Angiogenic responses were differential in vivo. In this case, it is mentioned that shear forces acting on capillaries may preferentially increase capillarity through intussusception [3]. On the other hand, as shown in **Figure 3**, overload by activation of MMPs and VEGF leads to angiogenesis via sprouting.

### **1.3 Arteriogenesis**

Arteriogenesis, formerly regarded as a variant of angiogenesis, is a relatively new term that was introduced to distinguish it from other mechanisms of vascular growth, such as angiogenesis and vasculogenesis. Vasculogenesis describes the

**5**

**Figure 2.**

**Figure 1.**

embryonic development of blood vessels from angioblasts, and angiogenesis is the formation of new capillaries by sprouting and intussusception from pre-existent capillaries. But, arteriogenesis describes the formation of mature arteries from preexistent interconnecting arterioles after an arterial occlusion. Arteriogenesis pointed

*Angiogenesis occurs by the processes of intussusception and sprouting.*

*Vascularisation of Skeletal Muscle*

*DOI: http://dx.doi.org/10.5772/intechopen.85903*

*Schematic representation of sprouting angiogenesis [4].*

*Vascularisation of Skeletal Muscle DOI: http://dx.doi.org/10.5772/intechopen.85903*

*Muscle Cells - Recent Advances and Future Perspectives*

Angiogenesis and inflammation are critical to the process of muscle regeneration, but the complex interactions between these multiple cell types are poorly understood [2]. Most normal angiogenesis occurs in the embryo where it establishes

Sprouting in angiogenesis refers to activated endothelial cells diverging from the existing vasculature, continuing through the encompassing matrix to form a cord-like structure (see **Figure 1**). The endothelial cell cord is changed into a tube and sticks to the extracellular matrix. It should be noted that the newly formed tube must reenter the capillary network via joining with another capillary or venule to become a functional capillary. Newly formed capillary in the beginning is leaky, but it blossoms to that of the original capillary when pericytes surround completely the endothelial cells. Like intussusception, sprouting needs to activate

Intussusceptions point to the process by which a mature capillary is divided into two separate capillaries from within, by the formation of a pillar-like structure or longitudinal divide on the luminal side of the capillary (**Figure 2**). Activated endothelial cells extend intraluminally, effectively forming two tubes through which blood can pass. Previous studies confirmed that intussusception is a main method of capillary formation during development. Angiogenic responses were differential in vivo. In this case, it is mentioned that shear forces acting on capillaries may preferentially increase capillarity through intussusception [3]. On the other hand, as shown in **Figure 3**, overload by activation of MMPs and VEGF leads to

Arteriogenesis, formerly regarded as a variant of angiogenesis, is a relatively new term that was introduced to distinguish it from other mechanisms of vascular growth, such as angiogenesis and vasculogenesis. Vasculogenesis describes the

the primary vascular tree as well as an adequate vasculature for growing and developing skeletal muscles. Angiogenesis involves the growth of new capillaries from existing blood vessels within the skeletal muscle and occurs in the adult during the ovarian cycle and in physiological repair processes such as wound healing. However, very little turnover of endothelial cells occurs in the adult vasculature. In order for new blood vessel sprouts to form, as previously described by Papetti et al. [1], mural cells (pericytes) must first be removed from the branching vessel. Endothelial cell basement membrane and extracellular matrix is then degraded and remodeled by specific proteases such as matrix metalloproteinase (MMPs), and the new matrix synthesized by stromal cells is then laid down. This new matrix, coupled with soluble growth factors, fosters the migration and proliferation of endothelial cells. After sufficient endothelial cell division has occurred, endothelial cells arrest in a monolayer and form a tube-like structure. Mural cells (pericytes in the microvasculature and smooth muscle cells in larger vessels) are recruited to the abluminal surface of the endothelium, and vessels uncovered by pericytes regress. Blood flow is then established in the new vessel [1]. Angiogenesis in the skeletal muscle can occur by two primary mechanisms: sprouting and intussusception.

**1.2 Angiogenesis**

*1.2.1 Sprouting angiogenesis*

endothelial cells [3].

*1.2.2 Capillary intussusceptions*

angiogenesis via sprouting.

**1.3 Arteriogenesis**

**4**

**Figure 1.** *Schematic representation of sprouting angiogenesis [4].*

### **Figure 2.**

*Angiogenesis occurs by the processes of intussusception and sprouting.*

embryonic development of blood vessels from angioblasts, and angiogenesis is the formation of new capillaries by sprouting and intussusception from pre-existent capillaries. But, arteriogenesis describes the formation of mature arteries from preexistent interconnecting arterioles after an arterial occlusion. Arteriogenesis pointed

### **Figure 3.**

*Differential angiogenic responses in vivo [5].*

to the enlargement of existing arterial vessels [6]. This enlargement indicates an increase in the caliber (diameter) and wall dimensions, resulting in a larger vessel. It is similar to angiogenesis in some features, but the pathways make it different [7].

Angiogenesis occurs under hypoxia/ischemia condition, which leads to the activation of the transcription factor HIF (hypoxia-inducible factor); in contrast, arteriogenesis is activated in an environment of normoxia [8].

Growth of arterioles in skeletal muscle has been documented during development.

Transformation of pericytes into smooth muscle cells and/or apposition of mesenchymal cells, most likely fibroblasts, to the abluminal surface of capillaries, followed by their gradual change into pericytes or smooth muscle cells lead to the growth of arteriolar known as "arteriolarization" [9]. Arteriolar growth accompanied rather than followed capillary growth.

### **1.4 Lymphogenesis**

Lymphatic vessels act in close relation with blood vessels. The formed lymph fluid is transported via initial lymphatic capillaries to collecting vessels, to lymph nodes, and finally back to the blood [10]. Hyperemia-induced increased filtration of skeletal muscle (during activity), promotes hydrostatic and colloid osmotic pressure and addresses the need for increased lymph flow to maintain optimal conditions in the muscle. It is clear that exercise increases skeletal muscle lymph flow significantly in both animals [11] and humans [12], especially at the beginning of exercise. Studies showed that skeletal muscles contain small capillary-sized lymphatic vessels, which are located next to blood capillaries between muscle fibers but are much fewer in number.

### **2. Stimulators of vascularization in skeletal muscle**

The studies showed that hypoxia, shear stress, adenosine, and muscle stretch are the most important stimulators of the angiogenesis process which are more fully described.

**7**

**Figure 4.**

*Vascularisation of Skeletal Muscle*

**2.1 Hypoxia**

**2.2 Shear stress**

membrane [14].

*DOI: http://dx.doi.org/10.5772/intechopen.85903*

Tissue hypoxia is thought to upregulate a series of local factors that contribute to angiogenesis. The status of tissue oxygenation determines blood vessels to undergo angiogenesis or stay quiescent. Accumulation of hypoxia-inducible factor (HIF)-α under hypoxia condition triggers tissue angiogenesis. HIF-α plays a pivotal role in the transcriptional activation of genes encoding angiogenic factors. Briefly, as previously described by Fong et al. [13], HIF-α abundance is negatively regulated by a subfamily of deoxygenates referred to as prolyl hydroxylase domain-containing proteins (PHDs), which use O2 as a substrate to hydroxylate HIF-α subunits and hence tag them for rapid degradation (**Figure 4**). Under hypoxic conditions, HIF-α subunits accumulate due to reduced hydroxylation efficiency and form transcriptionally active heterodimers with HIF-1β to activate the expression of angiogenic factors and other proteins important for cellular adaptation to hypoxia. Angiogenesis is regulated by a combination of at least two different mechanisms. The paracrine mechanism is mediated by nonendothelial expression of angiogenic factors such as vascular endothelial growth factor (VEGF)-A, which in turn interacts with endothelial cell surface receptors to initiate angiogenic activities. In the autocrine mechanism, endothelial cells themselves are induced to express VEGF-A, which collaborate with the paracrine mechanism to support angiogenesis and protect vascular integrity [13].

Relatively little is known about the importance of mechanical forces and the mechanisms of their transduction during the growth of vessels in skeletal muscle [14]. Repeated muscle contractions alter the local microcirculatory hemodynamics by dilatation of arterioles leading to increases in capillary flow velocity, shear stress (defined as shear stress = blood viscosity × (8 × mean flow velocity)/vessel diameter) [15] and, potentially, pressure. Capillary growth in response to shear stress proceeds by division of the lumen by endothelial cell protrusion and vessel splitting, without the requirement for disturbance and breakdown of the basement

According to the mechanotransduction hypothesis, shear-induced endothelial cell deformation, cytoskeletal perturbations, and increasing tension at the focal adhesion attachment sites activate the integrins, generating intracellular signals that

It is known that increased shear stress in endothelial cell cultures leads to an increase in protein expression of VEGF receptor 2 (Flk-1) [16]. Today it is clear that shear stress releases NO and increases VEGF and its receptor 2 expression during

*Regulatory mechanisms of PHD hydroxylase activities. Factors or processes with positive effects on PHD hydroxylase activities are shown in green, whereas those with inhibitory effects are shown in red [13].*

include transcription of genes involved in angiogenesis [15].

the initiation of endothelial cell proliferation and angiogenesis [14].

### **2.1 Hypoxia**

*Muscle Cells - Recent Advances and Future Perspectives*

to the enlargement of existing arterial vessels [6]. This enlargement indicates an increase in the caliber (diameter) and wall dimensions, resulting in a larger vessel. It is similar to angiogenesis in some features, but the pathways make it different [7]. Angiogenesis occurs under hypoxia/ischemia condition, which leads to the activation of the transcription factor HIF (hypoxia-inducible factor); in contrast,

Growth of arterioles in skeletal muscle has been documented during

Transformation of pericytes into smooth muscle cells and/or apposition of mesenchymal cells, most likely fibroblasts, to the abluminal surface of capillaries, followed by their gradual change into pericytes or smooth muscle cells lead to the growth of arteriolar known as "arteriolarization" [9]. Arteriolar growth accompa-

Lymphatic vessels act in close relation with blood vessels. The formed lymph fluid is transported via initial lymphatic capillaries to collecting vessels, to lymph nodes, and finally back to the blood [10]. Hyperemia-induced increased filtration of skeletal muscle (during activity), promotes hydrostatic and colloid osmotic pressure and addresses the need for increased lymph flow to maintain optimal conditions in the muscle. It is clear that exercise increases skeletal muscle lymph flow significantly in both animals [11] and humans [12], especially at the beginning of exercise. Studies showed that skeletal muscles contain small capillary-sized lymphatic vessels, which are located next to blood capillaries between muscle fibers but are much fewer in number.

The studies showed that hypoxia, shear stress, adenosine, and muscle stretch are the most important stimulators of the angiogenesis process which are more fully

arteriogenesis is activated in an environment of normoxia [8].

**2. Stimulators of vascularization in skeletal muscle**

nied rather than followed capillary growth.

*Differential angiogenic responses in vivo [5].*

**6**

described.

development.

**Figure 3.**

**1.4 Lymphogenesis**

Tissue hypoxia is thought to upregulate a series of local factors that contribute to angiogenesis. The status of tissue oxygenation determines blood vessels to undergo angiogenesis or stay quiescent. Accumulation of hypoxia-inducible factor (HIF)-α under hypoxia condition triggers tissue angiogenesis. HIF-α plays a pivotal role in the transcriptional activation of genes encoding angiogenic factors. Briefly, as previously described by Fong et al. [13], HIF-α abundance is negatively regulated by a subfamily of deoxygenates referred to as prolyl hydroxylase domain-containing proteins (PHDs), which use O2 as a substrate to hydroxylate HIF-α subunits and hence tag them for rapid degradation (**Figure 4**). Under hypoxic conditions, HIF-α subunits accumulate due to reduced hydroxylation efficiency and form transcriptionally active heterodimers with HIF-1β to activate the expression of angiogenic factors and other proteins important for cellular adaptation to hypoxia. Angiogenesis is regulated by a combination of at least two different mechanisms. The paracrine mechanism is mediated by nonendothelial expression of angiogenic factors such as vascular endothelial growth factor (VEGF)-A, which in turn interacts with endothelial cell surface receptors to initiate angiogenic activities. In the autocrine mechanism, endothelial cells themselves are induced to express VEGF-A, which collaborate with the paracrine mechanism to support angiogenesis and protect vascular integrity [13].

### **2.2 Shear stress**

Relatively little is known about the importance of mechanical forces and the mechanisms of their transduction during the growth of vessels in skeletal muscle [14]. Repeated muscle contractions alter the local microcirculatory hemodynamics by dilatation of arterioles leading to increases in capillary flow velocity, shear stress (defined as shear stress = blood viscosity × (8 × mean flow velocity)/vessel diameter) [15] and, potentially, pressure. Capillary growth in response to shear stress proceeds by division of the lumen by endothelial cell protrusion and vessel splitting, without the requirement for disturbance and breakdown of the basement membrane [14].

According to the mechanotransduction hypothesis, shear-induced endothelial cell deformation, cytoskeletal perturbations, and increasing tension at the focal adhesion attachment sites activate the integrins, generating intracellular signals that include transcription of genes involved in angiogenesis [15].

It is known that increased shear stress in endothelial cell cultures leads to an increase in protein expression of VEGF receptor 2 (Flk-1) [16]. Today it is clear that shear stress releases NO and increases VEGF and its receptor 2 expression during the initiation of endothelial cell proliferation and angiogenesis [14].

### **Figure 4.**

*Regulatory mechanisms of PHD hydroxylase activities. Factors or processes with positive effects on PHD hydroxylase activities are shown in green, whereas those with inhibitory effects are shown in red [13].*

### **2.3 Adenosine**

Of the major metabolites produced and released by exercising skeletal muscle, adenosine has received the greatest attention as an angiogenic factor. Adenosine is generated as ATP is catabolized when energy demands increase or oxygen supply decreases [17]. Feoktistov et al. demonstrated that stimulation of adenosine A2B receptors upregulates the angiogenic factors VEGF and IL-8 in human endothelial cells under normoxic conditions [18]. Studies in other cell culture models conducted under normoxic conditions have also indicated that adenosine upregulates proangiogenic and downregulates antiangiogenic factors [19]. Remarkably, chronic infusion of adenosine induced neovascularisation in the skeletal muscle and the heart muscle [20].

### **2.4 Muscle stretch and exercise**

Original studies demonstrated that stretch of cells promotes VEGF (mRNA and protein), which leads to enhanced endothelial cell migration and tube formation, and activates MT1-MMP, and upregulates Ang-2 and Tie-2 expression. Stretch also causes deformation of the extracellular matrix, and may result in the release of matrix-bound growth factors, that then can bind to and activate the surrounding cells. Furthermore, stretch induces tensional forces that initiate mechanotransduction signaling pathways through activation of integrin receptors [15].

When specific muscles are exercise trained, there can be an increase in flow capacity and an expected increase in the caliber of the large conduit vessels [21]. In this regard, Hounker et al. demonstrated that the subclavian arteries of the dominant arms of elite tennis players exhibited larger diameters than control group arteries [22]. Also, increased diameters of the femoral arteries were observed in elite cyclists, whereas decreases were observed with paraplegia compared with corresponding controls [21]. This increase in flow capacity to the major muscle groups of highly trained individuals could contribute to the greater exercise capacity exhibited by these individuals. Capillarity in active skeletal muscle is significantly increased by endurance exercise training although the increase in the heart muscle is less well established.

In this regard, laboratory studies indicate muscle mass loss [23], increased number of fast-twitch fibers, decreased slow-twitch fibers [24], and restriction of skeletal muscle blood flow in humans [25] and animals [26] after myocardial infarction. In addition, systemic blood flow in the skeletal muscle of mice with heart failure decreases at rest and during physical activity [27]. Experimental studies at the capillary level show that capillary density parameters [28] and capillary/fiber ratio (CF ratio) decrease following myocardial infarction. On the other hand, at the arteriole level, the arteriole tonic sympathetic vasoconstriction activity increases that decreases the arteriolar diameter and elasticity capacity [29].

A remaining question is what signaling cascade within the muscle fibers decodes muscle contractile activity signals in regulating VEGF expression. Mechanistically, the functional role of PGC-1α in exercise-induced VEGF expression and angiogenesis is dependent on the upstream p38 mitogen-activated protein kinase (p38ɤ MAPK) [30] and the downstream ERRα [31]. Interestingly, transgenic mice with muscle-specific expression of an inactive form of 5′-adenosine monophosphateactivated protein kinase (AMPK) have lower capillarity compared with the wildtype littermates, but have normal-induced angiogenesis in response to voluntary running exercise [32, 33].

The importance of other growth factor pathways remains to be elucidated. Although several signaling pathways have been identified (**Figure 5**), significant

**9**

and diabetes.

**Figure 5.**

blood flow.

**3. Fiber type and vascularization**

least mitochondrial volume and capillarization.

must be better supplied with a capillary network [35].

not accompanied by changes in oxidative metabolism [37, 38].

*Vascularisation of Skeletal Muscle*

*DOI: http://dx.doi.org/10.5772/intechopen.85903*

work remains on understanding the intracellular signaling pathways and transcrip-

*Proposed model including intracellular signaling and growth factor regulation in skeletal muscle. Solid lines* 

It is well known that the vascularization of skeletal muscle adapts to various physiological and pathological conditions such as fiber type, gender, aging, obesity,

The skeletal muscle is composed of a combination of different muscle fiber types: I, IIa, and IId/x. Fast- and slow-twitch fibers have different phenotypes with Type I fibers demonstrating the greatest and Type IId/x fibers demonstrating the

It has been confirmed that muscle fibers with a high oxidative potential are related to a denser capillary network. This would mean that highly oxidative fibers require a higher rate of oxygen delivery than nonoxidative fibers; therefore, they

Capillary density did not always correlate with oxidative capacity or maximal

Already, at first, some researchers showed that capillary density is not related to the type of muscles, but hypothesized that the mean number of capillary profiles around a fiber for red and white muscles due to the size of the fiber. Sullivan and Pitman confirmed that capillaries around glycolytic fibers must supply blood

Previous studies demonstrated that after triiodothyronine administration, rat soleus and white area of the medial head of gastrocnemius muscle capillarity are promoted, whereas the oxidative capacity increased in the soleus only [36]. Therefore, in strait skeletal muscles, oxidative capacity is not the only factor that determines capillarity. On the other hand, it is likely that anaerobic waste accumulation stimulates vascularization in glycolytic regions of rat skeletal muscles, which is

tion factors involved in basal- and exercise-induced angiogenesis [34].

*(—) are established pathways. Dashed lines (- -) are hypothesized pathways [34].*

*Vascularisation of Skeletal Muscle DOI: http://dx.doi.org/10.5772/intechopen.85903*

### **Figure 5.**

*Muscle Cells - Recent Advances and Future Perspectives*

Of the major metabolites produced and released by exercising skeletal muscle, adenosine has received the greatest attention as an angiogenic factor. Adenosine is generated as ATP is catabolized when energy demands increase or oxygen supply decreases [17]. Feoktistov et al. demonstrated that stimulation of adenosine A2B receptors upregulates the angiogenic factors VEGF and IL-8 in human endothelial cells under normoxic conditions [18]. Studies in other cell culture models conducted under normoxic conditions have also indicated that adenosine upregulates proangiogenic and downregulates antiangiogenic factors [19]. Remarkably, chronic infusion of adenosine induced neovascularisation in the skeletal muscle and the

Original studies demonstrated that stretch of cells promotes VEGF (mRNA and protein), which leads to enhanced endothelial cell migration and tube formation, and activates MT1-MMP, and upregulates Ang-2 and Tie-2 expression. Stretch also causes deformation of the extracellular matrix, and may result in the release of matrix-bound growth factors, that then can bind to and activate the surrounding cells. Furthermore, stretch induces tensional forces that initiate mechanotransduc-

When specific muscles are exercise trained, there can be an increase in flow capacity and an expected increase in the caliber of the large conduit vessels [21]. In this regard, Hounker et al. demonstrated that the subclavian arteries of the dominant arms of elite tennis players exhibited larger diameters than control group arteries [22]. Also, increased diameters of the femoral arteries were observed in elite cyclists, whereas decreases were observed with paraplegia compared with corresponding controls [21]. This increase in flow capacity to the major muscle groups of highly trained individuals could contribute to the greater exercise capacity exhibited by these individuals. Capillarity in active skeletal muscle is significantly increased by endurance exercise training although the increase in the heart muscle

In this regard, laboratory studies indicate muscle mass loss [23], increased number of fast-twitch fibers, decreased slow-twitch fibers [24], and restriction of skeletal muscle blood flow in humans [25] and animals [26] after myocardial infarction. In addition, systemic blood flow in the skeletal muscle of mice with heart failure decreases at rest and during physical activity [27]. Experimental studies at the capillary level show that capillary density parameters [28] and capillary/fiber ratio (CF ratio) decrease following myocardial infarction. On the other hand, at the arteriole level, the arteriole tonic sympathetic vasoconstriction activity increases

A remaining question is what signaling cascade within the muscle fibers decodes muscle contractile activity signals in regulating VEGF expression. Mechanistically, the functional role of PGC-1α in exercise-induced VEGF expression and angiogenesis is dependent on the upstream p38 mitogen-activated protein kinase (p38ɤ MAPK) [30] and the downstream ERRα [31]. Interestingly, transgenic mice with muscle-specific expression of an inactive form of 5′-adenosine monophosphateactivated protein kinase (AMPK) have lower capillarity compared with the wildtype littermates, but have normal-induced angiogenesis in response to voluntary

The importance of other growth factor pathways remains to be elucidated. Although several signaling pathways have been identified (**Figure 5**), significant

that decreases the arteriolar diameter and elasticity capacity [29].

tion signaling pathways through activation of integrin receptors [15].

**2.3 Adenosine**

heart muscle [20].

is less well established.

running exercise [32, 33].

**2.4 Muscle stretch and exercise**

**8**

*Proposed model including intracellular signaling and growth factor regulation in skeletal muscle. Solid lines (—) are established pathways. Dashed lines (- -) are hypothesized pathways [34].*

work remains on understanding the intracellular signaling pathways and transcription factors involved in basal- and exercise-induced angiogenesis [34].

It is well known that the vascularization of skeletal muscle adapts to various physiological and pathological conditions such as fiber type, gender, aging, obesity, and diabetes.

### **3. Fiber type and vascularization**

The skeletal muscle is composed of a combination of different muscle fiber types: I, IIa, and IId/x. Fast- and slow-twitch fibers have different phenotypes with Type I fibers demonstrating the greatest and Type IId/x fibers demonstrating the least mitochondrial volume and capillarization.

It has been confirmed that muscle fibers with a high oxidative potential are related to a denser capillary network. This would mean that highly oxidative fibers require a higher rate of oxygen delivery than nonoxidative fibers; therefore, they must be better supplied with a capillary network [35].

Capillary density did not always correlate with oxidative capacity or maximal blood flow.

Previous studies demonstrated that after triiodothyronine administration, rat soleus and white area of the medial head of gastrocnemius muscle capillarity are promoted, whereas the oxidative capacity increased in the soleus only [36]. Therefore, in strait skeletal muscles, oxidative capacity is not the only factor that determines capillarity. On the other hand, it is likely that anaerobic waste accumulation stimulates vascularization in glycolytic regions of rat skeletal muscles, which is not accompanied by changes in oxidative metabolism [37, 38].

Already, at first, some researchers showed that capillary density is not related to the type of muscles, but hypothesized that the mean number of capillary profiles around a fiber for red and white muscles due to the size of the fiber. Sullivan and Pitman confirmed that capillaries around glycolytic fibers must supply blood

stream to a greater volume of a muscle fiber than capillaries serving oxidative fibers [39]. In this regard, independent of fiber type in human muscles, there is a positive correlation between local capillary-to-fiber ratio (LCFR) and fiber area [40].

Cebasek and co-workers showed that the length of capillaries per unit fiber length was larger in Soleus (Slow muscle) than in Extensor Digitorum Longus (EDL, Fast muscle) muscle. On the other hand, these researchers showed that capillary length per unit fiber volume was larger in EDL muscle. There was no difference in the length of capillaries per unit fiber surface area between the two muscles. Oxidative and glycolytic fibers differ in the length of capillaries per unit fiber surface area. This parameter probably reflects the oxidative capacity of muscle fibers [41].

Ranjbar et al. showed that the pattern of vascularization in response to exercise training is different between fast- and slow-twitch muscles. We showed that the 10-week exercise training significantly increased capillary density and capillary-tofiber ratio (P < 0.05) in slow-twitch muscle, but did not change fast-twitch muscle capillary density and capillary-to-fiber ratio. Furthermore, arteriolar density in fast-twitch muscle increased remarkably (P < 0.05) in response to training, but slow-twitch muscle arteriolar density did not change in response to exercise in chronic heart failure rats. HIF-1 increased (P < 0.01) but VEGF and FGF-2 mRNA did not change in slow-twitch muscle after training. In fast-twitch muscle, HIF-1 mRNA increased (P < 0.05), and VEGF and angiostatin decreased (P < 0.01) significantly after training. We concluded that endurance training ameliorates fastand slow-twitch muscle revascularization nonuniformly in chronic heart failure rats by increasing capillary density in slow-twitch muscle and arteriolar density in fasttwitch muscle. The difference in revascularization at slow- and fast-twitch muscles may be induced by the difference in angiogenic and angiostatic gene expression response to endurance training [42].

In this regard, we showed that smaller arterioles decreased in cardiac after myocardial infarction. Aerobic training and l-arginine increased the number of cardiac arterioles with 11–25 and 26–50 μm diameters parallel to TGF-β overexpression. In gastrocnemius muscle, the number of arterioles/mm2 was only increased in the 11–25 μm in response to training with and without l-rginine parallel to angiostatin downregulation. Soleus arteriolar density with different sizes was not different between experimental groups. Results showed that 10 weeks aerobic exercise training and l-arginine supplementation promotes arteriogenesis of heart and gastrocnemius muscles parallel to overexpression of TGF-β and downregulation of angiostatin in myocardial infarction rats [43].

On the other hand, Panisello et al. showed that SO fibers are more sensitive to intermittent hypobaric hypoxia than both fast fiber types [44]. Furthermore, Murakami et al. showed that capillary-to-fiber ratio, Microvessel diameter, expression level of VEGF, and number of microvessels in the soleus were significantly higher than those in the EDL muscle [45].

In conclusion, capillary supply is evidently well adapted to different muscle fiber types; consequently, an average capillary supply of heterogeneous muscle depends on the muscle composition.

### **4. Gender**

Numerous studies have reported on the physiological differences among genders, but the effect of gender on neovascularization of skeletal muscle is not yet clear, and research in this field is limited.

**11**

muscle.

individuals [61].

**6. Obesity**

*Vascularisation of Skeletal Muscle*

**5. Aging**

*DOI: http://dx.doi.org/10.5772/intechopen.85903*

larity still remain controversial [49].

activity levels and gender [55].

to endothelial cell dysfunction with aging [21].

than in women (1.12 ± 0.03 endothelial cells × fiber<sup>−</sup><sup>1</sup>

Robbins et al. showed that men had a greater capillary-to-fiber ratio than women. However, capillary density per square millimeter was not different between men and women [46]. Also, Kyriakides et al. showed that gender does not influence angiogenesis and arteriogenesis in the rabbit model of chronic hind limb ischemia [47]. Opposite to these findings, Keteyian et al. showed that capillary density at baseline was significantly greater in men (1.42 ± 0.08 endothelial cells × fiber <sup>−</sup><sup>1</sup>

Aging effects on the structure and function of skeletal muscles have been intensely studied for decades; however, age-related changes in skeletal muscle capil-

Studies in both humans and animals have also produced conflicting results in skeletal muscle capillarity, with results in increases [50, 51], decreases [52, 53], or no change [54] in muscle capillaries with advancing age. These inconsistent reports could be due to a difference in factors, such as muscle type, fiber-type composition, and how this composition changes during aging, as well as to differences in subject

In general, it is believed that aging reduces the ability of an organism to respond

Obesity is a major health problem in the United States and many other developed countries. Several lines of evidence have demonstrated that the capillary density and oxidative capacity appear to be more strongly correlated with insulin sensitivity

Increasing adiposity is associated with lower skeletal muscle oxidative capacity and capillarization. Plasma insulin elevation does not result in insulin increment in the interstitial space concentration between the capillary and muscle fiber [60], suggesting that a limit may exist in the diffusional conductance of insulin in skeletal

Research studies have shown that there are associations between lower insulin action and a lower muscle capillary-to-fiber area ratio in obese muscle. It is likely that compared with lean individual, a delayed transport of insulin over the capillary wall may be attributed to lower skeletal muscle capillary density in obese

and body fatness than the prevalence of a specific fiber type [59].

to different types of stress [56]. For example, the angiogenic response to hind limb ischemia is impaired in aged compared with young mice [57, 58]. Researchers showed reduced angiogenic capacity in skeletal muscle with aging. Reduction of muscular oxidative capacity attainable through training in elderly people is related to the reduction in fitness that typically occurs in this population. The decrease in muscle blood flow in the aging period is related to altered reactivity of resistance arteries and arterioles, with impairment of both vasodilator and vasoconstrictor responses as a consequence of endothelial dysfunction [21]. Whilst aging and sedentary life style both lead to structural and functional impairment in skeletal muscle blood supply network, it is not yet clear whether impaired angiogenesis is an indirect response to reduced flow capacity. It is clear that reduction of VEGF secretion with maintaining of the ability to respond to exogenous cytokines is due

) [48].

)

### *Vascularisation of Skeletal Muscle DOI: http://dx.doi.org/10.5772/intechopen.85903*

Robbins et al. showed that men had a greater capillary-to-fiber ratio than women. However, capillary density per square millimeter was not different between men and women [46]. Also, Kyriakides et al. showed that gender does not influence angiogenesis and arteriogenesis in the rabbit model of chronic hind limb ischemia [47]. Opposite to these findings, Keteyian et al. showed that capillary density at baseline was significantly greater in men (1.42 ± 0.08 endothelial cells × fiber <sup>−</sup><sup>1</sup> ) than in women (1.12 ± 0.03 endothelial cells × fiber<sup>−</sup><sup>1</sup> ) [48].

### **5. Aging**

*Muscle Cells - Recent Advances and Future Perspectives*

muscle fibers [41].

response to endurance training [42].

angiostatin in myocardial infarction rats [43].

higher than those in the EDL muscle [45].

clear, and research in this field is limited.

on the muscle composition.

stream to a greater volume of a muscle fiber than capillaries serving oxidative fibers [39]. In this regard, independent of fiber type in human muscles, there is a positive correlation between local capillary-to-fiber ratio (LCFR) and fiber area [40]. Cebasek and co-workers showed that the length of capillaries per unit fiber length was larger in Soleus (Slow muscle) than in Extensor Digitorum Longus (EDL, Fast muscle) muscle. On the other hand, these researchers showed that capillary length per unit fiber volume was larger in EDL muscle. There was no difference in the length of capillaries per unit fiber surface area between the two muscles. Oxidative and glycolytic fibers differ in the length of capillaries per unit fiber surface area. This parameter probably reflects the oxidative capacity of

Ranjbar et al. showed that the pattern of vascularization in response to exercise training is different between fast- and slow-twitch muscles. We showed that the 10-week exercise training significantly increased capillary density and capillary-tofiber ratio (P < 0.05) in slow-twitch muscle, but did not change fast-twitch muscle capillary density and capillary-to-fiber ratio. Furthermore, arteriolar density in fast-twitch muscle increased remarkably (P < 0.05) in response to training, but slow-twitch muscle arteriolar density did not change in response to exercise in chronic heart failure rats. HIF-1 increased (P < 0.01) but VEGF and FGF-2 mRNA did not change in slow-twitch muscle after training. In fast-twitch muscle, HIF-1 mRNA increased (P < 0.05), and VEGF and angiostatin decreased (P < 0.01) significantly after training. We concluded that endurance training ameliorates fastand slow-twitch muscle revascularization nonuniformly in chronic heart failure rats by increasing capillary density in slow-twitch muscle and arteriolar density in fasttwitch muscle. The difference in revascularization at slow- and fast-twitch muscles may be induced by the difference in angiogenic and angiostatic gene expression

In this regard, we showed that smaller arterioles decreased in cardiac after myocardial infarction. Aerobic training and l-arginine increased the number of cardiac arterioles with 11–25 and 26–50 μm diameters parallel to TGF-β overexpression. In gastrocnemius muscle, the number of arterioles/mm2 was only increased in the 11–25 μm in response to training with and without l-rginine parallel to angiostatin downregulation. Soleus arteriolar density with different sizes was not different between experimental groups. Results showed that 10 weeks aerobic exercise training and l-arginine supplementation promotes arteriogenesis of heart and gastrocnemius muscles parallel to overexpression of TGF-β and downregulation of

On the other hand, Panisello et al. showed that SO fibers are more sensitive to intermittent hypobaric hypoxia than both fast fiber types [44]. Furthermore, Murakami et al. showed that capillary-to-fiber ratio, Microvessel diameter, expression level of VEGF, and number of microvessels in the soleus were significantly

In conclusion, capillary supply is evidently well adapted to different muscle fiber types; consequently, an average capillary supply of heterogeneous muscle depends

Numerous studies have reported on the physiological differences among genders, but the effect of gender on neovascularization of skeletal muscle is not yet

**10**

**4. Gender**

Aging effects on the structure and function of skeletal muscles have been intensely studied for decades; however, age-related changes in skeletal muscle capillarity still remain controversial [49].

Studies in both humans and animals have also produced conflicting results in skeletal muscle capillarity, with results in increases [50, 51], decreases [52, 53], or no change [54] in muscle capillaries with advancing age. These inconsistent reports could be due to a difference in factors, such as muscle type, fiber-type composition, and how this composition changes during aging, as well as to differences in subject activity levels and gender [55].

In general, it is believed that aging reduces the ability of an organism to respond to different types of stress [56]. For example, the angiogenic response to hind limb ischemia is impaired in aged compared with young mice [57, 58]. Researchers showed reduced angiogenic capacity in skeletal muscle with aging. Reduction of muscular oxidative capacity attainable through training in elderly people is related to the reduction in fitness that typically occurs in this population. The decrease in muscle blood flow in the aging period is related to altered reactivity of resistance arteries and arterioles, with impairment of both vasodilator and vasoconstrictor responses as a consequence of endothelial dysfunction [21]. Whilst aging and sedentary life style both lead to structural and functional impairment in skeletal muscle blood supply network, it is not yet clear whether impaired angiogenesis is an indirect response to reduced flow capacity. It is clear that reduction of VEGF secretion with maintaining of the ability to respond to exogenous cytokines is due to endothelial cell dysfunction with aging [21].

### **6. Obesity**

Obesity is a major health problem in the United States and many other developed countries. Several lines of evidence have demonstrated that the capillary density and oxidative capacity appear to be more strongly correlated with insulin sensitivity and body fatness than the prevalence of a specific fiber type [59].

Increasing adiposity is associated with lower skeletal muscle oxidative capacity and capillarization. Plasma insulin elevation does not result in insulin increment in the interstitial space concentration between the capillary and muscle fiber [60], suggesting that a limit may exist in the diffusional conductance of insulin in skeletal muscle.

Research studies have shown that there are associations between lower insulin action and a lower muscle capillary-to-fiber area ratio in obese muscle. It is likely that compared with lean individual, a delayed transport of insulin over the capillary wall may be attributed to lower skeletal muscle capillary density in obese individuals [61].

### **Figure 6.**

*Interactions between expanding adipose tissue and the endothelium [64].*

In this regard, researchers showed that VEGF circulation, myocardial VEGF expression, and VEGF receptor 2 [kinase insert domain-containing receptor (KDR) human/Flk-1 murine analog] were not different between lean and obese individuals, but VEGF receptor 1 (Flt-1) is lower in obese compared with lean Zucker rats [62, 63]. Gavin et al. showed lower capillary density but no difference in VEGF expression in obese versus lean young skeletal muscle in humans [61].

The interactions between expanding adipose tissue and the endothelium via adipokine secretions are shown in **Figure 6** [64].

### **7. Diabetes**

Another factor that affects the process of neovascularization in skeletal muscle is diabetes. Diabetes is a risk factor for peripheral vascular diseases, and it is associated with impaired collateral vessel growth in skeletal muscle. Diabetes, in turn, has been demonstrated to impair skeletal muscle and cardiac angiogenesis, and the mechanisms underlying this have generated much interest recently. Both type 1 and type 2 diabetes have been shown to affect angiogenic growth factors and inhibitors in skeletal muscle [65, 66].

Several proangiogenic protein gene expressions decreased and increased those of antiangiogenic ones in in diabetic mouse skeletal muscle [66]. Hence, the imbalance between stimulators and inhibitors may lead to peripheral cardiovascular complications in diabetes [67, 68].

### **8. Angiogenic factors**

To better understand the neovascularization of skeletal muscle, it is important to understand the current state of knowledge regarding different factors possessing angiogenic properties. The process of angiogenesis in skeletal muscle is controlled by a number of factors that are released in the tissues surrounding the small vessel involved, and it is thought to be controlled by net balance between pro-angiogenic (angiogenic) and anti-angiogenesis (angiostatic) factors. In skeletal muscle, the

**13**

*Vascularisation of Skeletal Muscle*

termed the "angiogenic switch".

is presented here.

**8.1 VEGF**

206 [72].

**8.2 FGFs**

**(PDGF-BB)**

*DOI: http://dx.doi.org/10.5772/intechopen.85903*

balance of proangiogenic factors with antiangiogenic factors controls the extent of microvascular growth. For angiogenesis to occur, the balance of proangiogenic and antiangiogenic factors must favor the proangiogenic factors, and this has been

Vascular endothelial growth factor (VEGF) is a 45 kDa secretable basic heparin-binding homodimeric glycoprotein. The human VEGF gene has been assigned to chromosome 6p21.3. Hypoxia is the main stimulus for VEGF production/expression. Observational studies support VEGF expression as an important factor for regulating skeletal muscle angiogenesis in both humans and animals [69, 70]. In patients with chronic disease conditions (e.g., chronic obstructive pulmonary disease, heart failure, and diabetes), as well as aging, locomotor skeletal muscle VEGF expression has also been reported to be lower, and these individuals often exhibit muscle weakness, reduced physical activity, and loss of skeletal muscle vascular density. A similar phenotype is found in sedentary (untrained) myocyte-specific VEGF gene ablated mice, which exhibit impaired exercise capacity and >50% loss of skeletal muscle microvessel density [71]. The human VEGF gene contains eight exons, seven introns, and a 14 kb coding region. VEGF has six different isoforms including VEGF165 (the predominant isoform), VEGF121, VEGF145, VEGF183, VEGF189, and VEGF

Binding of VEGF to the Flt-1 and KDR surface receptors activates their tyrosine kinase function resulting in enhanced endothelial cell proliferation, migration, vascular permeability, and protease activity [72, 73]. **Figure 7** shows several VEGF

Currently, the family of FGFs consists of more than 20 members with 30.70% homology. FGFs are multifunctional proteins that bind to five cell membrane tyrosine kinase receptors (FGFR-1.5) and stimulate proliferation of a variety of cell types, including endothelial cells (ECs) and smooth muscle cells (SMCs). FGFs play a role in development, tissue regeneration, hematopoiesis, angiogenesis, and tumorigenesis. In vitro FGF-1, -2, -4, and -9 exert the highest mitogenic activity. Of these, FGF-1 and FGF-2 have previously been shown to induce therapeutic angiogenesis in vivo [75]. To the best of our knowledge, there are no published data about

**8.3 Transforming growth factors (TGF-b) and platelet-derived growth factors** 

Unlike VEGF and FGF, which are directly involved in the angiogenic process, TGF-b and PDGF-BB indirectly contribute to the angiogenic process. Mice lacking these two indicators die in utero due to defects in the process of vascular maturation. TGF and PDGF shear stress elements mediate upregulation of these factors in

receptor signal transduction pathways that are known thus far.

the potency of FGF-4 and FGF-9 in animal models [76].

endothelial cells in response to increased shear stress in vitro.

Therefore, a brief summary about the most well-known angiogenic (VEGF, FGF, and TGF) and angiostatic factors (endostatin, angiostatin, and thrombospondin-1)

### *Vascularisation of Skeletal Muscle DOI: http://dx.doi.org/10.5772/intechopen.85903*

balance of proangiogenic factors with antiangiogenic factors controls the extent of microvascular growth. For angiogenesis to occur, the balance of proangiogenic and antiangiogenic factors must favor the proangiogenic factors, and this has been termed the "angiogenic switch".

Therefore, a brief summary about the most well-known angiogenic (VEGF, FGF, and TGF) and angiostatic factors (endostatin, angiostatin, and thrombospondin-1) is presented here.

### **8.1 VEGF**

*Muscle Cells - Recent Advances and Future Perspectives*

In this regard, researchers showed that VEGF circulation, myocardial VEGF expression, and VEGF receptor 2 [kinase insert domain-containing receptor (KDR) human/Flk-1 murine analog] were not different between lean and obese individuals, but VEGF receptor 1 (Flt-1) is lower in obese compared with lean Zucker rats [62, 63]. Gavin et al. showed lower capillary density but no difference in VEGF

The interactions between expanding adipose tissue and the endothelium via

Another factor that affects the process of neovascularization in skeletal muscle is diabetes. Diabetes is a risk factor for peripheral vascular diseases, and it is associated with impaired collateral vessel growth in skeletal muscle. Diabetes, in turn, has been demonstrated to impair skeletal muscle and cardiac angiogenesis, and the mechanisms underlying this have generated much interest recently. Both type 1 and type 2 diabetes have been shown to affect angiogenic growth factors and inhibitors

Several proangiogenic protein gene expressions decreased and increased those of antiangiogenic ones in in diabetic mouse skeletal muscle [66]. Hence, the imbalance between stimulators and inhibitors may lead to peripheral cardiovascular

To better understand the neovascularization of skeletal muscle, it is important to understand the current state of knowledge regarding different factors possessing angiogenic properties. The process of angiogenesis in skeletal muscle is controlled by a number of factors that are released in the tissues surrounding the small vessel involved, and it is thought to be controlled by net balance between pro-angiogenic (angiogenic) and anti-angiogenesis (angiostatic) factors. In skeletal muscle, the

expression in obese versus lean young skeletal muscle in humans [61].

adipokine secretions are shown in **Figure 6** [64].

*Interactions between expanding adipose tissue and the endothelium [64].*

**7. Diabetes**

**Figure 6.**

in skeletal muscle [65, 66].

**8. Angiogenic factors**

complications in diabetes [67, 68].

**12**

Vascular endothelial growth factor (VEGF) is a 45 kDa secretable basic heparin-binding homodimeric glycoprotein. The human VEGF gene has been assigned to chromosome 6p21.3. Hypoxia is the main stimulus for VEGF production/expression. Observational studies support VEGF expression as an important factor for regulating skeletal muscle angiogenesis in both humans and animals [69, 70]. In patients with chronic disease conditions (e.g., chronic obstructive pulmonary disease, heart failure, and diabetes), as well as aging, locomotor skeletal muscle VEGF expression has also been reported to be lower, and these individuals often exhibit muscle weakness, reduced physical activity, and loss of skeletal muscle vascular density. A similar phenotype is found in sedentary (untrained) myocyte-specific VEGF gene ablated mice, which exhibit impaired exercise capacity and >50% loss of skeletal muscle microvessel density [71]. The human VEGF gene contains eight exons, seven introns, and a 14 kb coding region. VEGF has six different isoforms including VEGF165 (the predominant isoform), VEGF121, VEGF145, VEGF183, VEGF189, and VEGF 206 [72].

Binding of VEGF to the Flt-1 and KDR surface receptors activates their tyrosine kinase function resulting in enhanced endothelial cell proliferation, migration, vascular permeability, and protease activity [72, 73]. **Figure 7** shows several VEGF receptor signal transduction pathways that are known thus far.

### **8.2 FGFs**

Currently, the family of FGFs consists of more than 20 members with 30.70% homology. FGFs are multifunctional proteins that bind to five cell membrane tyrosine kinase receptors (FGFR-1.5) and stimulate proliferation of a variety of cell types, including endothelial cells (ECs) and smooth muscle cells (SMCs). FGFs play a role in development, tissue regeneration, hematopoiesis, angiogenesis, and tumorigenesis. In vitro FGF-1, -2, -4, and -9 exert the highest mitogenic activity. Of these, FGF-1 and FGF-2 have previously been shown to induce therapeutic angiogenesis in vivo [75]. To the best of our knowledge, there are no published data about the potency of FGF-4 and FGF-9 in animal models [76].

### **8.3 Transforming growth factors (TGF-b) and platelet-derived growth factors (PDGF-BB)**

Unlike VEGF and FGF, which are directly involved in the angiogenic process, TGF-b and PDGF-BB indirectly contribute to the angiogenic process. Mice lacking these two indicators die in utero due to defects in the process of vascular maturation. TGF and PDGF shear stress elements mediate upregulation of these factors in endothelial cells in response to increased shear stress in vitro.

**Figure 7.**

*VEGF receptor signal transduction. Several signal transduction pathways are activated by the binding of VEGF to its receptor, leading to increased proliferation, survival, permeability, and migration of cells [74].*

PDGF also stimulates the proliferation of cultured smooth muscle cells and pericytes, both of which have been shown to express PDGF-b receptor [77].

TGF-b can control cell adhesion by regulating production of the extracellular matrix, stimulate or inhibit cell proliferation, protease inhibitors, and integrins, and induce cellular differentiation [78]. Much evidence points to an important role for TGF-b in the vasculature. It is clear that TGF-b recruits pericytes and smooth muscle cells in the arteriogenesis process [1].

### **8.4 Angiopoietin**

During development and angiogenesis in adult tissue, there is a close relationship between a new group of factors, angiopoietins, and VEGF. The angiopoietins include angiopoietin 1 (Ang-1) and angiopoietin 2 (Ang-2). Similar to VEGF, angiopoietin receptors are located on the endothelial cells. For this reason, their function has been observed on endothelial cells. [79, 80]. Binding of Ang-1 to its receptors (Tie-2) maintains and stabilizes mature vessels by promoting interaction between the endothelial cells and surrounding cells [73]. In contrast, Ang-2 is thought to block the Tie-2 receptor and leads to vessel regression [73, 81]. It should to be noted that angiopoietins function depending on the VEGF present.

### **8.5 MMPs**

Matrix metalloproteinases (MMPs) are a large family of protease enzymes and 26 members have been identified. Homeostasis of the extracellular matrix (ECM) in skeletal muscle dependent on MMP and TIMP balances [82]. The balance between angiogenic and anti-angiogenic factors interfere by MMPs via invoking angiogenic factors.

**15**

*Vascularisation of Skeletal Muscle*

the migration to cells [83].

blood flow [84].

**8.7 Follistatin-like 1**

**8.8 Angiomotin**

**8.6 Integrins**

*DOI: http://dx.doi.org/10.5772/intechopen.85903*

The ECM surrounds muscle fibers. ECM supports and protects muscle fiber and has a pivotal role in maintaining functional integrity of the fibers. Increased or decreased contractile activity of skeletal muscle promotes remodeling of the ECM. ECM degradation is a key step in the process of angiogenesis. Endothelial cells, stimulated by growth factors, produce MMPs that break down the cell membrane in physiological PH. Evidence is available that refers to the role of MMPs in the separation of smooth muscle cells from the extracellular matrix, and this allows

Although several MMPs are found in skeletal muscle, mainly MMP-2 and MMP-9 play a more important role in skeletal muscle. MMP-2 and MMP-9 play an important role in skeletal muscle adaptation to changing contractile demands and to response to injury. MMP-2 and MMP-9 degrade type IV collagen and have important functions in homeostasis of the ECM during morphogenesis, proliferation, and cell apoptosis in a wide range of tissues. Expression of MMP-2 in skeletal muscle was increased following administration of chronic electrical stimulation, and expression of MMP-9 was increased after exposure to a chronic increase in

The communication between endothelial cells and the surrounding tissue (extracellular matrix: ECM) is affected by stretching via various mechanisms. Integrins are heterodimeric cell-surface receptors that link the cytoskeleton to the ECM; therefore, according to their status, integrins are involved in the vascularization process. One member of the integrin family, integrin αvβ3, is expressed on the surface of newly formed cells but is barely detectable in mature vessels. Differential expression of the alpha v family members may play a role in EC migration and apoptosis, though their role appears to be more complex than at first thought due to bidirectional signaling properties. Furthermore, interfering with integrin αvβ<sup>3</sup> induces programmed cell death (apoptosis) in proliferating endothelial cells, which

Follistatin-like 1 (Fstl1), also referred to as TSC36, is an extracellular glycoprotein that, despite limited homology, has been grouped into the follistatin family of proteins. Fstl1 is poorly understood with regard to its functional significance. Studies indicated that Fstl1 is a secreted muscle protein or myokine that can function to promote endothelial cell function and stimulates revascularization in response to ischemic insult through its ability to activate Akt eNOS signaling [87].

Angiomotin was recently identified as a new pro-angiogenic molecule. Angiomotin was detected at the surface of blood vessels of both healthy and pathological tissues such as placenta, retina, Kaposi's sarcoma, and breast tumors. Alternative splicing of angiomotin mRNA results in two protein isoforms of 80 and 130 kDa that exert very distinct roles during angiogenesis [88]. The p80 angiomotin isoform strongly stimulates *in vitro* and *in vivo* the migration of endothelial cells, a key event of the angiogenic process. Interestingly, angiostatin binding to angiomotin extracellular domain strongly inhibits such an effect. In contrast, p130 angiomotin isoform only exerts a very weak stimulatory effect on endothelial cell migration. The p130 protein was identified as tightly associated to cytoskeleton actin filaments

suggests its importance for the angiogenic process [80, 85, 86].

### *Vascularisation of Skeletal Muscle DOI: http://dx.doi.org/10.5772/intechopen.85903*

The ECM surrounds muscle fibers. ECM supports and protects muscle fiber and has a pivotal role in maintaining functional integrity of the fibers. Increased or decreased contractile activity of skeletal muscle promotes remodeling of the ECM. ECM degradation is a key step in the process of angiogenesis. Endothelial cells, stimulated by growth factors, produce MMPs that break down the cell membrane in physiological PH. Evidence is available that refers to the role of MMPs in the separation of smooth muscle cells from the extracellular matrix, and this allows the migration to cells [83].

Although several MMPs are found in skeletal muscle, mainly MMP-2 and MMP-9 play a more important role in skeletal muscle. MMP-2 and MMP-9 play an important role in skeletal muscle adaptation to changing contractile demands and to response to injury. MMP-2 and MMP-9 degrade type IV collagen and have important functions in homeostasis of the ECM during morphogenesis, proliferation, and cell apoptosis in a wide range of tissues. Expression of MMP-2 in skeletal muscle was increased following administration of chronic electrical stimulation, and expression of MMP-9 was increased after exposure to a chronic increase in blood flow [84].

### **8.6 Integrins**

*Muscle Cells - Recent Advances and Future Perspectives*

muscle cells in the arteriogenesis process [1].

**8.4 Angiopoietin**

**Figure 7.**

on the VEGF present.

**8.5 MMPs**

PDGF also stimulates the proliferation of cultured smooth muscle cells and pericytes, both of which have been shown to express PDGF-b receptor [77].

*VEGF receptor signal transduction. Several signal transduction pathways are activated by the binding of VEGF to its receptor, leading to increased proliferation, survival, permeability, and migration of cells [74].*

TGF-b can control cell adhesion by regulating production of the extracellular matrix, stimulate or inhibit cell proliferation, protease inhibitors, and integrins, and induce cellular differentiation [78]. Much evidence points to an important role for TGF-b in the vasculature. It is clear that TGF-b recruits pericytes and smooth

During development and angiogenesis in adult tissue, there is a close relationship between a new group of factors, angiopoietins, and VEGF. The angiopoietins include angiopoietin 1 (Ang-1) and angiopoietin 2 (Ang-2). Similar to VEGF, angiopoietin receptors are located on the endothelial cells. For this reason, their function has been observed on endothelial cells. [79, 80]. Binding of Ang-1 to its receptors (Tie-2) maintains and stabilizes mature vessels by promoting interaction between the endothelial cells and surrounding cells [73]. In contrast, Ang-2 is thought to block the Tie-2 receptor and leads to vessel regression [73, 81]. It should to be noted that angiopoietins function depending

Matrix metalloproteinases (MMPs) are a large family of protease enzymes and 26 members have been identified. Homeostasis of the extracellular matrix (ECM) in skeletal muscle dependent on MMP and TIMP balances [82]. The balance between angiogenic and anti-angiogenic factors interfere by MMPs via invoking angiogenic

**14**

factors.

The communication between endothelial cells and the surrounding tissue (extracellular matrix: ECM) is affected by stretching via various mechanisms. Integrins are heterodimeric cell-surface receptors that link the cytoskeleton to the ECM; therefore, according to their status, integrins are involved in the vascularization process. One member of the integrin family, integrin αvβ3, is expressed on the surface of newly formed cells but is barely detectable in mature vessels. Differential expression of the alpha v family members may play a role in EC migration and apoptosis, though their role appears to be more complex than at first thought due to bidirectional signaling properties. Furthermore, interfering with integrin αvβ<sup>3</sup> induces programmed cell death (apoptosis) in proliferating endothelial cells, which suggests its importance for the angiogenic process [80, 85, 86].

### **8.7 Follistatin-like 1**

Follistatin-like 1 (Fstl1), also referred to as TSC36, is an extracellular glycoprotein that, despite limited homology, has been grouped into the follistatin family of proteins. Fstl1 is poorly understood with regard to its functional significance.

Studies indicated that Fstl1 is a secreted muscle protein or myokine that can function to promote endothelial cell function and stimulates revascularization in response to ischemic insult through its ability to activate Akt eNOS signaling [87].

### **8.8 Angiomotin**

Angiomotin was recently identified as a new pro-angiogenic molecule. Angiomotin was detected at the surface of blood vessels of both healthy and pathological tissues such as placenta, retina, Kaposi's sarcoma, and breast tumors. Alternative splicing of angiomotin mRNA results in two protein isoforms of 80 and 130 kDa that exert very distinct roles during angiogenesis [88]. The p80 angiomotin isoform strongly stimulates *in vitro* and *in vivo* the migration of endothelial cells, a key event of the angiogenic process. Interestingly, angiostatin binding to angiomotin extracellular domain strongly inhibits such an effect. In contrast, p130 angiomotin isoform only exerts a very weak stimulatory effect on endothelial cell migration. The p130 protein was identified as tightly associated to cytoskeleton actin filaments

and highly involved in vessel stabilization and maturation [89]. Interestingly, skeletal muscles represent the most abundant tissue of the body and they express high levels of angiomotin. Moreover, microcirculation is a critical component of muscle function since capillaries provide myofibers with oxygen and nutriments, and remove carbon dioxide and metabolic waste. As myofibers respond to physiological or pathological conditions with a remarkable plasticity, it is crucial that the microcirculation remains well matched with the myofibers' needs in order to preserve muscle function. Depending on conditions, such muscle angio-adaptation can involve either angiogenesis or some vascular regression. Given its role not only during angiogenesis but also for vessel stabilization and maturation, angiomotin might thus represent an important factor in muscle angio-adaptation. To date, angiomotin expression in skeletal muscle has never been investigated in response to physiological or pathological conditionings [89].

### **8.9 EPH-B4/EPHRIN-B2**

A unique class of receptor/ligand pair, Eph receptors and ephrin ligands, plays a prominent role in blood vessel development. Interestingly, not only does an ephrin expressed on the surface of one cell bind and activate its cognate Eph receptor on another cell, but through a reciprocal signaling mechanism the ephrin is also activated upon receptor engagement [90].

Ephrin-B2 as a member of the ephrin family is explicit on arterial endothelial cells, and its receptor eph-B4 is localized to venous endothelial cells.

Interaction of ephrin-B2 and its receptor has determined the primary capillary plexus after vasculogenesis [91]. The exact role of this interaction in the angiogenic process is not completely clear. But what has been approved is that establishment of contact and signaling between arterial and venous compartments mediated by ephrin-B2 and eph-4B is necessary for remodeling of the established primary capillary plexus [1].

### **9. Angiostatic factors**

Vascularization can be suppressed at any of a number of key steps in this process by endothelial growth cycle disruption in which a quiescent vessel becomes an actively growing and invading endothelial tube.

In this regard, vasculogenesis can be blocked by different factors. Degradation of the extracellular matrix by activated endothelial cells can also be prevented, which suppresses sprouting and invasion of growing capillaries into their surroundings. Alternatively, endothelial cell proliferation can be inhibited by agents that block signaling within the cell and arrest its division cycle or by agents that prevent the maturation of nascent endothelial cells into functional tubes .Finally, endothelial cells can be forced to apoptose, which destroys the existing vessels and thereby impairs survival of vessel and metastasis through the vascular route.

In addition to the numerous factors that stimulate angiogenesis, both physiologically and pathologically, many substances including those mentioned above can inhibit blood vessel growth.

### **9.1 Angiostatin**

Angiostatin is an internal fragment of plasminogen. Angiostatin was the first proteolytic fragment described with anti-angiogenic activity, derived from plasminogen via MMP degradation of plasmin.

**17**

muscles [95].

**9.4 TIMPs**

*Vascularisation of Skeletal Muscle*

**9.2 Endostatin**

KDR/Flk-1 [93, 94].

**9.3 Thrombospondin-1**

*DOI: http://dx.doi.org/10.5772/intechopen.85903*

Inhibition of NOS increased the expression of angiostatin and activities of MMP-2 and MMP-9 which generate it, suggesting that compromised NO production may lead to impaired angiogenesis during endothelial dysfunction. Whether there is any role in modulating flow-mediated angiogenesis is unknown. Four potential "receptors" for angiostatin have been identified: integrin αvβ3, ATP synthase, angiomotin, and the NG2 chondroitin sulfate proteoglycan (CSPG).

Endostatin is a 20-kDa carboxyl terminal proteolytic cleavage fragment of collagen type XVIII [92]. It is thought to be generated through a two-step process, as follows: a metal-dependent early cleavage of collagen type XVIII, followed by cleavage at an Ala-His site. It is not entirely clear which protease(s) is responsible for endostatin generation from collagen type XVIII in vivo, as multiple proteases can cleave recombinant fragments of human collagen type XVIII to generate endostatin-like fragments. In a study testing approximately 12 different proteases, elastase and cathepsin-L were the only two proteases found to efficiently cleave fragments of recombinant human collagen type XVIII, generating endostatin-like fragments. In support of a role for cathepsin-L in the generation of endostatin, cathepsin-L can proteolytically generate endostatin from its precursor in murine hemangioendothelioma cells propagated in vitro. Endostatin was originally reported to inhibit the proliferation of bovine capillary endothelial cells, but not the proliferation of cells of nonendothelial origin, and to also inhibit angiogenesis in the chick chorioallantoic membrane model [92] . Endostatin receptors that mediate the biological effects of endostatin are not yet clear. In this regard, research studies have shown that endostatin has the ability to bind to various cell surface molecules such as glypican, integrin α5β1, tropomyosin, and the VEGF receptor

Thrombospondin-1 (TSP-1) is a large (∼450 kDa) multifunctional homotrimeric matrix glycoprotein whose action primarily serves to inhibit angiogenesis. Originally found to be stored and secreted in platelet *α*-granules, TSP-1 is now known to be produced by a wide variety of cells, including fibroblasts, keratinocytes, neutrophils, and macrophages and is believed to be a major secretory product of vascular smooth muscle and endothelial cells. The actions of TSP-1 include participation in platelet aggregation, inhibition of proteolytic enzymes, inhibition of endothelial cell proliferation, diminution of cell spreading, disruption of focal (cell-to-matrix) adhesions, and inhibition of angiogenesis *in vitro* and *in vivo*. However, the physiological relevance of TSP-1 in regulating skeletal muscle angiogenesis is not known. In these regard, Malek et al. showed that TSP-1 is an important endogenous negative regulator of angiogenesis that prevents excessive capillarization in the heart and skeletal

TIMPs inhibit MMP activities. Of the four TIMPs (TIMP-1, TIMP-2, TIMP-3, and TIMP-4) identified so far, TIMP not only has a direct effect on the growth and migration of endothelial cells, but also affects the extracellular matrix, which is an essential component of angiogenesis responses. The balance between stimulants and inhibitors of MMPs is a key step in the angiogenesis regulation. TIMP-1 and

### *Vascularisation of Skeletal Muscle DOI: http://dx.doi.org/10.5772/intechopen.85903*

Inhibition of NOS increased the expression of angiostatin and activities of MMP-2 and MMP-9 which generate it, suggesting that compromised NO production may lead to impaired angiogenesis during endothelial dysfunction. Whether there is any role in modulating flow-mediated angiogenesis is unknown. Four potential "receptors" for angiostatin have been identified: integrin αvβ3, ATP synthase, angiomotin, and the NG2 chondroitin sulfate proteoglycan (CSPG).

### **9.2 Endostatin**

*Muscle Cells - Recent Advances and Future Perspectives*

physiological or pathological conditionings [89].

activated upon receptor engagement [90].

actively growing and invading endothelial tube.

**8.9 EPH-B4/EPHRIN-B2**

**9. Angiostatic factors**

the vascular route.

**9.1 Angiostatin**

inhibit blood vessel growth.

minogen via MMP degradation of plasmin.

and highly involved in vessel stabilization and maturation [89]. Interestingly, skeletal muscles represent the most abundant tissue of the body and they express high levels of angiomotin. Moreover, microcirculation is a critical component of muscle function since capillaries provide myofibers with oxygen and nutriments, and remove carbon dioxide and metabolic waste. As myofibers respond to physiological or pathological conditions with a remarkable plasticity, it is crucial that the microcirculation remains well matched with the myofibers' needs in order to preserve muscle function. Depending on conditions, such muscle angio-adaptation can involve either angiogenesis or some vascular regression. Given its role not only during angiogenesis but also for vessel stabilization and maturation, angiomotin might thus represent an important factor in muscle angio-adaptation. To date, angiomotin expression in skeletal muscle has never been investigated in response to

A unique class of receptor/ligand pair, Eph receptors and ephrin ligands, plays a prominent role in blood vessel development. Interestingly, not only does an ephrin expressed on the surface of one cell bind and activate its cognate Eph receptor on another cell, but through a reciprocal signaling mechanism the ephrin is also

Ephrin-B2 as a member of the ephrin family is explicit on arterial endothelial

Interaction of ephrin-B2 and its receptor has determined the primary capillary plexus after vasculogenesis [91]. The exact role of this interaction in the angiogenic process is not completely clear. But what has been approved is that establishment of contact and signaling between arterial and venous compartments mediated by ephrin-B2 and eph-4B is necessary for remodeling of the established primary capillary plexus [1].

Vascularization can be suppressed at any of a number of key steps in this process

by endothelial growth cycle disruption in which a quiescent vessel becomes an

Degradation of the extracellular matrix by activated endothelial cells can also be prevented, which suppresses sprouting and invasion of growing capillaries into their surroundings. Alternatively, endothelial cell proliferation can be inhibited by agents that block signaling within the cell and arrest its division cycle or by agents that prevent the maturation of nascent endothelial cells into functional tubes .Finally, endothelial cells can be forced to apoptose, which destroys the existing vessels and thereby impairs survival of vessel and metastasis through

In addition to the numerous factors that stimulate angiogenesis, both physiologically and pathologically, many substances including those mentioned above can

Angiostatin is an internal fragment of plasminogen. Angiostatin was the first proteolytic fragment described with anti-angiogenic activity, derived from plas-

In this regard, vasculogenesis can be blocked by different factors.

cells, and its receptor eph-B4 is localized to venous endothelial cells.

**16**

Endostatin is a 20-kDa carboxyl terminal proteolytic cleavage fragment of collagen type XVIII [92]. It is thought to be generated through a two-step process, as follows: a metal-dependent early cleavage of collagen type XVIII, followed by cleavage at an Ala-His site. It is not entirely clear which protease(s) is responsible for endostatin generation from collagen type XVIII in vivo, as multiple proteases can cleave recombinant fragments of human collagen type XVIII to generate endostatin-like fragments. In a study testing approximately 12 different proteases, elastase and cathepsin-L were the only two proteases found to efficiently cleave fragments of recombinant human collagen type XVIII, generating endostatin-like fragments. In support of a role for cathepsin-L in the generation of endostatin, cathepsin-L can proteolytically generate endostatin from its precursor in murine hemangioendothelioma cells propagated in vitro. Endostatin was originally reported to inhibit the proliferation of bovine capillary endothelial cells, but not the proliferation of cells of nonendothelial origin, and to also inhibit angiogenesis in the chick chorioallantoic membrane model [92] . Endostatin receptors that mediate the biological effects of endostatin are not yet clear. In this regard, research studies have shown that endostatin has the ability to bind to various cell surface molecules such as glypican, integrin α5β1, tropomyosin, and the VEGF receptor KDR/Flk-1 [93, 94].

### **9.3 Thrombospondin-1**

Thrombospondin-1 (TSP-1) is a large (∼450 kDa) multifunctional homotrimeric matrix glycoprotein whose action primarily serves to inhibit angiogenesis. Originally found to be stored and secreted in platelet *α*-granules, TSP-1 is now known to be produced by a wide variety of cells, including fibroblasts, keratinocytes, neutrophils, and macrophages and is believed to be a major secretory product of vascular smooth muscle and endothelial cells. The actions of TSP-1 include participation in platelet aggregation, inhibition of proteolytic enzymes, inhibition of endothelial cell proliferation, diminution of cell spreading, disruption of focal (cell-to-matrix) adhesions, and inhibition of angiogenesis *in vitro* and *in vivo*. However, the physiological relevance of TSP-1 in regulating skeletal muscle angiogenesis is not known. In these regard, Malek et al. showed that TSP-1 is an important endogenous negative regulator of angiogenesis that prevents excessive capillarization in the heart and skeletal muscles [95].

### **9.4 TIMPs**

TIMPs inhibit MMP activities. Of the four TIMPs (TIMP-1, TIMP-2, TIMP-3, and TIMP-4) identified so far, TIMP not only has a direct effect on the growth and migration of endothelial cells, but also affects the extracellular matrix, which is an essential component of angiogenesis responses. The balance between stimulants and inhibitors of MMPs is a key step in the angiogenesis regulation. TIMP-1 and

TIMP-2 block the release of MMP-2 and MMP-9 zymogens, respectively. This factor reduces VEGF gene expression.

TIMP-1 is the most abundant type of TIMPs. TIMP-1 is a glycoprotein with a weight of 25.8 kDa and 184 amino acids, which has a variety of functions, such as growth factor activity, stimulation of morphological changes in cells, and prevention of angiogenesis. TIMP-1 with different gravities interacts with all known MMPs.

TIMP-2 is a nonglycolytic protein weighing 21 kDa and 196 amino acids, which prevents tumor growth. This protein mainly attaches to MMP-2. Of course, this factor, like TIMP-1, has the ability to connect to all MMPs.

TIMP-3 is a protein with a weight of 41 kDa and 188 amino acids, which causes cell death. This angiostatic factor specifically results in the deactivation of MMP-1, MMP-2, MMP3, and MMP-9. In this regard, TIMP-4 specifically connects to the MMP-2 and inhibits biological effects of MMP-2 [94].

### **9.5 Interferons**

Interferons (INF-a, b, and g) are members of a family of secreted glycoproteins that were initially characterized for their antiviral effect. Interferons inhibit angiogenesis [96]. It is likely that IFN-a and IFN-b lead to downregulation of bFGF mRNA and protein levels [97] as well as its inhibitory effect on endothelial cell migration [1].

### **10. Conclusions**

It is clear that a single paradigm cannot be used to encompass the unique patterns of capillary growth observed in response to various angiogenic stimuli. Over the past decade, novel markers of neovascularization in skeletal muscle have been identified, both at molecular and genetic levels, consequently leading our understanding of the molecular mechanisms involved in neovascularization of skeletal muscle to new heights. We have reviewed that the EC response during physiological angiogenesis within skeletal muscle is potentially sensitive to the hypoxia, shear stress, mechanical stimulus, and adenosine. A complex mix of humoral/metabolic and mechanical stimuli works coordinately within muscle to provide the cues that stimulate vascularization. The intricacies of these signaling pathways and levels of crosstalk between pathways remain to be elucidated. Although we still require delineation of signaling pathways evoked by individual angiogenic stimuli, a major goal for future research will be to determine how multiple stimuli are integrated within the capillary to determine a particular pattern of capillary growth. As mentioned, the process of vascularization in skeletal muscle depends on many factors such as angiogenic and angiostatic factors, but how their participation in the process of vascularization in the type of skeletal muscle fibers is not yet clear. Several key questions about the process of vascularization in skeletal muscle still remain to be addressed. For example: what is the relationship between intussusception, sprouting, angiogenic, and angiostatic factors and the type of skeletal muscle fibers? What is the relationship between angiogenesis factors secreted from muscle fibers and myosin heavy chains?

**19**

Iran

**Author details**

Kamal Ranjbar1

provided the original work is properly cited.

\* and Bayan Fayazi2

Islamic Azad University, Bandar Abbas, Iran

*Vascularisation of Skeletal Muscle*

*DOI: http://dx.doi.org/10.5772/intechopen.85903*

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 Department of Physical Education and Sport Science, Bandar Abbas Branch,

2 Faculty of Physical Education and Sport Science, Razi University, Kermanshah,

\*Address all correspondence to: kamal\_ranjbar2010@yahoo.com

*Vascularisation of Skeletal Muscle DOI: http://dx.doi.org/10.5772/intechopen.85903*

*Muscle Cells - Recent Advances and Future Perspectives*

factor, like TIMP-1, has the ability to connect to all MMPs.

MMP-2 and inhibits biological effects of MMP-2 [94].

reduces VEGF gene expression.

MMPs.

**9.5 Interferons**

migration [1].

**10. Conclusions**

TIMP-2 block the release of MMP-2 and MMP-9 zymogens, respectively. This factor

TIMP-1 is the most abundant type of TIMPs. TIMP-1 is a glycoprotein with a weight of 25.8 kDa and 184 amino acids, which has a variety of functions, such as growth factor activity, stimulation of morphological changes in cells, and prevention of angiogenesis. TIMP-1 with different gravities interacts with all known

TIMP-2 is a nonglycolytic protein weighing 21 kDa and 196 amino acids, which prevents tumor growth. This protein mainly attaches to MMP-2. Of course, this

TIMP-3 is a protein with a weight of 41 kDa and 188 amino acids, which causes cell death. This angiostatic factor specifically results in the deactivation of MMP-1, MMP-2, MMP3, and MMP-9. In this regard, TIMP-4 specifically connects to the

Interferons (INF-a, b, and g) are members of a family of secreted glycoproteins that were initially characterized for their antiviral effect. Interferons inhibit angiogenesis [96]. It is likely that IFN-a and IFN-b lead to downregulation of bFGF mRNA and protein levels [97] as well as its inhibitory effect on endothelial cell

It is clear that a single paradigm cannot be used to encompass the unique patterns of capillary growth observed in response to various angiogenic stimuli. Over the past decade, novel markers of neovascularization in skeletal muscle have been identified, both at molecular and genetic levels, consequently leading our understanding of the molecular mechanisms involved in neovascularization of skeletal muscle to new heights. We have reviewed that the EC response during physiological angiogenesis within skeletal muscle is potentially sensitive to the hypoxia, shear stress, mechanical stimulus, and adenosine. A complex mix of humoral/metabolic and mechanical stimuli works coordinately within muscle to provide the cues that stimulate vascularization. The intricacies of these signaling pathways and levels of crosstalk between pathways remain to be elucidated. Although we still require delineation of signaling pathways evoked by individual angiogenic stimuli, a major goal for future research will be to determine how multiple stimuli are integrated within the capillary to determine a particular pattern of capillary growth. As mentioned, the process of vascularization in skeletal muscle depends on many factors such as angiogenic and angiostatic factors, but how their participation in the process of vascularization in the type of skeletal muscle fibers is not yet clear. Several key questions about the process of vascularization in skeletal muscle still remain to be addressed. For example: what is the relationship between intussusception, sprouting, angiogenic, and angiostatic factors and the type of skeletal muscle fibers? What is the relationship between angiogenesis factors secreted from muscle

**18**

fibers and myosin heavy chains?

### **Author details**

Kamal Ranjbar1 \* and Bayan Fayazi2

1 Department of Physical Education and Sport Science, Bandar Abbas Branch, Islamic Azad University, Bandar Abbas, Iran

2 Faculty of Physical Education and Sport Science, Razi University, Kermanshah, Iran

\*Address all correspondence to: kamal\_ranjbar2010@yahoo.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[2] Shireman PK. The chemokine system in arteriogenesis and hind limb ischemia. Journal of Vascular Surgery. 2007;**45**(Suppl A):A48-A56

[3] Prior BM, Yang HT, Terjung RL. What makes vessels grow with exercise training? Journal of Applied Physiology. 2004;**97**(3):1119-1128

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[10] Kivela R et al. Effects of acute exercise, exercise training, and diabetes on the expression of lymphangiogenic growth factors and lymphatic vessels

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[11] Coates G, O'Brodovich H, Goeree G. Hindlimb and lung lymph flows during prolonged exercise. Journal of Applied Physiology. 1993;**75**(2):633-638

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[13] Fong GH. Regulation of angiogenesis by oxygen sensing mechanisms. Journal of Molecular Medicine. 2009;**87**(6):549-560

[14] Hudlicka O, Brown MD. Adaptation of skeletal muscle microvasculature to increased or decreased blood flow: Role of shear stress, nitric oxide and vascular endothelial growth factor. Journal of Vascular Research. 2009;**46**(5):504-512

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[17] Fredholm BB et al. International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacological Reviews. 2001;**53**(4):527-552

[18] Feoktistov I et al. Differential expression of adenosine receptors in human endothelial cells: Role of A2B receptors in angiogenic factor

**21**

*Vascularisation of Skeletal Muscle*

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*DOI: http://dx.doi.org/10.5772/intechopen.85903*

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2008;**586**(Pt 24):6021-6035

[34] Gavin TP. Basal and exerciseinduced regulation of skeletal muscle capillarization. Exercise and Sport Sciences Reviews. 2009;**37**(2):86-92

[35] Janacek J et al. 3D visualization and measurement of capillaries supplying metabolically different fiber types in the rat extensor digitorum longus muscle

[28] Schieffer B et al. Development and prevention of skeletal muscle structural alterations after experimental myocardial infarction. American Journal of Physiology - Heart and Circulatory Physiology. 1995;**269**(5):H1507-H1513

*Vascularisation of Skeletal Muscle DOI: http://dx.doi.org/10.5772/intechopen.85903*

regulation. Circulation Research. 2002;**90**(5):531-538

[19] Adair TH. Growth regulation of the vascular system: An emerging role for adenosine. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 2005;**289**(2):R283-R296

[20] Ryzhov S et al. Role of adenosine receptors in the regulation of angiogenic factors and neovascularization in hypoxia. The Journal of Pharmacology and Experimental Therapeutics. 2007;**320**(2):565-572

[21] Bloor CM. Angiogenesis during exercise and training. Angiogenesis. 2005;**8**(3):263-271

[22] Huonker M et al. Size and blood flow of central and peripheral arteries in highly trained able-bodied and disabled athletes. Journal of Applied Physiology. 2003;**95**(2):685-691

[23] Magnusson G et al. Exercise capacity in heart failure patients: Relative importance of heart and skeletal muscle. Clinical Physiology. 1996;**16**(2):183-195

[24] Sullivan MJ, Green HJ, Cobb FR. Skeletal muscle biochemistry and histology in ambulatory patients with long-term heart failure. Circulation. 1990;**81**(2):518-527

[25] Drexler H et al. Alterations of skeletal muscle in chronic heart failure. Circulation. 1992;**85**(5):1751-1759

[26] Didion SP et al. Enhanced constrictor responses of skeletal muscle arterioles during chronic myocardial infarction. American Journal of Physiology - Heart and Circulatory Physiology. 1997;**273**(3):H1502-H1508

[27] Kindig CA et al. Impaired capillary hemodynamics in skeletal muscle of

rats in chronic heart failure. Journal of Applied Physiology. 1999;**87**(2):652-660

[28] Schieffer B et al. Development and prevention of skeletal muscle structural alterations after experimental myocardial infarction. American Journal of Physiology - Heart and Circulatory Physiology. 1995;**269**(5):H1507-H1513

[29] Thomas DP, Hudlická O. Arteriolar reactivity and capillarization in chronically stimulated rat limb skeletal muscle post-MI. Journal of Applied Physiology. 1999;**87**(6):2259-2265

[30] Pogozelski AR et al. p38γ mitogenactivated protein kinase is a key regulator in skeletal muscle metabolic adaptation in mice. PLoS One. 2009;**4**(11):e7934

[31] Chinsomboon J et al. The transcriptional coactivator PGC-1alpha mediates exercise-induced angiogenesis in skeletal muscle. Proceedings of the National Academy of Sciences of the United States of America. 2009;**106**(50):21401-21406

[32] Yan Z et al. Regulation of exerciseinduced fiber type transformation, mitochondrial biogenesis, and angiogenesis in skeletal muscle. Journal of Applied Physiology. 2011;**110**(1):264-274

[33] Zwetsloot KA et al. AMPK regulates basal skeletal muscle capillarization and VEGF expression, but is not necessary for the angiogenic response to exercise. The Journal of Physiology. 2008;**586**(Pt 24):6021-6035

[34] Gavin TP. Basal and exerciseinduced regulation of skeletal muscle capillarization. Exercise and Sport Sciences Reviews. 2009;**37**(2):86-92

[35] Janacek J et al. 3D visualization and measurement of capillaries supplying metabolically different fiber types in the rat extensor digitorum longus muscle

**20**

*Muscle Cells - Recent Advances and Future Perspectives*

in skeletal muscle. American Journal of Physiology. Heart and Circulatory Physiology. 2007;**293**(4):H2573-H2579

[11] Coates G, O'Brodovich H, Goeree G. Hindlimb and lung lymph flows during prolonged exercise. Journal of Applied

Physiology. 1993;**75**(2):633-638

from human skeletal muscle during prolonged steady-state running. Experimental Physiology.

[13] Fong GH. Regulation of angiogenesis by oxygen sensing mechanisms. Journal of Molecular Medicine. 2009;**87**(6):549-560

2000;**85**(6):863-868

2009;**46**(5):504-512

2002;**27**(5):491-515

2002;**22**(6):907-913

Union of Pharmacology.

Reviews. 2001;**53**(4):527-552

[18] Feoktistov I et al. Differential expression of adenosine receptors in human endothelial cells: Role of A2B receptors in angiogenic factor

[12] Havas E et al. Albumin clearance

[14] Hudlicka O, Brown MD. Adaptation of skeletal muscle microvasculature to increased or decreased blood flow: Role of shear stress, nitric oxide and vascular endothelial growth factor. Journal of Vascular Research.

[15] Haas TL. Molecular control of capillary growth in skeletal muscle. Canadian Journal of Applied Physiology.

[16] Abumiya T et al. Shear stress induces expression of vascular endothelial growth factor receptor Flk-1/KDR through the CT-rich Sp1 binding site. Arteriosclerosis, Thrombosis, and Vascular Biology.

[17] Fredholm BB et al. International

XXV. Nomenclature and classification of adenosine receptors. Pharmacological

[1] Papetti M, Herman IM. Mechanisms

of normal and tumor-derived angiogenesis. American Journal of Physiology. Cell Physiology. 2002;**282**(5):C947-C970

**References**

[2] Shireman PK. The chemokine system in arteriogenesis and hind limb ischemia. Journal of Vascular Surgery.

[3] Prior BM, Yang HT, Terjung RL. What makes vessels grow with exercise training? Journal of Applied Physiology.

[4] Dufraine J, Funahashi Y, Kitajewski J. Notch signaling regulates tumor angiogenesis by diverse mechanisms.

[5] Egginton S. Physiological factors influencing capillary growth. Acta Physiologica (Oxford, England).

[6] Egginton S. Invited review: Activityinduced angiogenesis. Pflügers Archiv.

[7] Cai W, Schaper W. Mechanisms of arteriogenesis. Acta Biochimica et Biophysica Sinica Shanghai.

[8] Helisch A et al. Impact of mouse strain differences in innate hindlimb collateral vasculature. Arteriosclerosis, Thrombosis, and Vascular Biology.

[9] Hansen-Smith F et al. Growth of arterioles precedes that of capillaries in stretch-induced angiogenesis in skeletal muscle. Microvascular Research.

[10] Kivela R et al. Effects of acute exercise, exercise training, and diabetes on the expression of lymphangiogenic growth factors and lymphatic vessels

2007;**45**(Suppl A):A48-A56

Oncogene. 2008;**27**(38):5132

2004;**97**(3):1119-1128

2011;**202**(3):225-239

2009;**457**(5):963-977

2008;**40**(8):681-692

2006;**26**(3):520-526

2001;**62**(1):1-14

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[43] Ranjbar K, Rahmani-Nia F, Shahabpour E. Aerobic training and l-arginine supplementation promotes rat heart and hindleg muscles arteriogenesis after myocardial infarction. Journal of Physiology and Biochemistry. 2016;**72**(3):393-404

[44] Panisello P et al. Capillary supply, fibre types and fibre morphometry in rat tibialis anterior and diaphragm muscles after intermittent exposure to hypobaric hypoxia. European Journal of Applied Physiology. 2008;**103**(2):203-213

[45] Murakami S et al. Comparison of capillary architecture between slow and fast muscles in rats using a confocal laser scanning microscope. Acta Medica Okayama. 2010;**64**(1):11-18

[46] Robbins JL et al. A sex-specific relationship between capillary density and anaerobic threshold. Journal of Applied Physiology. 2009;**106**(4):1181-1186

[47] Kyriakides ZS et al. Gender does not influence angiogenesis and arteriogenesis in a rabbit model of chronic hind limb ischemia. International Journal of Cardiology. 2003;**92**(1):83-91

[48] Keteyian SJ et al. Differential effects of exercise training in men and women with chronic heart failure. American Heart Journal. 2003;**145**(5):912-918

[49] Cui L et al. Arteriolar and venular capillary distribution in skeletal muscles of old rats. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences. 2008;**63**(9):928-935

[50] Davidson YS et al. The effect of aging on skeletal muscle capillarization in a murine model. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences. 1999;**54**(10):B448-B451

[51] Coggan AR et al. Histochemical and enzymatic characteristics of skeletal muscle in master athletes. Journal of Applied Physiology. 1990;**68**(5):1896-1901

[52] Degens H et al. Capillary proliferation related to fibre types in

**23**

*Vascularisation of Skeletal Muscle*

Biology. 1994;**345**:669-676

1971;**31**(3):323-325

2007;**103**(5):1815-1823

aerobic capacity, and density of muscle capillaries in young and old men. Journal of Applied Physiology.

[54] Kano Y et al. Effects of aging on capillary number and luminal size in rat soleus and plantaris muscles. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences. 2002;**57**(12):B422-B427

[55] Lyon MJ, Steer LM, Malmgren LT. Stereological estimates indicate that aging does not alter the capillary length density in the human posterior cricoarytenoid muscle. Journal of Applied Physiology.

[56] Gavin TP et al. No difference in the skeletal muscle angiogenic response to aerobic exercise training between young and aged men. The Journal of Physiology. 2007;**585**(Pt 1):231-239

[57] Shimada T et al. Angiogenesis and vasculogenesis are impaired in the precocious-aging klotho mouse. Circulation. 2004;**110**(9):1148-1155

[58] Yu J et al. An engineered

1999;**48**(10):1267-1271

VEGF-activating zinc finger protein transcription factor improves blood flow and limb salvage in advanced-age mice. The FASEB Journal. 2006;**20**(3):479-481

[59] Shono N et al. Decreased skeletal muscle capillary density is related to higher serum levels of lowdensity lipoprotein cholesterol and apolipoprotein B in men. Metabolism.

[60] Gudbjornsdottir S et al. Direct measurements of the permeability surface area for insulin and glucose in human skeletal muscle. The Journal of

*DOI: http://dx.doi.org/10.5772/intechopen.85903*

Clinical Endocrinology and Metabolism.

[61] Gavin TP et al. Lower capillary density but no difference in VEGF expression in obese vs. lean young skeletal muscle in humans. Journal of Applied Physiology.

[62] Chou E et al. Decreased cardiac expression of vascular endothelial growth factor and its receptors in insulin-resistant and diabetic states: A possible explanation for impaired collateral formation in cardiac tissue. Circulation. 2002;**105**(3):373-379

[63] Toblli JE et al. Angiotensinconverting enzyme inhibition and angiogenesis in myocardium of obese Zucker rats. American Journal of Hypertension. 2004;**17**(2):172-180

[64] Rutkowski JM, Davis KE, Scherer PE. Mechanisms of obesity and related pathologies: The macro- and microcirculation of adipose tissue. The FEBS Journal. 2009;**276**(20):5738-5746

angiogenesis in glutathione peroxidase-

[65] Galasso G et al. Impaired

1-deficient mice is associated with endothelial progenitor cell dysfunction. Circulation Research.

[66] Kivela R et al. Effects of experimental type 1 diabetes and exercise training on angiogenic gene expression and capillarization in skeletal muscle. The FASEB Journal.

[67] Kivela R et al. Exercise-induced expression of angiogenic growth factors in skeletal muscle and in

[68] Boodhwani M et al. Functional, cellular, and molecular characterization of the angiogenic response to chronic

capillaries of healthy and diabetic mice. Cardiovascular Diabetology. 2008;**7**:13

2006;**98**(2):254-261

2006;**20**(9):1570-1572

2003;**88**(10):4559-4564

2005;**98**(1):315-321

hypertrophied aging rat M. Plantaris. Advances in Experimental Medicine and

[53] Parizkova J et al. Body composition,

*Vascularisation of Skeletal Muscle DOI: http://dx.doi.org/10.5772/intechopen.85903*

*Muscle Cells - Recent Advances and Future Perspectives*

[44] Panisello P et al. Capillary supply, fibre types and fibre morphometry in rat tibialis anterior and diaphragm muscles after intermittent exposure to hypobaric hypoxia. European Journal of Applied Physiology.

[45] Murakami S et al. Comparison of capillary architecture between slow and fast muscles in rats using a confocal laser scanning microscope. Acta Medica

[46] Robbins JL et al. A sex-specific relationship between capillary density and anaerobic threshold. Journal of Applied Physiology.

[47] Kyriakides ZS et al. Gender does not influence angiogenesis and arteriogenesis in a rabbit model of chronic hind limb ischemia. International Journal of Cardiology.

[48] Keteyian SJ et al. Differential effects of exercise training in men and women with chronic heart failure. American Heart Journal. 2003;**145**(5):912-918

[49] Cui L et al. Arteriolar and venular capillary distribution in skeletal muscles of old rats. The Journals of Gerontology.

Series A, Biological Sciences and Medical Sciences. 2008;**63**(9):928-935

[50] Davidson YS et al. The effect of aging on skeletal muscle capillarization in a murine model. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences.

[51] Coggan AR et al. Histochemical and enzymatic characteristics of skeletal muscle in master athletes. Journal of Applied Physiology.

proliferation related to fibre types in

1999;**54**(10):B448-B451

1990;**68**(5):1896-1901

[52] Degens H et al. Capillary

2008;**103**(2):203-213

Okayama. 2010;**64**(1):11-18

2009;**106**(4):1181-1186

2003;**92**(1):83-91

during denervation and reinnervation. The Journal of Histochemistry and Cytochemistry. 2009;**57**(5):437-447

[36] Charifi N et al. Enhancement of microvessel tortuosity in the vastus lateralis muscle of old men in response to endurance training. The Journal of Physiology. 2004;**554**(Pt 2):559-569

[37] Badr I et al. Differences in local environment determine the site of physiological angiogenesis in rat skeletal muscle. Experimental Physiology.

[38] Egginton S, Hudlicka O. Selective long-term electrical stimulation of fast glycolytic fibres increases capillary supply but not oxidative enzyme activity in rat skeletal muscles. Experimental Physiology. 2000;**85**(5):567-573

[39] Sullivan SM, Pittman RN. In vitro O2 uptake and histochemical fiber type of resting hamster muscles. Journal of Applied Physiology. 1984;**57**(1):246-253

[41] Cebasek V et al. Nerve injury affects the capillary supply in rat slow and fast muscles differently. Cell and Tissue Research. 2006;**323**(2):305-312

[42] Ranjbar K, Ardakanizade M, Nazem F. Endurance training induces fiber type-specific revascularization in hindlimb skeletal muscles of rats with chronic heart failure. Iranian journal of basic medical sciences. 2017;**20**(1):90

[43] Ranjbar K, Rahmani-Nia F, Shahabpour E. Aerobic training and l-arginine supplementation promotes

rat heart and hindleg muscles arteriogenesis after myocardial infarction. Journal of Physiology and Biochemistry. 2016;**72**(3):393-404

[40] Ahmed SK et al. Is human skeletal muscle capillary supply modelled according to fibre size or fibre type? Experimental Physiology.

1997;**82**(1):231-234

2003;**88**(5):565-568

**22**

hypertrophied aging rat M. Plantaris. Advances in Experimental Medicine and Biology. 1994;**345**:669-676

[53] Parizkova J et al. Body composition, aerobic capacity, and density of muscle capillaries in young and old men. Journal of Applied Physiology. 1971;**31**(3):323-325

[54] Kano Y et al. Effects of aging on capillary number and luminal size in rat soleus and plantaris muscles. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences. 2002;**57**(12):B422-B427

[55] Lyon MJ, Steer LM, Malmgren LT. Stereological estimates indicate that aging does not alter the capillary length density in the human posterior cricoarytenoid muscle. Journal of Applied Physiology. 2007;**103**(5):1815-1823

[56] Gavin TP et al. No difference in the skeletal muscle angiogenic response to aerobic exercise training between young and aged men. The Journal of Physiology. 2007;**585**(Pt 1):231-239

[57] Shimada T et al. Angiogenesis and vasculogenesis are impaired in the precocious-aging klotho mouse. Circulation. 2004;**110**(9):1148-1155

[58] Yu J et al. An engineered VEGF-activating zinc finger protein transcription factor improves blood flow and limb salvage in advanced-age mice. The FASEB Journal. 2006;**20**(3):479-481

[59] Shono N et al. Decreased skeletal muscle capillary density is related to higher serum levels of lowdensity lipoprotein cholesterol and apolipoprotein B in men. Metabolism. 1999;**48**(10):1267-1271

[60] Gudbjornsdottir S et al. Direct measurements of the permeability surface area for insulin and glucose in human skeletal muscle. The Journal of Clinical Endocrinology and Metabolism. 2003;**88**(10):4559-4564

[61] Gavin TP et al. Lower capillary density but no difference in VEGF expression in obese vs. lean young skeletal muscle in humans. Journal of Applied Physiology. 2005;**98**(1):315-321

[62] Chou E et al. Decreased cardiac expression of vascular endothelial growth factor and its receptors in insulin-resistant and diabetic states: A possible explanation for impaired collateral formation in cardiac tissue. Circulation. 2002;**105**(3):373-379

[63] Toblli JE et al. Angiotensinconverting enzyme inhibition and angiogenesis in myocardium of obese Zucker rats. American Journal of Hypertension. 2004;**17**(2):172-180

[64] Rutkowski JM, Davis KE, Scherer PE. Mechanisms of obesity and related pathologies: The macro- and microcirculation of adipose tissue. The FEBS Journal. 2009;**276**(20):5738-5746

[65] Galasso G et al. Impaired angiogenesis in glutathione peroxidase-1-deficient mice is associated with endothelial progenitor cell dysfunction. Circulation Research. 2006;**98**(2):254-261

[66] Kivela R et al. Effects of experimental type 1 diabetes and exercise training on angiogenic gene expression and capillarization in skeletal muscle. The FASEB Journal. 2006;**20**(9):1570-1572

[67] Kivela R et al. Exercise-induced expression of angiogenic growth factors in skeletal muscle and in capillaries of healthy and diabetic mice. Cardiovascular Diabetology. 2008;**7**:13

[68] Boodhwani M et al. Functional, cellular, and molecular characterization of the angiogenic response to chronic

myocardial ischemia in diabetes. Circulation. 2007;**116**(11 Suppl):I31-I37

[69] Olfert IM et al. Myocyte vascular endothelial growth factor is required for exercise-induced skeletal muscle angiogenesis. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 2010;**299**(4):R1059-R1067

[70] Prior BM et al. Exercise-induced vascular remodeling. Exercise and Sport Sciences Reviews. 2003;**31**(1):26-33

[71] Olfert IM et al. Muscle-specific VEGF deficiency greatly reduces exercise endurance in mice. The Journal of Physiology. 2009;**587**(Pt 8):1755-1767

[72] Lockwood CJ, Schatz F, Krikun G. Angiogenic factors and the endometrium following long term progestin only contraception. Histology and Histopathology. 2004;**19**(1):167-172

[73] Qazi Y, Maddula S, Ambati BK. Mediators of ocular angiogenesis. Journal of Genetics. 2009;**88**(4):495-515

[74] Hoeben A et al. Vascular endothelial growth factor and angiogenesis. Pharmacological Reviews. 2004;**56**(4):549-580

[75] Muhlhauser J et al. In vivo angiogenesis induced by recombinant adenovirus vectors coding either for secreted or nonsecreted forms of acidic fibroblast growth factor. Human Gene Therapy. 1995;**6**(11):1457-1465

[76] Rissanen TT et al. Fibroblast growth factor 4 induces vascular permeability, angiogenesis and arteriogenesis in a rabbit hindlimb ischemia model. The FASEB Journal. 2003;**17**(1):100-102

[77] Nicosia RF, Nicosia SV, Smith M. Vascular endothelial growth factor, platelet-derived growth factor, and insulin-like growth factor-1 promote rat aortic angiogenesis in vitro. The

American Journal of Pathology. 1994;**145**(5):1023-1029

[78] Massague J. The transforming growth factor-beta family. Annual Review of Cell Biology. 1990;**6**:597-641

[79] Sato TN et al. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature. 1995;**376**(6535):70-74

[80] Gustafsson T, Kraus WE. Exerciseinduced angiogenesis-related growth and transcription factors in skeletal muscle, and their modification in muscle pathology. Frontiers in Bioscience. 2001;**6**:D75-D89

[81] Holash J et al. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF. Science. 1999;**284**(5422):1994-1998

[82] Carmeli E et al. Matrix metalloproteinases and skeletal muscle: A brief review. Muscle & Nerve. 2004;**29**(2):191-197

[83] John A, Tuszynski G. The role of matrix metalloproteinases in tumor angiogenesis and tumor metastasis. Pathology Oncology Research. 2001;**7**(1):14

[84] Carmeli E et al. High intensity exercise increases expression of matrix metalloproteinases in fast skeletal muscle fibres. Experimental Physiology. 2005;**90**(4):613-619

[85] Brooks PC, Clark RA, Cheresh DA. Requirement of vascular integrin alpha v beta 3 for angiogenesis. Science. 1994;**264**(5158):569-571

[86] Brooks PC et al. Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell. 1994;**79**(7):1157-1164

**25**

*Vascularisation of Skeletal Muscle*

[87] Ouchi N et al. Follistatinlike 1, a secreted muscle protein, promotes endothelial cell function and revascularization in ischemic tissue through a nitric-oxide synthase-dependent mechanism. The Journal of Biological Chemistry.

2008;**283**(47):32802-32811

2008;**1783**(3):429-437

[88] Ernkvist M et al. Differential roles of p80- and p130-angiomotin in the switch between migration and stabilization of endothelial cells. Biochimica et Biophysica Acta.

[89] Roudier E et al. Angiomotin p80/ p130 ratio: A new indicator of exerciseinduced angiogenic activity in skeletal muscles from obese and non-obese rats? The Journal of Physiology. 2009;**587**(Pt 16):4105-4119

[90] Holland SJ et al. Bidirectional signalling through the EPHfamily receptor Nuk and its transmembrane ligands. Nature.

[91] Wang HU, Chen ZF, Anderson DJ. Molecular distinction and angiogenic interaction between embryonic arteries

and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell.

[92] O'Reilly MS et al. Endostatin: An endogenous inhibitor of

and human endostatin exhibit distinct antiangiogenic activities mediated by alpha v beta 3 and alpha 5 beta 1 integrins. Proceedings of the National Academy of Sciences of the United States of America.

2003;**100**(8):4766-4771

angiogenesis and tumor growth. Cell.

[93] Sudhakar A et al. Human tumstatin

[94] Rege TA, Fears CY, Gladson CL. Endogenous inhibitors of angiogenesis

in malignant gliomas: Nature's

1996;**383**(6602):722-725

1998;**93**(5):741-753

1997;**88**(2):277-285

*DOI: http://dx.doi.org/10.5772/intechopen.85903*

antiangiogenic therapy. Neuro-Oncology. 2005;**7**(2):106-121

[95] Malek MH, Olfert IM. Global deletion of thrombospondin-1 increases cardiac and skeletal muscle capillarity and exercise capacity in mice. Experimental Physiology.

[96] Ribatti D et al. Human recombinant

angiogenesis of chick area vasculosa in shell-less culture. International Journal of Microcirculation, Clinical and Experimental. 1996;**16**(4):165-169

[97] Singh RK et al. Interferons alpha and beta down-regulate the expression of basic fibroblast growth factor in human carcinomas. Proceedings of the National Academy of Sciences of the United States of America.

2009;**94**(6):749-760

interferon alpha-2a inhibits

1995;**92**(10):4562-4566

*Vascularisation of Skeletal Muscle DOI: http://dx.doi.org/10.5772/intechopen.85903*

*Muscle Cells - Recent Advances and Future Perspectives*

American Journal of Pathology.

growth factor-beta family. Annual Review of Cell Biology.

[78] Massague J. The transforming

[79] Sato TN et al. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature.

[80] Gustafsson T, Kraus WE. Exerciseinduced angiogenesis-related growth and transcription factors in skeletal muscle, and their modification in muscle pathology. Frontiers in Bioscience. 2001;**6**:D75-D89

1994;**145**(5):1023-1029

1990;**6**:597-641

1995;**376**(6535):70-74

[81] Holash J et al. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF. Science. 1999;**284**(5422):1994-1998

[82] Carmeli E et al. Matrix

2004;**29**(2):191-197

2005;**90**(4):613-619

1994;**264**(5158):569-571

1994;**79**(7):1157-1164

2001;**7**(1):14

metalloproteinases and skeletal muscle: A brief review. Muscle & Nerve.

[83] John A, Tuszynski G. The role of matrix metalloproteinases in tumor angiogenesis and tumor metastasis. Pathology Oncology Research.

[84] Carmeli E et al. High intensity exercise increases expression of matrix metalloproteinases in fast skeletal muscle fibres. Experimental Physiology.

[85] Brooks PC, Clark RA, Cheresh DA. Requirement of vascular integrin alpha v beta 3 for angiogenesis. Science.

[86] Brooks PC et al. Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell.

myocardial ischemia in diabetes. Circulation. 2007;**116**(11 Suppl):I31-I37

[69] Olfert IM et al. Myocyte vascular endothelial growth factor is required for exercise-induced skeletal muscle angiogenesis. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 2010;**299**(4):R1059-R1067

[70] Prior BM et al. Exercise-induced vascular remodeling. Exercise and Sport Sciences Reviews. 2003;**31**(1):26-33

[71] Olfert IM et al. Muscle-specific VEGF deficiency greatly reduces exercise endurance in mice. The Journal of Physiology. 2009;**587**(Pt 8):1755-1767

[72] Lockwood CJ, Schatz F, Krikun G.

[73] Qazi Y, Maddula S, Ambati BK. Mediators of ocular angiogenesis. Journal of Genetics. 2009;**88**(4):495-515

angiogenesis. Pharmacological Reviews.

angiogenesis induced by recombinant adenovirus vectors coding either for secreted or nonsecreted forms of acidic fibroblast growth factor. Human Gene

[76] Rissanen TT et al. Fibroblast growth factor 4 induces vascular permeability, angiogenesis and arteriogenesis in a rabbit hindlimb ischemia model. The FASEB Journal. 2003;**17**(1):100-102

[77] Nicosia RF, Nicosia SV, Smith M. Vascular endothelial growth factor, platelet-derived growth factor, and insulin-like growth factor-1 promote rat aortic angiogenesis in vitro. The

[74] Hoeben A et al. Vascular endothelial growth factor and

[75] Muhlhauser J et al. In vivo

Therapy. 1995;**6**(11):1457-1465

2004;**56**(4):549-580

Angiogenic factors and the endometrium following long term progestin only contraception. Histology and Histopathology. 2004;**19**(1):167-172

**24**

[87] Ouchi N et al. Follistatinlike 1, a secreted muscle protein, promotes endothelial cell function and revascularization in ischemic tissue through a nitric-oxide synthase-dependent mechanism. The Journal of Biological Chemistry. 2008;**283**(47):32802-32811

[88] Ernkvist M et al. Differential roles of p80- and p130-angiomotin in the switch between migration and stabilization of endothelial cells. Biochimica et Biophysica Acta. 2008;**1783**(3):429-437

[89] Roudier E et al. Angiomotin p80/ p130 ratio: A new indicator of exerciseinduced angiogenic activity in skeletal muscles from obese and non-obese rats? The Journal of Physiology. 2009;**587**(Pt 16):4105-4119

[90] Holland SJ et al. Bidirectional signalling through the EPHfamily receptor Nuk and its transmembrane ligands. Nature. 1996;**383**(6602):722-725

[91] Wang HU, Chen ZF, Anderson DJ. Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell. 1998;**93**(5):741-753

[92] O'Reilly MS et al. Endostatin: An endogenous inhibitor of angiogenesis and tumor growth. Cell. 1997;**88**(2):277-285

[93] Sudhakar A et al. Human tumstatin and human endostatin exhibit distinct antiangiogenic activities mediated by alpha v beta 3 and alpha 5 beta 1 integrins. Proceedings of the National Academy of Sciences of the United States of America. 2003;**100**(8):4766-4771

[94] Rege TA, Fears CY, Gladson CL. Endogenous inhibitors of angiogenesis in malignant gliomas: Nature's

antiangiogenic therapy. Neuro-Oncology. 2005;**7**(2):106-121

[95] Malek MH, Olfert IM. Global deletion of thrombospondin-1 increases cardiac and skeletal muscle capillarity and exercise capacity in mice. Experimental Physiology. 2009;**94**(6):749-760

[96] Ribatti D et al. Human recombinant interferon alpha-2a inhibits angiogenesis of chick area vasculosa in shell-less culture. International Journal of Microcirculation, Clinical and Experimental. 1996;**16**(4):165-169

[97] Singh RK et al. Interferons alpha and beta down-regulate the expression of basic fibroblast growth factor in human carcinomas. Proceedings of the National Academy of Sciences of the United States of America. 1995;**92**(10):4562-4566

**27**

**Chapter 2**

**Abstract**

Muscle

*Alexandra V. Ulyanova*

voltage-gated Ca2+ and Na+

the largest pressure drop.

**2. Myogenic tone**

**1. Introduction**

Excitability of Vascular Smooth

Regulation of pressure and local blood flow occurs at the level of resistance arteries and arterioles. Under physiological conditions, these small vessels exist in a state of partial constriction, termed myogenic tone. Myogenic tone is considered to be an intrinsic property of arteriolar smooth muscle cells, which membranes depolarize in response to increase in the intraluminal pressure. Oscillations of membrane potential in smooth muscles are mediated by the activity of voltage-gated L-type Ca2+ channels, which provide an influx of Ca2+ to activate various voltage-gated and Ca2+-sensitive channels of smooth muscle cells and to initiate endothelial Ca2+ signaling needed for vasodilation. Although a relationship between change in membrane potential and myogenic response is considered to be universal throughout various smooth muscle tissues, it may be regulated differently based on autoregulatory responses and channels expression. Here we review electrophysiological signature of arteriolar smooth muscle in various tissues, with an emphases and specific examples

of the excitability of 4th order arterioles isolated from skeletal muscle.

channels

**Keywords:** vasculature, arteriolar smooth muscle, excitability, electrophysiology,

Regulation of pressure and local blood flow occurs at the level of resistance arteries and arterioles. Once blood exits the heart, it first flows into large elastic arteries, followed by smaller distributing arteries, which branch further into small resistance arteries and, finally, arterioles. The branching and reduction in vessel diameter actually results in the increase in total cross-sectional area of circulation. Because of their small diameter, resistance arteries and arterioles are the place of

Resistance arteries and arterioles typically exhibit a state of partial constriction termed myogenic tone [1]. Myogenic tone is related to the level of the intraluminal pressure and provides a level of tone that vasodilators can act upon [2]. When the intraluminal pressure increases, resistance arteries and arterioles first dilate due to their elastic properties and then constrict to the new steady-state level. Myogenic tone is an intrinsic property of arteriolar smooth muscle cells and does not require endothelium [3, 4]. Resistance to blood flow is actively controlled by contraction

### **Chapter 2**

## Excitability of Vascular Smooth Muscle

*Alexandra V. Ulyanova*

### **Abstract**

Regulation of pressure and local blood flow occurs at the level of resistance arteries and arterioles. Under physiological conditions, these small vessels exist in a state of partial constriction, termed myogenic tone. Myogenic tone is considered to be an intrinsic property of arteriolar smooth muscle cells, which membranes depolarize in response to increase in the intraluminal pressure. Oscillations of membrane potential in smooth muscles are mediated by the activity of voltage-gated L-type Ca2+ channels, which provide an influx of Ca2+ to activate various voltage-gated and Ca2+-sensitive channels of smooth muscle cells and to initiate endothelial Ca2+ signaling needed for vasodilation. Although a relationship between change in membrane potential and myogenic response is considered to be universal throughout various smooth muscle tissues, it may be regulated differently based on autoregulatory responses and channels expression. Here we review electrophysiological signature of arteriolar smooth muscle in various tissues, with an emphases and specific examples of the excitability of 4th order arterioles isolated from skeletal muscle.

**Keywords:** vasculature, arteriolar smooth muscle, excitability, electrophysiology, voltage-gated Ca2+ and Na+ channels

### **1. Introduction**

Regulation of pressure and local blood flow occurs at the level of resistance arteries and arterioles. Once blood exits the heart, it first flows into large elastic arteries, followed by smaller distributing arteries, which branch further into small resistance arteries and, finally, arterioles. The branching and reduction in vessel diameter actually results in the increase in total cross-sectional area of circulation. Because of their small diameter, resistance arteries and arterioles are the place of the largest pressure drop.

### **2. Myogenic tone**

Resistance arteries and arterioles typically exhibit a state of partial constriction termed myogenic tone [1]. Myogenic tone is related to the level of the intraluminal pressure and provides a level of tone that vasodilators can act upon [2]. When the intraluminal pressure increases, resistance arteries and arterioles first dilate due to their elastic properties and then constrict to the new steady-state level. Myogenic tone is an intrinsic property of arteriolar smooth muscle cells and does not require endothelium [3, 4]. Resistance to blood flow is actively controlled by contraction

or relaxation of arteriolar smooth muscle cells wrapped around the vessel so that their tone regulates the vessel diameter [2]. Arterial walls are made up of three layers: the tunica intima, tunica media and tunica adventitia. While the tunica intima contains endothelial cells and a thin layer of connective tissue, the tunica media supplies mechanical strength and contractile power. It is composed of several layers of spindle-shaped smooth muscle cells arranged helically in a matrix of elastin and collagen fibers. In some places, endothelial cells make contacts with smooth muscle cells to transmit signals between the intima and media. The tunica adventitia is mostly a connective tissue sheath with no distinct outer border. Its role is to tether the vessel loosely to the surrounding tissue [5]. Arterioles, the smallest resistance arteries placed right before capillaries, have a single layer of spindleshaped smooth muscle cells [6]. Endothelial cells frame the lumen of arterioles. The shape and orientation within the vessels help to distinguish between these two distinct cells types (**Figure 1**).

Development of the tone is associated with depolarization of smooth muscle cells. While the mechanisms underlying depolarization are not completely understood, it is known that development of myogenic tone depends on extracellular Ca2+. At 0 Ca2+, pressurized resistance arteries and arterioles are fully dilated. Adding Ca2+ up to ≈ 2 mM to the extracellular space causes maximal tone [7]. In addition to the development of basal tone, the myogenic mechanism is believed to underlie the response to acute changes in pressure and contribute to spontaneous vasomotor activity [7].

Myogenic tone is controlled by intrinsic as well as extrinsic mechanisms. Intrinsic mechanisms include constriction of arterioles in response to pressure increase (Bayliss effect), endothelial secretions (nitric oxide, EDHF, prostacyclin, endothelin), vasoactive metabolites (e.g. adenosine in exercising muscle), autacoids (local vasoactive paracrine secretions such as histamine), and temperature. Important physiological responses mediated entirely by intrinsic regulation include the autoregulation of flow, and functional and reactive hyperemia. Intrinsic regulation also contributes to pathological responses such as inflammation and arterial vasospasm. Extrinsic regulation is brought about by factors originating outside the organ, namely the vasomotor nerves (sympathetic, parasympathetic and others) and circulating hormones such as adrenaline, vasopressin and insulin (for review, see [8]).

### **Figure 1.**

*4th order skeletal muscle arteriole loaded with 10 mM Fluo-4. A single layer of spindle-like vascular smooth muscle cells (VSMC) runs perpendicular to the vessel's length (left panel). Long endothelial cells (EC) frame the lumen of skeletal muscle arteriole (right panel, adapted from [6]). Scale bar = 50 μm.*

**29**

*Excitability of Vascular Smooth Muscle DOI: http://dx.doi.org/10.5772/intechopen.85053*

**3. Skeletal muscle vasculature**

Skeletal muscle is the largest organ in the body that receives about 20% of cardiac output at rest and up to 80% during exercise. In skeletal muscle, the local blood flow is regulated over a 20× range to meet the demands of exercise. Therefore, vascular resistance of skeletal muscle is a critical determinant of total peripheral resistance and blood pressure. Arterioles of skeletal muscle have a high myogenic tone at rest as they are subject to major hyperemia (increased blood flow) during exercise [8]. In case of orthostatic hypotension, a potential contributor to cardiovascular adaptation to prolonged periods of bed rest or microgravity, skeletal muscle arterioles may develop functional or structural adaptations. Despite the physiological importance of skeletal muscle arterioles, little is known about ionic mechanisms underlying their excitability. Even within the same skeletal muscle, arterioles might respond differently to pressure changes. For instance, the first-order arterioles isolated from fast-twitch (e.g., white gastrocnemius) skeletal muscle fiber demonstrated both functional and structural changes such as reduced myogenic tone, decreased contractile responsiveness, and reduced wall thickness with no change in luminal diameter [9, 10]. In contrast, arterioles isolated from slow-twitch (e.g., soleus) fibers show no difference in myogenic tone or contractile responsiveness but rather a

structural remodeling resulting in a decreased arteriole diameter [11].

After development of isolated vessel techniques, the electrophysiology of smooth muscle cells was extensively studied in small arteries isolated from various

Both slow changes (myogenic tone) and fast spikes in the membrane potential have been observed in arteriolar smooth muscle cells. Arteriolar smooth muscle cells can generate rhythmical contractions (vasomotion) over an extended period of time. Vasomotion occurs spontaneously or in response to vasoactive stimulation with a frequency of 3–20 per minute. The exact physiological role of vasomotion is not clear. In cases of ischemia and hypertension, it serves as a protective mechanism. Vasomotion is not a consequence of heartbeat, respiration, or neuronal input. It is generated within arteriolar smooth muscle cells by an endogenous pacemaker mechanism driven by a cytosolic Ca2+ oscillator. The cytosolic Ca2+ oscillator depends on Ca2+ entry and is regulated by transmitters and hormones, which increase the formation of InsP3 and diacylglycerol (DAG) and promote oscillatory activity (for review, see [13]). From the early studies, it has been appreciated that an increase in intraluminal pressure leads to depolarization and consequent contraction of vascular smooth muscle cell (electromechanical coupling). Mechanisms of depolarization are still not well understood. Depolarization increases Ca2+ concentration inside the cell, which leads to activation of myosin light chain kinase by Ca2+/calmodulin and consequent contraction. Propagation of depolarization is achieved through gap junctions between neighboring smooth muscle cells. The signaling between endothelial and smooth muscle cells via gap junctions is essential for normal vascular function. Gap junctions in arteriolar smooth muscle cells and endothelial cells are formed by connexins Cx37, Cx40, and Cx43. Coupling between arteriolar smooth muscle cells appears to be essential for the maintenance of oscillations in membrane potential,

**4. Excitability of vascular smooth muscle**

**4.1 Membrane potential of vascular smooth muscle**

the intracellular [Ca2+], and vasomotion.

vascular beds [12].

### **3. Skeletal muscle vasculature**

*Muscle Cells - Recent Advances and Future Perspectives*

distinct cells types (**Figure 1**).

vasomotor activity [7].

and insulin (for review, see [8]).

or relaxation of arteriolar smooth muscle cells wrapped around the vessel so that their tone regulates the vessel diameter [2]. Arterial walls are made up of three layers: the tunica intima, tunica media and tunica adventitia. While the tunica intima contains endothelial cells and a thin layer of connective tissue, the tunica media supplies mechanical strength and contractile power. It is composed of several layers of spindle-shaped smooth muscle cells arranged helically in a matrix of elastin and collagen fibers. In some places, endothelial cells make contacts with smooth muscle cells to transmit signals between the intima and media. The tunica adventitia is mostly a connective tissue sheath with no distinct outer border. Its role is to tether the vessel loosely to the surrounding tissue [5]. Arterioles, the smallest resistance arteries placed right before capillaries, have a single layer of spindleshaped smooth muscle cells [6]. Endothelial cells frame the lumen of arterioles. The shape and orientation within the vessels help to distinguish between these two

Development of the tone is associated with depolarization of smooth muscle cells. While the mechanisms underlying depolarization are not completely understood, it is known that development of myogenic tone depends on extracellular Ca2+. At 0 Ca2+, pressurized resistance arteries and arterioles are fully dilated. Adding Ca2+ up to ≈ 2 mM to the extracellular space causes maximal tone [7]. In addition to the development of basal tone, the myogenic mechanism is believed to underlie the response to acute changes in pressure and contribute to spontaneous

Myogenic tone is controlled by intrinsic as well as extrinsic mechanisms. Intrinsic mechanisms include constriction of arterioles in response to pressure increase (Bayliss effect), endothelial secretions (nitric oxide, EDHF, prostacyclin, endothelin), vasoactive metabolites (e.g. adenosine in exercising muscle), autacoids (local vasoactive paracrine secretions such as histamine), and temperature. Important physiological responses mediated entirely by intrinsic regulation include the autoregulation of flow, and functional and reactive hyperemia. Intrinsic regulation also contributes to pathological responses such as inflammation and arterial vasospasm. Extrinsic regulation is brought about by factors originating outside the organ, namely the vasomotor nerves (sympathetic, parasympathetic and others) and circulating hormones such as adrenaline, vasopressin

*4th order skeletal muscle arteriole loaded with 10 mM Fluo-4. A single layer of spindle-like vascular smooth muscle cells (VSMC) runs perpendicular to the vessel's length (left panel). Long endothelial cells (EC) frame the* 

*lumen of skeletal muscle arteriole (right panel, adapted from [6]). Scale bar = 50 μm.*

**28**

**Figure 1.**

Skeletal muscle is the largest organ in the body that receives about 20% of cardiac output at rest and up to 80% during exercise. In skeletal muscle, the local blood flow is regulated over a 20× range to meet the demands of exercise. Therefore, vascular resistance of skeletal muscle is a critical determinant of total peripheral resistance and blood pressure. Arterioles of skeletal muscle have a high myogenic tone at rest as they are subject to major hyperemia (increased blood flow) during exercise [8]. In case of orthostatic hypotension, a potential contributor to cardiovascular adaptation to prolonged periods of bed rest or microgravity, skeletal muscle arterioles may develop functional or structural adaptations. Despite the physiological importance of skeletal muscle arterioles, little is known about ionic mechanisms underlying their excitability. Even within the same skeletal muscle, arterioles might respond differently to pressure changes. For instance, the first-order arterioles isolated from fast-twitch (e.g., white gastrocnemius) skeletal muscle fiber demonstrated both functional and structural changes such as reduced myogenic tone, decreased contractile responsiveness, and reduced wall thickness with no change in luminal diameter [9, 10]. In contrast, arterioles isolated from slow-twitch (e.g., soleus) fibers show no difference in myogenic tone or contractile responsiveness but rather a structural remodeling resulting in a decreased arteriole diameter [11].

### **4. Excitability of vascular smooth muscle**

After development of isolated vessel techniques, the electrophysiology of smooth muscle cells was extensively studied in small arteries isolated from various vascular beds [12].

### **4.1 Membrane potential of vascular smooth muscle**

Both slow changes (myogenic tone) and fast spikes in the membrane potential have been observed in arteriolar smooth muscle cells. Arteriolar smooth muscle cells can generate rhythmical contractions (vasomotion) over an extended period of time. Vasomotion occurs spontaneously or in response to vasoactive stimulation with a frequency of 3–20 per minute. The exact physiological role of vasomotion is not clear. In cases of ischemia and hypertension, it serves as a protective mechanism. Vasomotion is not a consequence of heartbeat, respiration, or neuronal input. It is generated within arteriolar smooth muscle cells by an endogenous pacemaker mechanism driven by a cytosolic Ca2+ oscillator. The cytosolic Ca2+ oscillator depends on Ca2+ entry and is regulated by transmitters and hormones, which increase the formation of InsP3 and diacylglycerol (DAG) and promote oscillatory activity (for review, see [13]).

From the early studies, it has been appreciated that an increase in intraluminal pressure leads to depolarization and consequent contraction of vascular smooth muscle cell (electromechanical coupling). Mechanisms of depolarization are still not well understood. Depolarization increases Ca2+ concentration inside the cell, which leads to activation of myosin light chain kinase by Ca2+/calmodulin and consequent contraction. Propagation of depolarization is achieved through gap junctions between neighboring smooth muscle cells. The signaling between endothelial and smooth muscle cells via gap junctions is essential for normal vascular function. Gap junctions in arteriolar smooth muscle cells and endothelial cells are formed by connexins Cx37, Cx40, and Cx43. Coupling between arteriolar smooth muscle cells appears to be essential for the maintenance of oscillations in membrane potential, the intracellular [Ca2+], and vasomotion.

The exact signaling mechanisms that underlie detection of the mechanical stimulus and membrane depolarization are not completely understood (for review, see [7, 14]). Depolarization of vascular smooth muscle's membrane activates various voltage-gated ion channels, pumps and exchangers, including voltage-gated Ca2+ and K+ channels, Ca2+-activated BKCa and ClCa channels, Na+ /Ca2+ exchanger, Ca2+-ATP pump and N+ /K+ -ATPase, ATP-sensitive P2X receptor, and transient receptor potential (TRP) ion channels. Voltage-gated L-type Ca2+ channels are activated upon depolarization and increase Ca2+ concentration inside the cell. BKCa K+ channels hyperpolarize smooth muscle cell back to its resting potential [15, 16]. Some vascular myocytes, particularly in large vessels, contract via pathways that appear to be unrelated to significant changes in the membrane potential (pharmacomechanical coupling).

Membrane potential of arteriolar smooth muscle cells is difficult to measure because of the changes in the intraluminal pressure and active vasomotor responses. Resting membrane potentials range from approximately *−*60 to *−*35 mV for physiological pressures (for review, see [17, 18]). Resting membrane potentials between *−*80 to *−*60 mV were previously reported for un-pressurized mice mesenteric artery [19], submucosal arterioles [20], and cerebral arterioles [21, 22]. A range of membrane potentials was reported for un-pressurized mice skeletal muscle arterioles (**Figure 2**). The average resting potential was reported to be around −68 mV (**Figure 2**, adapted from [6]). However, it's significantly more negative than −55 mV previously reported for un-pressurized arterioles in rat cremaster muscle [23]. Resting membrane potential in vascular smooth muscle cells is determined to a large extent by K+ conductance [18]. Intracellular [Cl<sup>−</sup> ] in vascular smooth muscle cells is around 55 mM, which is unusually high and mediated probably through Cl<sup>−</sup> ▬HCO3 − exchanger. A small influx of Ca2+ and efflux of Cl<sup>−</sup> ions at rest reduce the membrane potential from the Nernst equilibrium for K+ [24].

### **4.2 Excitability of vascular smooth muscle**

Most of the arteriolar smooth muscle cells are quiescent. In some cases, a drop in the intraluminal pressure (during trauma to a vessel) that leads to hyperpolarization and dilation generates membrane action potentials. Physiological role of membrane action potentials in arteriolar smooth muscle is unclear, as relatively small changes in the membrane potential (between −55 and − 35 mV) are sufficient to control Ca2+ entry and to initiate contraction in response to stimulation by mechanical stress of the blood flow [14]. Spontaneous action potentials could be associated either with rhythmic vasomotor activity [23]; follow K<sup>+</sup> -induced hyperpolarization and dilation [25], or precede injury-induced constriction [26].

### **Figure 2.**

*Resting membrane potential of un-pressurized 4th order arterioles isolated from mice skeletal muscle. Distribution of the resting membrane potential values was fitted by a single Gaussian function peaking at −77 ± 2 mV (n = 81, smooth line). The average resting potential was Vrest = −68 ± 2 mV (n = 81). Adapted from [6].*

**31**

for voltage-gated K+

*Excitability of Vascular Smooth Muscle DOI: http://dx.doi.org/10.5772/intechopen.85053*

*solution containing 150 KCl. Adapted from [6].*

in **Figure 3** [6, 21].

**Figure 3.**

*4.3.1 Voltage-gated Ca2+ channels*

Only a few recordings of action potentials in skeletal muscle arterioles were made so far [6, 23, 25, 27]. Spontaneous action potential spikes could be observed in pressurized small arteries [23, 25], as well as in un-pressurized arterioles as shown

*Spontaneous action potentials were observed in un-pressurized skeletal muscle arterioles (2 mM Ca bath solution). Smooth muscle cells were current-clamped using gramicidin-perforated configuration, with intracellular* 

Unlike in many other excitable tissues, action potentials in smooth muscle cells of arteries [28] and arterioles [21] are thought to be independent of voltage-gated Na+ channels, as they could not be blocked by the application of voltage-gated Na+

nel blocker tetrodotoxin (TTX). Depolarization of smooth muscle cells induced by the change in intraluminal pressure and/or tissue-stretch produces an increase in the intracellular [Ca2+], consequent myosin light chain phosphorylation and contraction (for review, see [14]). Since specific blockers of L-type voltage-gated Ca2+ channels suppress both the upstroke and the after-depolarizing components of action potentials, these channels are thought to be the main pathway for the depolarizing current.

present in arterial beds and are activated upon membrane depolarization [6, 29].

Voltage-gated Ca2+ channels mediate influx of Ca2+ ions in response to membrane depolarization and regulate intracellular processes such as contraction, secretion, neurotransmission, and gene expression in many different cell types. Their activity is essential to couple electrical signals in the cell surface to physiological events in cells. Voltage-gated Ca2+ channels are comprised of the pore-forming α1 subunit in complex with auxiliary subunits. A transmembrane disulfide-linked α2δ, an intracellular β, and a γ subunit are components of most types of calcium channels. The α<sup>1</sup> subunit contains four repeats of a domain with six transmembrane segments, the fourth of which is the voltage-sensing S4 segment. The pore loop between transmembrane segments S5 and S6 in each domain determines ion conductance and selectivity (for review, see [30]). Ten distinct genes encode mammalian α1 subunits of three subfamilies of voltage-gated Ca2+ channels. The amino acid sequences of these α1 subunits are more than 70% identical within a subfamily but less than 40% identical among subfamilies. Voltage-gated Ca2+ channels are named using the chemical symbol of the principal permeating ion with the principal physiological regulator (voltage) indicated as a subscript according to the nomenclature developed

channels [31]. The CaV1 subfamily (CaV1.1–CaV1.4) represents

high-voltage activated L-type Ca2+ channels, the CaV2 subfamily (CaV2.1–CaV2.3) represents neuronal N-, P/Q-, and R-types Ca2+ channels, and CaV3 subfamily (CaV3.1–CaV3.3) represents low-voltage activated T-type Ca2+ channels.

Nevertheless, several groups have demonstrated that voltage-gated Na+

chan-

channels are

**4.3 Voltage-gated channels of arterial smooth muscle**

*Excitability of Vascular Smooth Muscle DOI: http://dx.doi.org/10.5772/intechopen.85053*

### **Figure 3.**

*Muscle Cells - Recent Advances and Future Perspectives*

Ca2+-activated BKCa and ClCa channels, Na+

increase Ca2+ concentration inside the cell. BKCa K+

smooth muscle cells is determined to a large extent by K+

either with rhythmic vasomotor activity [23]; follow K<sup>+</sup>

and dilation [25], or precede injury-induced constriction [26].

**4.2 Excitability of vascular smooth muscle**

mediated probably through Cl<sup>−</sup>

N+ /K+

[Cl<sup>−</sup>

Cl<sup>−</sup>

The exact signaling mechanisms that underlie detection of the mechanical stimulus and membrane depolarization are not completely understood (for review, see [7, 14]). Depolarization of vascular smooth muscle's membrane activates various voltage-gated


Membrane potential of arteriolar smooth muscle cells is difficult to measure because of the changes in the intraluminal pressure and active vasomotor responses. Resting membrane potentials range from approximately *−*60 to *−*35 mV for physiological pressures (for review, see [17, 18]). Resting membrane potentials between *−*80 to *−*60 mV were previously reported for un-pressurized mice mesenteric artery [19], submucosal arterioles [20], and cerebral arterioles [21, 22]. A range of membrane potentials was reported for un-pressurized mice skeletal muscle arterioles (**Figure 2**). The average resting potential was reported to be around −68 mV (**Figure 2**, adapted from [6]). However, it's significantly more negative than −55 mV previously reported for un-pressurized arterioles in rat cremaster muscle [23]. Resting membrane potential in vascular

] in vascular smooth muscle cells is around 55 mM, which is unusually high and

Most of the arteriolar smooth muscle cells are quiescent. In some cases, a drop in the intraluminal pressure (during trauma to a vessel) that leads to hyperpolarization and dilation generates membrane action potentials. Physiological role of membrane action potentials in arteriolar smooth muscle is unclear, as relatively small changes in the membrane potential (between −55 and − 35 mV) are sufficient to control Ca2+ entry and to initiate contraction in response to stimulation by mechanical stress of the blood flow [14]. Spontaneous action potentials could be associated

*Resting membrane potential of un-pressurized 4th order arterioles isolated from mice skeletal muscle. Distribution of the resting membrane potential values was fitted by a single Gaussian function peaking at −77 ± 2 mV (n = 81,* 

*smooth line). The average resting potential was Vrest = −68 ± 2 mV (n = 81). Adapted from [6].*

ions at rest reduce the membrane potential from the Nernst equilibrium for K+

channels. Voltage-gated L-type Ca2+ channels are activated upon depolarization and

muscle cell back to its resting potential [15, 16]. Some vascular myocytes, particularly in large vessels, contract via pathways that appear to be unrelated to significant

channels,

/Ca2+ exchanger, Ca2+-ATP pump and

channels hyperpolarize smooth

conductance [18]. Intracellular


[24].

exchanger. A small influx of Ca2+ and efflux of

ion channels, pumps and exchangers, including voltage-gated Ca2+ and K+

changes in the membrane potential (pharmacomechanical coupling).

▬HCO3 −

**30**

**Figure 2.**

*Spontaneous action potentials were observed in un-pressurized skeletal muscle arterioles (2 mM Ca bath solution). Smooth muscle cells were current-clamped using gramicidin-perforated configuration, with intracellular solution containing 150 KCl. Adapted from [6].*

Only a few recordings of action potentials in skeletal muscle arterioles were made so far [6, 23, 25, 27]. Spontaneous action potential spikes could be observed in pressurized small arteries [23, 25], as well as in un-pressurized arterioles as shown in **Figure 3** [6, 21].

### **4.3 Voltage-gated channels of arterial smooth muscle**

Unlike in many other excitable tissues, action potentials in smooth muscle cells of arteries [28] and arterioles [21] are thought to be independent of voltage-gated Na+ channels, as they could not be blocked by the application of voltage-gated Na+ channel blocker tetrodotoxin (TTX). Depolarization of smooth muscle cells induced by the change in intraluminal pressure and/or tissue-stretch produces an increase in the intracellular [Ca2+], consequent myosin light chain phosphorylation and contraction (for review, see [14]). Since specific blockers of L-type voltage-gated Ca2+ channels suppress both the upstroke and the after-depolarizing components of action potentials, these channels are thought to be the main pathway for the depolarizing current. Nevertheless, several groups have demonstrated that voltage-gated Na+ channels are present in arterial beds and are activated upon membrane depolarization [6, 29].

### *4.3.1 Voltage-gated Ca2+ channels*

Voltage-gated Ca2+ channels mediate influx of Ca2+ ions in response to membrane depolarization and regulate intracellular processes such as contraction, secretion, neurotransmission, and gene expression in many different cell types. Their activity is essential to couple electrical signals in the cell surface to physiological events in cells. Voltage-gated Ca2+ channels are comprised of the pore-forming α1 subunit in complex with auxiliary subunits. A transmembrane disulfide-linked α2δ, an intracellular β, and a γ subunit are components of most types of calcium channels. The α<sup>1</sup> subunit contains four repeats of a domain with six transmembrane segments, the fourth of which is the voltage-sensing S4 segment. The pore loop between transmembrane segments S5 and S6 in each domain determines ion conductance and selectivity (for review, see [30]). Ten distinct genes encode mammalian α1 subunits of three subfamilies of voltage-gated Ca2+ channels. The amino acid sequences of these α1 subunits are more than 70% identical within a subfamily but less than 40% identical among subfamilies. Voltage-gated Ca2+ channels are named using the chemical symbol of the principal permeating ion with the principal physiological regulator (voltage) indicated as a subscript according to the nomenclature developed for voltage-gated K+ channels [31]. The CaV1 subfamily (CaV1.1–CaV1.4) represents high-voltage activated L-type Ca2+ channels, the CaV2 subfamily (CaV2.1–CaV2.3) represents neuronal N-, P/Q-, and R-types Ca2+ channels, and CaV3 subfamily (CaV3.1–CaV3.3) represents low-voltage activated T-type Ca2+ channels.

The pharmacology and biophysics of Ca2+ channels subfamilies are quite distinct. L-type Ca2+ calcium channels typically require a strong depolarization for activation and do not inactivate at positive potentials. They are the main pathway for Ca2+ currents recorded in muscle cells, where they initiate contraction and secretion. The CaV1 subfamily is the molecular target of the organic Ca2+ channel blockers used widely in the therapy of cardiovascular diseases. L-type Ca2+ channels are blocked by the organic antagonists, including dihydropyridines (e.g., nifedipine), phenylalkylamines, and benzothiazepines. Dihydropyridines can be channel activators or inhibitors and therefore are thought to act allosterically to shift the channel toward the open or closed state rather than by occluding the pore. T-type Ca2+ channels are activated by weak depolarization and are transient at sustained depolarization. They are expressed in a wide variety of cell types, where they are involved in shaping the action potential and controlling patterns of repetitive firing. The CaV3 subfamily of voltage-gated Ca2+ channels is insensitive to both the dihydropyridines that block CaV1 channels and the spider and cone snail toxins that block the CaV2 channels. There are no widely useful pharmacological agents that block T-type Ca2+ currents specifically. Mibefradil blocks both T-type Ca2+ channels and with less potency L-type Ca2+ channels. Currents through expressed CaV3.2 channels could be selectively blocked by application of 40 μM of Ni2+ [32].

Currents through dihydropyridine-sensitive Ca2+ channels were recorded in arteries of various vascular beds [21, 33–36], including from smooth muscle cells of skeletal muscle arterioles (**Figure 4**, left panel). CaV1.2 considered to be the principal sub-type of voltage-gated Ca2+ channels involved in excitation-contraction coupling of vascular smooth muscle cells [37]. Since L-type Ca2+ blocker nifedipine did not eliminate basal tone in skeletal muscle arterioles, other Ca2+ entry mechanisms are believed to contribute to myogenic tone along with L-type Ca2+ channels [38]. In addition to L-type, vascular smooth muscle cells also express T-type Ca2+ channels (for review, see [30, 39]). Currents through T-type Ca2+ channels have been found in smooth muscle cells of arteries and arterioles of cerebral [21], mesenteric [40], renal [41], coronary [35], and skeletal [6] vascular beds as shown in **Figure 4** (right panel). Although the messenger RNAs for both, CaV3.1 and CaV3.2 T-type Ca2+ channels were found in rat cremaster arterioles [42], only L-type Ca2+ currents were recorded in single smooth muscle cells isolated from resistance arteries of hamster cremaster muscle [43]. The pressure/stretch stimulus initiates a depolarization that

### **Figure 4.**

*Voltage-gated T-type and L-type Ca2+ channels are present in arteriolar smooth muscle. Whole-cell Ba2+ currents were recorded in the presence of 1 μM tetrodotoxin (TTX), voltage-gated Na+ channel blocker as described in more detail in [6]. Two kinetically different components were observed: while voltage steps from −30 up to −10 mV produced fast inactivating T-type Ca2+ current (left panel), further depolarization activated slowly inactivating L-type Ca2+ current (right panel). Adapted from [6].*

**33**

**Figure 5.**

*TTX-sensitive voltage-gated Na+*

*Excitability of Vascular Smooth Muscle DOI: http://dx.doi.org/10.5772/intechopen.85053*

dependent K<sup>+</sup>

channels [40–43, 50, 51].

When voltage-gated Ca2+ and Na+

Voltage-gated Na<sup>+</sup>

current that brings the cell to threshold for Na<sup>+</sup>

 *channels*

*4.3.2 Voltage-gated Na<sup>+</sup>*

causes Ca2+ influx through voltage-gated L-type Ca2+ channels and initiates Ca2+ sparklets [44–46]. Both, Ca2+ sparklets and Ca2+ sparks (Ca2+ release from intracellular stores) signaling events, activate negative feedback mechanisms via Ca2+-

Ca2+ influx through L-type Ca2+ channels are believed to be involved in contraction of vascular smooth muscle, T-type Ca2+ channels, particularly CaV3.2 appear to be mostly involved in relaxation of coronary arteries, acting through activation of BKCa

In addition to voltage-gated Ca2+ channels, significant TTX-sensitive Na+

rents were found in smooth muscle cells from rabbit main pulmonary artery [52], human aorta [53], murine mesenteric arteries [29], and skeletal muscle arterioles [6] as shown in **Figure 5**. The above observations suggest that more complex mechanisms are likely to generate action potential in vascular smooth muscle cells.

tance is thought to be responsible for generation and propagation of action potential in excitable cells such as neurons, striated muscle, and neuroendocrine cells. It has

rapidly inactivate, L-type voltage-gated Ca2+ channels open and further depolarize the cell, thus prolonging the plateau of an action potential. In contrast, T-type voltage-gated Ca2+ channels may open at resting potential and produce depolarizing

subunits. The pore-forming subunit is sufficient for functional expression, but the kinetics and voltage dependence of channel gating are modified by the subunits. These auxiliary subunits are involved in channel localization and interaction with cell adhesion molecules, extracellular matrix, and intracellular cytoskeleton.

organized in four homologous domains, each composed of six transmembrane segments. S4 segment is a voltage sensor, and a pore loop located between the S5 and S6 segments determines ion conductance and selectivity (for review, see [56]).

lower threshold and faster rise time than Ca2+ conductance. After Na+

Similar to the voltage-gated Ca2+ channels, the subunits of Na+

currents thus preventing unrestrained depolarization [47–49]. While

channels coexist in the same cell, Na+

spike [54, 55].

 *channels are present in arteriolar smooth muscle. In arteriolar smooth muscle of* 

*skeletal muscle arterioles, voltage steps produced inward currents with at least two kinetically distinct components* 

*in 2 mM Ca solution (left panel). The fast component was through voltage-gated Na+*

*by application of 1 μM TTX (right panel). Adapted from [6].*

channels consist of the subunit associated with auxiliary

cur-

conduc-

channels

channels are

 *channels as it was blocked* 

### *Excitability of Vascular Smooth Muscle DOI: http://dx.doi.org/10.5772/intechopen.85053*

*Muscle Cells - Recent Advances and Future Perspectives*

The pharmacology and biophysics of Ca2+ channels subfamilies are quite distinct. L-type Ca2+ calcium channels typically require a strong depolarization for activation and do not inactivate at positive potentials. They are the main pathway for Ca2+ currents recorded in muscle cells, where they initiate contraction and secretion. The CaV1 subfamily is the molecular target of the organic Ca2+ channel blockers used widely in the therapy of cardiovascular diseases. L-type Ca2+ channels are blocked by the organic antagonists, including dihydropyridines (e.g., nifedipine), phenylalkylamines, and benzothiazepines. Dihydropyridines can be channel activators or inhibitors and therefore are thought to act allosterically to shift the channel toward the open or closed state rather than by occluding the pore. T-type Ca2+ channels are activated by weak depolarization and are transient at sustained depolarization. They are expressed in a wide variety of cell types, where they are involved in shaping the action potential and controlling patterns of repetitive firing. The CaV3 subfamily of voltage-gated Ca2+ channels is insensitive to both the dihydropyridines that block CaV1 channels and the spider and cone snail toxins that block the CaV2 channels. There are no widely useful pharmacological agents that block T-type Ca2+ currents specifically. Mibefradil blocks both T-type Ca2+ channels and with less potency L-type Ca2+ channels. Currents through expressed CaV3.2 channels could be selectively blocked by application of 40 μM of Ni2+ [32].

Currents through dihydropyridine-sensitive Ca2+ channels were recorded in arteries of various vascular beds [21, 33–36], including from smooth muscle cells of skeletal muscle arterioles (**Figure 4**, left panel). CaV1.2 considered to be the principal sub-type of voltage-gated Ca2+ channels involved in excitation-contraction coupling of vascular smooth muscle cells [37]. Since L-type Ca2+ blocker nifedipine did not eliminate basal tone in skeletal muscle arterioles, other Ca2+ entry mechanisms are believed to contribute to myogenic tone along with L-type Ca2+ channels [38]. In addition to L-type, vascular smooth muscle cells also express T-type Ca2+ channels (for review, see [30, 39]). Currents through T-type Ca2+ channels have been found in smooth muscle cells of arteries and arterioles of cerebral [21], mesenteric [40], renal [41], coronary [35], and skeletal [6] vascular beds as shown in **Figure 4** (right panel). Although the messenger RNAs for both, CaV3.1 and CaV3.2 T-type Ca2+ channels were found in rat cremaster arterioles [42], only L-type Ca2+ currents were recorded in single smooth muscle cells isolated from resistance arteries of hamster cremaster muscle [43]. The pressure/stretch stimulus initiates a depolarization that

**32**

**Figure 4.**

*Voltage-gated T-type and L-type Ca2+ channels are present in arteriolar smooth muscle. Whole-cell Ba2+ currents* 

*detail in [6]. Two kinetically different components were observed: while voltage steps from −30 up to −10 mV produced fast inactivating T-type Ca2+ current (left panel), further depolarization activated slowly inactivating* 

 *channel blocker as described in more* 

*were recorded in the presence of 1 μM tetrodotoxin (TTX), voltage-gated Na+*

*L-type Ca2+ current (right panel). Adapted from [6].*

causes Ca2+ influx through voltage-gated L-type Ca2+ channels and initiates Ca2+ sparklets [44–46]. Both, Ca2+ sparklets and Ca2+ sparks (Ca2+ release from intracellular stores) signaling events, activate negative feedback mechanisms via Ca2+ dependent K<sup>+</sup> currents thus preventing unrestrained depolarization [47–49]. While Ca2+ influx through L-type Ca2+ channels are believed to be involved in contraction of vascular smooth muscle, T-type Ca2+ channels, particularly CaV3.2 appear to be mostly involved in relaxation of coronary arteries, acting through activation of BKCa channels [40–43, 50, 51].

### *4.3.2 Voltage-gated Na<sup>+</sup> channels*

In addition to voltage-gated Ca2+ channels, significant TTX-sensitive Na+ currents were found in smooth muscle cells from rabbit main pulmonary artery [52], human aorta [53], murine mesenteric arteries [29], and skeletal muscle arterioles [6] as shown in **Figure 5**. The above observations suggest that more complex mechanisms are likely to generate action potential in vascular smooth muscle cells. When voltage-gated Ca2+ and Na+ channels coexist in the same cell, Na+ conductance is thought to be responsible for generation and propagation of action potential in excitable cells such as neurons, striated muscle, and neuroendocrine cells. It has lower threshold and faster rise time than Ca2+ conductance. After Na+ channels rapidly inactivate, L-type voltage-gated Ca2+ channels open and further depolarize the cell, thus prolonging the plateau of an action potential. In contrast, T-type voltage-gated Ca2+ channels may open at resting potential and produce depolarizing current that brings the cell to threshold for Na<sup>+</sup> spike [54, 55].

Voltage-gated Na<sup>+</sup> channels consist of the subunit associated with auxiliary subunits. The pore-forming subunit is sufficient for functional expression, but the kinetics and voltage dependence of channel gating are modified by the subunits. These auxiliary subunits are involved in channel localization and interaction with cell adhesion molecules, extracellular matrix, and intracellular cytoskeleton. Similar to the voltage-gated Ca2+ channels, the subunits of Na+ channels are organized in four homologous domains, each composed of six transmembrane segments. S4 segment is a voltage sensor, and a pore loop located between the S5 and S6 segments determines ion conductance and selectivity (for review, see [56]).

### **Figure 5.**

*TTX-sensitive voltage-gated Na+ channels are present in arteriolar smooth muscle. In arteriolar smooth muscle of skeletal muscle arterioles, voltage steps produced inward currents with at least two kinetically distinct components in 2 mM Ca solution (left panel). The fast component was through voltage-gated Na+ channels as it was blocked by application of 1 μM TTX (right panel). Adapted from [6].*

According to the nomenclature, the NaV1 superfamily (NaV1.1–NaV1.9) represents voltage-gated Na<sup>+</sup> channels. Unlike the different classes of voltage-gated Ca2+ channels, the functional properties of Na<sup>+</sup> channels are relatively similar. Amino acid sequences of the nine mammalian Na+ channel isoforms are more than 50% identical to each other, but separate families are difficult to define. By this criterion, nine isoforms are considered to be members of one NaV1 gene subfamily. Some voltagegated Na<sup>+</sup> channels (NaV1.1, NaV1.2, NaV1.3, NaV1.4, NaV1.6, and NaV1.7) could be blocked by tetrodotoxin that binds to the extracellular side of the pore.

Several isoforms of TTX-sensitive voltage-gated channels have been found previously smooth muscle cells of in vasa recta (NaV1.3), portal vein (NaV1.6 and NaV1.8), and vas deference (NaV1.6) [57–59]. The activity of these voltage-gated Na+ channels may be tightly controlled by elevations of intracellular Ca2+, potentially suppressing activity of Na+ channels [6]. For instance, Ca2+/CaM binding was shown to downregulates skeletal muscle isoform NaV1.4 by shifting its steady-state inactivation curve in the hyperpolarizing direction [60]. NaV1.3 channels in descending vasa recta are suppressed by calmodulin inhibitors, while elevation of the intracellular [Ca2+] shifts the voltage-dependence of their activation to more positive voltages [57]. In addition, Ca2+-dependent down-regulation of Na+ channels can also occur via the PKC pathway since activation of PKC decreases peak sodium currents through brain NaV1.2 and skeletal muscle NaV1.4 channels by up to 80% [61, 62].

### **5. Conclusion**

L-type voltage-gated Ca2+ channels are involved in excitation-contraction coupling of vascular smooth muscle cells. In addition, T-type Ca2+ channels were detected in arteriolar smooth muscle cells. These two types of voltage-gated Ca2+ channels together play an important role in the constriction/relaxation of smooth muscle cells by regulating Ca2+ signaling during myogenic tone. However, there are indications that other type of voltage-gated channels, specifically Na+ channels are present in various vascular beds. While the role of voltage-gated Ca2+ channels is well established, contribution of voltage-gated Na+ channel remains to be determined.

### **Acknowledgements**

I would like to thank Dr. Roman E. Shirokov for invaluable discussion related to this work.

**35**

**Author details**

Alexandra V. Ulyanova

provided the original work is properly cited.

*Excitability of Vascular Smooth Muscle DOI: http://dx.doi.org/10.5772/intechopen.85053*

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Center for Brain Injury and Repair, Department of Neurosurgery, Perelman School

of Medicine, University of Pennsylvania, Philadelphia, USA

\*Address all correspondence to: ulyanova@pennmedicine.upenn.edu.

### **Conflict of interest**

The author declares no conflict of interest.

### **Abbreviations**


*Excitability of Vascular Smooth Muscle DOI: http://dx.doi.org/10.5772/intechopen.85053*

*Muscle Cells - Recent Advances and Future Perspectives*

addition, Ca2+-dependent down-regulation of Na+

established, contribution of voltage-gated Na+

The author declares no conflict of interest.

InsP3 inositol 1,4,5-triphosphate

VSMC vascular smooth muscle cell

EDHF endothelium-derived hyperpolarizing factor

NaV1.2 and skeletal muscle NaV1.4 channels by up to 80% [61, 62].

indications that other type of voltage-gated channels, specifically Na+

nels, the functional properties of Na<sup>+</sup>

sequences of the nine mammalian Na+

voltage-gated Na<sup>+</sup>

gated Na<sup>+</sup>

activity of Na+

**5. Conclusion**

**Acknowledgements**

**Conflict of interest**

**Abbreviations**

DAG diacylglycerol EC endothelial cell

TTX tetrodotoxin

this work.

According to the nomenclature, the NaV1 superfamily (NaV1.1–NaV1.9) represents

cal to each other, but separate families are difficult to define. By this criterion, nine isoforms are considered to be members of one NaV1 gene subfamily. Some voltage-

blocked by tetrodotoxin that binds to the extracellular side of the pore.

and vas deference (NaV1.6) [57–59]. The activity of these voltage-gated Na+

channels (NaV1.1, NaV1.2, NaV1.3, NaV1.4, NaV1.6, and NaV1.7) could be

channels [6]. For instance, Ca2+/CaM binding was shown to down-

Several isoforms of TTX-sensitive voltage-gated channels have been found previously smooth muscle cells of in vasa recta (NaV1.3), portal vein (NaV1.6 and NaV1.8),

may be tightly controlled by elevations of intracellular Ca2+, potentially suppressing

PKC pathway since activation of PKC decreases peak sodium currents through brain

L-type voltage-gated Ca2+ channels are involved in excitation-contraction coupling of vascular smooth muscle cells. In addition, T-type Ca2+ channels were detected in arteriolar smooth muscle cells. These two types of voltage-gated Ca2+ channels together play an important role in the constriction/relaxation of smooth muscle cells by regulating Ca2+ signaling during myogenic tone. However, there are

present in various vascular beds. While the role of voltage-gated Ca2+ channels is well

I would like to thank Dr. Roman E. Shirokov for invaluable discussion related to

regulates skeletal muscle isoform NaV1.4 by shifting its steady-state inactivation curve in the hyperpolarizing direction [60]. NaV1.3 channels in descending vasa recta are suppressed by calmodulin inhibitors, while elevation of the intracellular [Ca2+] shifts the voltage-dependence of their activation to more positive voltages [57]. In

channels. Unlike the different classes of voltage-gated Ca2+ chan-

channels are relatively similar. Amino acid

channel isoforms are more than 50% identi-

channels can also occur via the

channel remains to be determined.

channels

channels are

**34**

### **Author details**

Alexandra V. Ulyanova

Center for Brain Injury and Repair, Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA

\*Address all correspondence to: ulyanova@pennmedicine.upenn.edu.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*Muscle Cells - Recent Advances and Future Perspectives*

T-type calcium channels in myogenic tone of skeletal muscle resistance arteries. American Journal of Physiology. Heart and Circulatory Physiology. 2002;**283**(6):H2239-H2H43

[43] Cohen KD, Jackson WF. Hypoxia inhibits contraction but not calcium channel currents or changes in intracellular calcium in arteriolar muscle cells. Microcirculation.

[44] Amberg GC, Navedo MF, Nieves-Cintron M, Molkentin JD, Santana LF. Calcium sparklets regulate local and global calcium in murine arterial smooth muscle. Journal of Physiology (London). 2007;**579**(1):187-201

[45] Navedo MF, Amberg GC, Nieves M, Molkentin JD, Santana LF. Mechanisms underlying heterogeneous Ca2+ sparklet activity in arterial smooth muscle. The Journal of General Physiology.

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[47] Jaggar JH, Wellman GC, Heppner TJ, Porter VA, Perez GJ, Gollasch M, et al. Ca2+ channels, ryanodine receptors

functional unit for regulating arterial tone. Acta Physiologica Scandinavica.

[48] Ledoux J, Werner ME, Brayden JE, Nelson MT. Calcium-activated potassium channels and the regulation of vascular tone. Physiology. 2006;**21**(1):69-78

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channels: A

2003;**10**(2):133-141

2006;**127**(6):611-622

and Ca2+-activated K+

2008;**164**(4):577-587

1995;**270**(5236):633

arteries: Modulation by dihydropyridine

drugs. Circulation Research.

[35] Ganitkevich VY, Isenberg G. Contribution of two types of calcium channels to membrane conductance of single myocytes from guinea-pig coronary

artery. The Journal of Physiology.

[36] Smirnov SV, Aaronson PI. Ca2+ currents in single myocytes from human mesenteric arteries: Evidence for a physiological role of L-type channels. The Journal of Physiology.

[37] Koch W, Ellinor P, Schwartz A. cDNA cloning of a dihydropyridinesensitive calcium channel from rat aorta. Evidence for the existence of alternatively spliced forms. The Journal of Biological Chemistry. 1990;**265**(29):17786-17791

[38] Hill MA, Meininger GA. Calcium entry and myogenic phenomena in skeletal muscle arterioles. American Journal of Physiology. Heart and Circulatory Physiology. 1994;**267**(3):H1085-H1H92

[39] Perez-Reyes E. Molecular physiology of low voltage-activated T-type calcium channels. Physiological

Reviews. 2003;**83**(1):117-161

of nifedipine-insensitive, high voltage-activated Ca2+ channels in the terminal mesenteric artery of guinea pig. Circulation Research.

[41] Gordienko DV, Clausen C, Goligorsky MS. Ionic currents and endothelin signaling in smooth muscle cells from rat renal resistance arteries. American Journal of Physiology. Renal Physiology. 1994;**266**(2):F325-F341

[42] VanBavel E, Sorop O, Andreasen D, Pfaffendorf M, Jensen BL. Role of

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[40] Morita H, Cousins H, Onoue H, Ito Y, Inoue R. Predominant distribution

1986;**59**(2):229-235

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Abnormal coronary function in mice deficient in alpha1H T-type Ca2+ channels. Science. 2003;**302**(5649):1416-1418

[51] Morita H, Shi J, Ito Y, Inoue R. T-channel-like pharmacological properties of high voltage-activated, nifedipine-insensitive Ca2+ currents in the rat terminal mesenteric artery. British Journal of Pharmacology. 2002;**137**(4):467-476

[52] Okabe K, Kitamura K, Kuriyama H. The existence of a highly tetrodotoxin sensitive Na channel in freshly dispersed smooth muscle cells of the rabbit main pulmonary artery. Pflügers Archiv— European Journal of Physiology. 1988;**411**(4):423-428

[53] Cox RH, Zhou Z, Tulenko TN. Voltage-gated sodium channels in human aortic smooth muscle cells. Journal of Vascular Research. 1998;**35**(5):310-317

[54] Llinás R, Yarom Y. Electrophysiology of mammalian inferior olivary neurones in vitro. Different types of voltage-dependent ionic conductances. The Journal of Physiology. 1981;**315**:549-567

[55] Llinás R, Yarom Y. Properties and distribution of ionic conductances generating electroresponsiveness of mammalian inferior olivary neurones in vitro. The Journal of Physiology. 1981;**315**:569-584

[56] Catterall WA, Goldin AL, Waxman SG. International union of pharmacology. XLVII. Nomenclature and structure-function relationships of voltage-gated sodium channels. Pharmacological Reviews. 2005;**57**(4):397

[57] Lee-Kwon W, Goo JH, Zhang Z, Silldorff EP, Pallone TL. Vasa recta voltage-gated Na<sup>+</sup> channel NaV1.3 is regulated by calmodulin. American Journal of Physiology. Renal Physiology. 2007;**292**(1):F404-F414

[58] Saleh S, Yeung SYM, Prestwich S, Pucovsky V, Greenwood I. Electrophysiological and molecular identification of voltage-gated sodium channels in murine vascular myocytes. The Journal of Physiology. 2005;**568**(1):155-169

[59] Zhu H-L, Shibata A, Inai T, Nomura M, Shibata Y, Brock JA, et al. Characterization of NaV1.6-mediated Na<sup>+</sup> currents in smooth muscle cells isolated from mouse vas deferens. Journal of Cellular Physiology. 2010;**223**(1):234-243

[60] Biswas S, Deschenes I, DiSilvestre D, Tian Y, Halperin VL, Tomaselli GF. Calmodulin regulation of NaV1.4 current: Role of binding to the carboxyl terminus. The Journal of General Physiology. 2008;**131**(3):197-209

[61] Numann R, Catterall WA, Scheuer T. Functional modulation of brain sodium channels by protein kinase C phosphorylation. Science. 1991;**254**(5028):115-118

[62] Numann R, Hauschka S, Catterall W, Scheuer T. Modulation of skeletal muscle sodium channels in a satellite cell line by protein kinase C. The Journal of Neuroscience. 1994;**14**(7):4226-4236

**41**

**Chapter 3**

**Abstract**

**1. Introduction**

Mitochondria

*Kentu Lassiter and Sami Dridi*

of orexin's action on avian muscle mitochondria.

the poultry industry, but also to the biomedical field.

Orexin System and Avian Muscle

In mammals, orexin A and B (also known as hypocretin 1 and 2) are two orexigenic peptides produced primarily by the lateral hypothalamus that signal through two G-protein-coupled receptors, orexin receptors 1/2, and have been implicated in the regulation of several physiological processes. However, the physiological roles of orexin are not well defined in avian (non-mammalian vertebrate) species. Recently, we made a breakthrough by identifying that orexin and its related receptors 1/2 (ORXR1/2) are expressed in avian muscle tissue and cell line, and appears to be a secretory protein. Functional in vitro studies showed that orexin A and B differentially regulated expression of the orexin system, suggesting that orexins might have autocrine, paracrine, and/or endocrine roles. Administration of recombinant orexin modulated mitochondrial biogenesis, dynamics, function, and bioenergetics. In this chapter, we include a brief overview of the (patho) physiological role of orexin, comparative findings between mammalian and avian orexin, and in-depth analysis

**Keywords:** orexin system, muscle, avian species, mitochondria, gene expression

Orexin/hypocretin was originally identified by two different groups and reported in 1998 as an orexigenic feeding-related neuropeptide mainly produced in the rat hypothalamus [1, 2]. Numerous subsequent studies conducted in mammals have shown that orexin and its receptors regulate several physiological processes including food and water intake [3], control of wakefulness [4], circadian clock [5], energy and glucose homeostasis [6–8], lipid metabolism [9, 10], heart rate and blood pressure [11, 12], and neuroendocrine response to stress [13]. Despite these advancements in understanding the orexin system, studies investigating its distribution and function in avian (non-mammalian vertebrate) species are very limited and merit more consideration and further in-depth explorations. Understanding orexin distribution and unraveling its function in avian tissues, particularly in broiler (meat-type chickens) skeletal muscle is of particular importance not only to

The poultry (meat and egg) industry supports the livelihoods and food security of billions of people worldwide with an average annual production of 118 million metric tons of meat and 1360 billion eggs in 2017 [14]. This success was mainly achieved by intensive genetic selection for important economic and agricultural traits such as high growth rate, improved feed efficiency (conversion of feed to muscle mass), and increased muscle mass [15]. Today, indeed, modern broilers are

### **Chapter 3**

## Orexin System and Avian Muscle Mitochondria

*Kentu Lassiter and Sami Dridi*

### **Abstract**

In mammals, orexin A and B (also known as hypocretin 1 and 2) are two orexigenic peptides produced primarily by the lateral hypothalamus that signal through two G-protein-coupled receptors, orexin receptors 1/2, and have been implicated in the regulation of several physiological processes. However, the physiological roles of orexin are not well defined in avian (non-mammalian vertebrate) species. Recently, we made a breakthrough by identifying that orexin and its related receptors 1/2 (ORXR1/2) are expressed in avian muscle tissue and cell line, and appears to be a secretory protein. Functional in vitro studies showed that orexin A and B differentially regulated expression of the orexin system, suggesting that orexins might have autocrine, paracrine, and/or endocrine roles. Administration of recombinant orexin modulated mitochondrial biogenesis, dynamics, function, and bioenergetics. In this chapter, we include a brief overview of the (patho) physiological role of orexin, comparative findings between mammalian and avian orexin, and in-depth analysis of orexin's action on avian muscle mitochondria.

**Keywords:** orexin system, muscle, avian species, mitochondria, gene expression

### **1. Introduction**

Orexin/hypocretin was originally identified by two different groups and reported in 1998 as an orexigenic feeding-related neuropeptide mainly produced in the rat hypothalamus [1, 2]. Numerous subsequent studies conducted in mammals have shown that orexin and its receptors regulate several physiological processes including food and water intake [3], control of wakefulness [4], circadian clock [5], energy and glucose homeostasis [6–8], lipid metabolism [9, 10], heart rate and blood pressure [11, 12], and neuroendocrine response to stress [13]. Despite these advancements in understanding the orexin system, studies investigating its distribution and function in avian (non-mammalian vertebrate) species are very limited and merit more consideration and further in-depth explorations. Understanding orexin distribution and unraveling its function in avian tissues, particularly in broiler (meat-type chickens) skeletal muscle is of particular importance not only to the poultry industry, but also to the biomedical field.

The poultry (meat and egg) industry supports the livelihoods and food security of billions of people worldwide with an average annual production of 118 million metric tons of meat and 1360 billion eggs in 2017 [14]. This success was mainly achieved by intensive genetic selection for important economic and agricultural traits such as high growth rate, improved feed efficiency (conversion of feed to muscle mass), and increased muscle mass [15]. Today, indeed, modern broilers are

marketed in about half the time and at about twice the body weight compared to 50–60 years ago [16]. This fast growth is largely allocated to the beast muscle [17].

Chickens are naturally hyperglycemic compared with mammals [18], insulinresistant [19], lack the glucose transporter GLUT4 [20], lack functional brown adipose tissue [21], and are prone to obesity [22], and thereby they represent a highly relevant animal model for biomedical researches.

The proper function of mitochondria is invariably related to muscle growth since these organelles produce a vast majority (~90%) of the ATP needed for tissue growth and maintenance of energy metabolism homeostasis. Previous studies in broiler chickens have shown that mitochondrial perturbations are directly related to a decrease in feed efficiency. Additionally, the absence of orexin's effect on feed intake in chickens [23] suggest that this neuropeptide may be more involved in other peripheral metabolic and physiological processes rather than food/water intake and wakefulness that is seen in mammals. Therefore, if orexin and its receptors are shown to be present in skeletal muscle tissue of avian species, they may be exerting control of energy metabolism and muscle growth (i.e., myogenesis, insulin sensitivity, and glucose transport) in the cell in part by regulating mitochondrial dynamics. The role of orexin in skeletal muscle function could be of interest not only in improving health and feed efficiency of agricultural animals, but also in molecular medicine for pathophysiological understanding and therapeutic perspectives.

### **2. Identification of orexin and its receptors**

Orexin, which regulates wakefulness, energy homeostasis, and appetite/feeding behavior based on nutritional status, is a neuropeptide that was originally discovered in the hypothalamus of rats by two different research groups in 1998. De Lecea's group isolated cDNAs selectively expressed within the hypothalamus and found that two peptides showed high amino acid sequence homology with secretin (the gut peptide hormone), therefore they named the two peptides hypocretin 1 and 2 [1]. Sakurai's group, on the other hand, used reverse pharmacology to identify ligands of orphan G-protein-coupled receptors. Orphan receptors are those whose ligand and physiological actions are unknown [24]. They identified a novel family of neuropeptides that induced feeding behavior, so they named them orexin A and B [2]. The term orexin originates from the Greek word "orexis," meaning appetite. Both orexin A and B are formed by proteolytic cleavage of the precursor prepro-orexin [2]. When initially discovered in rats, the precursor peptide prepro-orexin was shown to be a 130-residue polypeptide from which the mature peptides ORX-A and ORX-B were formed, with ORX-A containing 33 amino acids and a molecular weight 3.562 kDa and ORX-B containing 28 amino acids and a molecular weight of 2.937 kDa [2]. When comparing the two peptides, ORX-B was shown to be 56% identical in amino acid sequence to ORX-A. However, when comparing mammalian species (human, rat, mouse, pig, and cow), the sequence and structure of both peptides is highly conserved [2]. A number of studies have also shown that the structures of ORX-A and ORX-B in chicken and certain types of fish are conserved when compared to their mammalian counterparts [25–28].

ORX-A and ORX-B signal through the G-protein-coupled receptors orexin receptor 1 (ORXR1) and orexin receptor 2 (ORXR2). These two ubiquitously expressed receptors were first identified in human brain tissue through expressed sequence tags combined with database searching using tBLASTn [2, 29]. In humans it has been shown that the amino acid sequence for ORXR1 and ORXR2 is more than

**43**

broiler chickens.

*Orexin System and Avian Muscle Mitochondria DOI: http://dx.doi.org/10.5772/intechopen.85177*

death pathways [35, 36].

**3. Orexin system in avian species**

testis and ovary [38], and the stomach and intestine [41].

60% identical, making them more similar to each other than to other G-proteincoupled receptors [2]. The same study also showed that both receptors are highly conserved between humans and rats, with the sequence identity being greater than 90% for both. The two orexin peptides have different binding affinities for the two orexin receptors. ORX-A is able to bind to both receptors but has a higher affinity for ORXR1, while ORX-B binds to ORXR2 with the same affinity as ORX-A [28]. Several studies have indicated that the binding of orexins to orexin receptors activates multiple G-proteins. In studies conducted using humans [30, 31] and rats [32], it was shown that the binding of ORXR2 activates Gi, Gs, Go, and Gq proteins in adrenal cortical tissue. It appears that the responses to orexin receptor signaling are highly diverse. The activation of the various G-proteins can lead to a variety of cellular responses such as the regulation of protein/lipid kinases [33, 34]. In the case of orexin stimulation, activation of G-proteins can lead to the excitation of neurons that affect the regulation of ion channels, the activation of signaling cascades that regulate the activity of adenylyl cyclase and phospholipases, and activation of cell

Significantly fewer studies concerning the orexin system have been conducted in avian species when compared to mammals. Chicken prepro-orexin was first cloned, sequenced, and characterized in 2002 [37]. In that study, chicken orexin cDNA was shown to be expressed in the periventricular and lateral hypothalamic areas and consisting of 658 bp that encode 148 amino acids. Also, chicken ORX-A and ORX-B are evolutionary conserved with their mammalian counterparts, showing ~85 and 65% similarity at the amino acid level [37]. Characterization of the chicken orexin receptor shows that its cDNA has a length of 1869 bp that encode 501 amino acids, which corresponds to mammalian ORXR2 with an 80% homology [37]. Studies looking at tissue distribution of orexin and orexin receptors in chickens show that the peptides are expressed in the brain [37–40], pituitary gland, adrenal gland,

Orexin does not appear to elicit the same responses in birds as it does in mammals. One of the most noted actions that centrally administered orexin has in mammals is that it stimulates feeding/food intake [2, 3, 28, 42]. However, central administration of ORX-A or ORX-B did not stimulate feed intake in neonatal broiler and layer chicks [23, 43] or adult pigeons [44]. Studies examining mRNA expression of prepro-orexin in the hypothalamus of chicken [37] and quail [45] following 24 h fasting showed no increase in expression, providing further evidence for the lack of a stimulatory effect on feeding behavior in birds. Another feeding behavior study did show an increase in prepro-orexin mRNA [46], but this was measured after 48 h fasting, which would be an extreme fasting condition for

As stated previously, another hallmark of orexin function in mammals is its effects on the regulation of sleep/wakefulness, where a dysfunction in the orexin system is associated with the sleep condition narcolepsy [4]. Studies investigating the effects of orexin on arousal in birds have been conducted with mixed results. It has been concluded that either hypothalamic orexin does not play a role in arousal of the sleep/wake cycle [39], or that only ORX-A in conjunction with the enzyme monoamine oxidase-A (MAO-A) increases arousal in layer chicks only and not broiler chicks [43, 47]. Multiple studies investigating orexin in avian species theorize that the peptide appears to be more involved in the regulation of energy

metabolism than feed intake and sleep/wake cycles [39, 40, 48].

### *Orexin System and Avian Muscle Mitochondria DOI: http://dx.doi.org/10.5772/intechopen.85177*

*Muscle Cells - Recent Advances and Future Perspectives*

peutic perspectives.

highly relevant animal model for biomedical researches.

**2. Identification of orexin and its receptors**

marketed in about half the time and at about twice the body weight compared to 50–60 years ago [16]. This fast growth is largely allocated to the beast muscle [17]. Chickens are naturally hyperglycemic compared with mammals [18], insulinresistant [19], lack the glucose transporter GLUT4 [20], lack functional brown adipose tissue [21], and are prone to obesity [22], and thereby they represent a

The proper function of mitochondria is invariably related to muscle growth since these organelles produce a vast majority (~90%) of the ATP needed for tissue growth and maintenance of energy metabolism homeostasis. Previous studies in broiler chickens have shown that mitochondrial perturbations are directly related to a decrease in feed efficiency. Additionally, the absence of orexin's effect on feed intake in chickens [23] suggest that this neuropeptide may be more involved in other peripheral metabolic and physiological processes rather than food/water intake and wakefulness that is seen in mammals. Therefore, if orexin and its receptors are shown to be present in skeletal muscle tissue of avian species, they may be exerting control of energy metabolism and muscle growth (i.e., myogenesis, insulin sensitivity, and glucose transport) in the cell in part by regulating mitochondrial dynamics. The role of orexin in skeletal muscle function could be of interest not only in improving health and feed efficiency of agricultural animals, but also in molecular medicine for pathophysiological understanding and thera-

Orexin, which regulates wakefulness, energy homeostasis, and appetite/feeding behavior based on nutritional status, is a neuropeptide that was originally discovered in the hypothalamus of rats by two different research groups in 1998. De Lecea's group isolated cDNAs selectively expressed within the hypothalamus and found that two peptides showed high amino acid sequence homology with secretin (the gut peptide hormone), therefore they named the two peptides hypocretin 1 and 2 [1]. Sakurai's group, on the other hand, used reverse pharmacology to identify ligands of orphan G-protein-coupled receptors. Orphan receptors are those whose ligand and physiological actions are unknown [24]. They identified a novel family of neuropeptides that induced feeding behavior, so they named them orexin A and B [2]. The term orexin originates from the Greek word "orexis," meaning appetite. Both orexin A and B are formed by proteolytic cleavage of the precursor prepro-orexin [2]. When initially discovered in rats, the precursor peptide prepro-orexin was shown to be a 130-residue polypeptide from which the mature peptides ORX-A and ORX-B were formed, with ORX-A containing 33 amino acids and a molecular weight 3.562 kDa and ORX-B containing 28 amino acids and a molecular weight of 2.937 kDa [2]. When comparing the two peptides, ORX-B was shown to be 56% identical in amino acid sequence to ORX-A. However, when comparing mammalian species (human, rat, mouse, pig, and cow), the sequence and structure of both peptides is highly conserved [2]. A number of studies have also shown that the structures of ORX-A and ORX-B in chicken and certain types of fish are conserved when compared to their mamma-

ORX-A and ORX-B signal through the G-protein-coupled receptors orexin receptor 1 (ORXR1) and orexin receptor 2 (ORXR2). These two ubiquitously expressed receptors were first identified in human brain tissue through expressed sequence tags combined with database searching using tBLASTn [2, 29]. In humans it has been shown that the amino acid sequence for ORXR1 and ORXR2 is more than

**42**

lian counterparts [25–28].

60% identical, making them more similar to each other than to other G-proteincoupled receptors [2]. The same study also showed that both receptors are highly conserved between humans and rats, with the sequence identity being greater than 90% for both. The two orexin peptides have different binding affinities for the two orexin receptors. ORX-A is able to bind to both receptors but has a higher affinity for ORXR1, while ORX-B binds to ORXR2 with the same affinity as ORX-A [28]. Several studies have indicated that the binding of orexins to orexin receptors activates multiple G-proteins. In studies conducted using humans [30, 31] and rats [32], it was shown that the binding of ORXR2 activates Gi, Gs, Go, and Gq proteins in adrenal cortical tissue. It appears that the responses to orexin receptor signaling are highly diverse. The activation of the various G-proteins can lead to a variety of cellular responses such as the regulation of protein/lipid kinases [33, 34]. In the case of orexin stimulation, activation of G-proteins can lead to the excitation of neurons that affect the regulation of ion channels, the activation of signaling cascades that regulate the activity of adenylyl cyclase and phospholipases, and activation of cell death pathways [35, 36].

### **3. Orexin system in avian species**

Significantly fewer studies concerning the orexin system have been conducted in avian species when compared to mammals. Chicken prepro-orexin was first cloned, sequenced, and characterized in 2002 [37]. In that study, chicken orexin cDNA was shown to be expressed in the periventricular and lateral hypothalamic areas and consisting of 658 bp that encode 148 amino acids. Also, chicken ORX-A and ORX-B are evolutionary conserved with their mammalian counterparts, showing ~85 and 65% similarity at the amino acid level [37]. Characterization of the chicken orexin receptor shows that its cDNA has a length of 1869 bp that encode 501 amino acids, which corresponds to mammalian ORXR2 with an 80% homology [37]. Studies looking at tissue distribution of orexin and orexin receptors in chickens show that the peptides are expressed in the brain [37–40], pituitary gland, adrenal gland, testis and ovary [38], and the stomach and intestine [41].

Orexin does not appear to elicit the same responses in birds as it does in mammals. One of the most noted actions that centrally administered orexin has in mammals is that it stimulates feeding/food intake [2, 3, 28, 42]. However, central administration of ORX-A or ORX-B did not stimulate feed intake in neonatal broiler and layer chicks [23, 43] or adult pigeons [44]. Studies examining mRNA expression of prepro-orexin in the hypothalamus of chicken [37] and quail [45] following 24 h fasting showed no increase in expression, providing further evidence for the lack of a stimulatory effect on feeding behavior in birds. Another feeding behavior study did show an increase in prepro-orexin mRNA [46], but this was measured after 48 h fasting, which would be an extreme fasting condition for broiler chickens.

As stated previously, another hallmark of orexin function in mammals is its effects on the regulation of sleep/wakefulness, where a dysfunction in the orexin system is associated with the sleep condition narcolepsy [4]. Studies investigating the effects of orexin on arousal in birds have been conducted with mixed results. It has been concluded that either hypothalamic orexin does not play a role in arousal of the sleep/wake cycle [39], or that only ORX-A in conjunction with the enzyme monoamine oxidase-A (MAO-A) increases arousal in layer chicks only and not broiler chicks [43, 47]. Multiple studies investigating orexin in avian species theorize that the peptide appears to be more involved in the regulation of energy metabolism than feed intake and sleep/wake cycles [39, 40, 48].

### **4. Orexin system in avian muscle**

Studies investigating the orexin-producing neurons in the rat/mouse brain report that central administration of the neuropeptide facilitates changes in

### **Figure 1.**

*Prepro-orexin and its related receptors are expressed in broiler chicken muscle (a, b) and QM7 cell line (c–f). (a) RT-PCR. Total RNA (1 μg) was reverse transcribed and subjected to RT-PCR. Brain, testis, and ovary were used as positive controls. (b) Western blot. 70 μg total protein extracted from each tissue were electrophoresed and blotted onto polyvinylidene difluoride membrane. Prepro-orexin (ORX) and orexin receptor 1 (ORXR1) expression was detected by immunoblot using rabbit anti-mouse ORX and rabbit anti-rat ORXR1 antibodies. Hypothalamus and brain were used as positive controls. The figure is a representative picture from one animal. (c) RT-PCR. Total RNA (1 μg) was isolated from chicken brain and QM7 cells and subjected to RT-PCR. (d) Western blot. Total protein extracted from the cells were electrophoresed and blotted onto polyvinylidene difluoride membrane. Prepro-orexin (ORX) and ORXR1/2 expression was detected by immunoblot using rabbit anti-mouse ORX and rabbit anti-rat ORXR1/2 antibodies. Brain was used as positive control. (e) Northern blot. Total RNA (10 μg) was separated by agarose gel electrophoresis and transferred to a nylon membrane and hybridized with specific biotinlabeled DNA probe toward chicken ORX, and 18S. Hybridization signals were detected by FluorChem M MultiFluor system. (f) Immunofluorescence staining. Intracellular ORX system distribution visualized by fluorescent microscope in the presence of a secondary antibody conjugated with Alexa Fluor 488 (green) and DAPI (blue).*

**45**

expression.

*Orexin System and Avian Muscle Mitochondria DOI: http://dx.doi.org/10.5772/intechopen.85177*

quail [54–56].

endocrine roles.

skeletal muscle tone [49, 50] in addition to increasing glucose metabolism and insulin sensitivity of skeletal muscle [51]. This indicates that orexin signaling in the central nervous system regulates muscle glucose metabolism by activating muscle sympathetic nerves. Even though the orexin system has been identified in the peripheral tissues of a variety of vertebrate species [52], very few studies have been conducted to determine whether orexin and its related receptors are expressed in vertebrate skeletal muscle and if so, what effects it's presence may have on vertebrate muscle function and physiology. Review of the current literature shows that to date orexin has only been identified as being expressed in the heart muscle of zebrafish [53] and skeletal muscle of goldfish, chicken, and

We have conducted studies using RT-PCR and Western blot analysis to identify both orexin and its related receptors as being expressed in broiler chicken muscle (**Figure 1a, b**). In addition, RT-PCR, Western blot analysis, Northern blot analysis, and immunofluorescence have been used to illustrate that orexin and its receptors are also expressed in the cytoplasmic compartment of a spontaneously immortalized quail muscle (QM7) cell line (**Figure 1c**–**f**). In humans, scientific evidence indicates that orexin is a secretory peptide due to its presence in the blood circulation. Additional support for orexin being secreted is given by the first 33 amino acids of human prepro-orexin containing a hydrophobic core followed by residues with small polar side chains, which are characteristics of a secretory signal sequence [57]. Cell culture experiments using the QM7 cell line have been carried out in order to determine whether orexin is also secreted in avian muscle. When QM7 cells are incubated in serum-free growth media in the presence of recombinant human orexin B (rORX-B), an increase in the level of prepro-orexin in the growth media is evident as seen in **Figure 2a**. Further support for the secretion of prepro-orexin is illustrated in **Figure 2b** where the levels of prepro-orexin in the serum-free growth media accumulate over a 72 h period, and in **Figure 2c** where application of the anti-secretory compound brefeldin A causes a buildup of prepro-orexin in the QM7 cell lysate and the subsequent absence of the peptide in the growth media. The expression and secretion of prepro-orexin from avian muscle cells suggests that avian orexin could be a myokine that probably functions in autocrine, paracrine, and/or

Subsequent experiments were conducted in order to determine whether the orexin system is capable of self-regulation. The effects of 10 and 100 nM of recombinant human orexin A (rORX-A) and rORX-B on mRNA and protein expression of ORX and its related receptors ORXR1 and ORXR2 in QM7 cells are shown in **Figure 2d–g**. A 24 h treatment with either 10 or 100 nM rORX-A upregulated ORX and ORXR1, but not ORXR2 mRNA expression (**Figure 2f**). Treating cells with rORX-B downregulated ORX and ORXR2 and increased ORXR1 mRNA levels (**Figure 2g**). Protein expression levels of ORX, ORXR1, and ORXR2 showed the same patterns as their corresponding genes (**Figure 2d, e**) with the effects on expression levels appearing to be dose-dependent. The ability of rORX-A and rORX-B to differentially regulate gene and protein expression of the orexin system supports the idea that orexins function in an autocrine, paracrine, and/or endocrine role in avian muscle. Although the underlying mechanisms behind the differential regulation of the orexin system is still unknown, the divergent effects of rORX-A and rORX-B on orexin system expression might be related to their structure (presence of disulfide bonds in orexin A and not in orexin B) and their different binding affinity to ORXR2, since they had similar effects on ORXR1

*Orexin System and Avian Muscle Mitochondria DOI: http://dx.doi.org/10.5772/intechopen.85177*

*Muscle Cells - Recent Advances and Future Perspectives*

Studies investigating the orexin-producing neurons in the rat/mouse brain report that central administration of the neuropeptide facilitates changes in

*Prepro-orexin and its related receptors are expressed in broiler chicken muscle (a, b) and QM7 cell line (c–f). (a) RT-PCR. Total RNA (1 μg) was reverse transcribed and subjected to RT-PCR. Brain, testis, and ovary were used as positive controls. (b) Western blot. 70 μg total protein extracted from each tissue were electrophoresed and blotted onto polyvinylidene difluoride membrane. Prepro-orexin (ORX) and orexin receptor 1 (ORXR1) expression was detected by immunoblot using rabbit anti-mouse ORX and rabbit anti-rat ORXR1 antibodies. Hypothalamus and brain were used as positive controls. The figure is a representative picture from one animal. (c) RT-PCR. Total RNA (1 μg) was isolated from chicken brain and QM7 cells and subjected to RT-PCR. (d) Western blot. Total protein extracted from the cells were electrophoresed and blotted onto polyvinylidene difluoride membrane. Prepro-orexin (ORX) and ORXR1/2 expression was detected by immunoblot using rabbit anti-mouse ORX and rabbit anti-rat ORXR1/2 antibodies. Brain was used as positive control. (e) Northern blot. Total RNA (10 μg) was separated by agarose gel electrophoresis and transferred to a nylon membrane and hybridized with specific biotinlabeled DNA probe toward chicken ORX, and 18S. Hybridization signals were detected by FluorChem M MultiFluor system. (f) Immunofluorescence staining. Intracellular ORX system distribution visualized by fluorescent microscope in the presence of a secondary antibody conjugated with Alexa Fluor 488 (green)* 

**4. Orexin system in avian muscle**

**44**

*and DAPI (blue).*

**Figure 1.**

skeletal muscle tone [49, 50] in addition to increasing glucose metabolism and insulin sensitivity of skeletal muscle [51]. This indicates that orexin signaling in the central nervous system regulates muscle glucose metabolism by activating muscle sympathetic nerves. Even though the orexin system has been identified in the peripheral tissues of a variety of vertebrate species [52], very few studies have been conducted to determine whether orexin and its related receptors are expressed in vertebrate skeletal muscle and if so, what effects it's presence may have on vertebrate muscle function and physiology. Review of the current literature shows that to date orexin has only been identified as being expressed in the heart muscle of zebrafish [53] and skeletal muscle of goldfish, chicken, and quail [54–56].

We have conducted studies using RT-PCR and Western blot analysis to identify both orexin and its related receptors as being expressed in broiler chicken muscle (**Figure 1a, b**). In addition, RT-PCR, Western blot analysis, Northern blot analysis, and immunofluorescence have been used to illustrate that orexin and its receptors are also expressed in the cytoplasmic compartment of a spontaneously immortalized quail muscle (QM7) cell line (**Figure 1c**–**f**). In humans, scientific evidence indicates that orexin is a secretory peptide due to its presence in the blood circulation. Additional support for orexin being secreted is given by the first 33 amino acids of human prepro-orexin containing a hydrophobic core followed by residues with small polar side chains, which are characteristics of a secretory signal sequence [57]. Cell culture experiments using the QM7 cell line have been carried out in order to determine whether orexin is also secreted in avian muscle. When QM7 cells are incubated in serum-free growth media in the presence of recombinant human orexin B (rORX-B), an increase in the level of prepro-orexin in the growth media is evident as seen in **Figure 2a**. Further support for the secretion of prepro-orexin is illustrated in **Figure 2b** where the levels of prepro-orexin in the serum-free growth media accumulate over a 72 h period, and in **Figure 2c** where application of the anti-secretory compound brefeldin A causes a buildup of prepro-orexin in the QM7 cell lysate and the subsequent absence of the peptide in the growth media. The expression and secretion of prepro-orexin from avian muscle cells suggests that avian orexin could be a myokine that probably functions in autocrine, paracrine, and/or endocrine roles.

Subsequent experiments were conducted in order to determine whether the orexin system is capable of self-regulation. The effects of 10 and 100 nM of recombinant human orexin A (rORX-A) and rORX-B on mRNA and protein expression of ORX and its related receptors ORXR1 and ORXR2 in QM7 cells are shown in **Figure 2d–g**. A 24 h treatment with either 10 or 100 nM rORX-A upregulated ORX and ORXR1, but not ORXR2 mRNA expression (**Figure 2f**). Treating cells with rORX-B downregulated ORX and ORXR2 and increased ORXR1 mRNA levels (**Figure 2g**). Protein expression levels of ORX, ORXR1, and ORXR2 showed the same patterns as their corresponding genes (**Figure 2d, e**) with the effects on expression levels appearing to be dose-dependent. The ability of rORX-A and rORX-B to differentially regulate gene and protein expression of the orexin system supports the idea that orexins function in an autocrine, paracrine, and/or endocrine role in avian muscle. Although the underlying mechanisms behind the differential regulation of the orexin system is still unknown, the divergent effects of rORX-A and rORX-B on orexin system expression might be related to their structure (presence of disulfide bonds in orexin A and not in orexin B) and their different binding affinity to ORXR2, since they had similar effects on ORXR1 expression.

### **Figure 2.**

*Secretion of orexin by QM7 cells (a–c) and effect of orexin treatment on orexin system expression in QM7 cells (d–g). (a) Cell monolayers were incubated in serum-free medium with rORX-B (100 nM) for up to 72 h prior to Western blotting. (b) Percent change in ORX levels when compared to control (2 h post-treatment). (c) Cells were incubated in serum-free medium with or without brefeldin A (0.3 μg/ml) for 12 h. Medium and/or cell lysates were subjected to immunoblot analysis using an orexin antibody. (d–g) Cells were treated with 0 (control), 10, or 100 nM of recombinant orexin A and B for 24 h. Total protein and RNA were isolated and relative expression of ORX and ORXR1/2 was determined. Protein levels were measured by Western blot analysis (d, e). mRNA expression was measured by QPCR using 2−ΔΔCt method (f, g). Data are expressed as means ± SE (n = 6). \* Significant difference between orexin-treated and control cells (P < 0.05).*

### **4.1 The orexin system differentially regulates mitochondrial-related genes and mitochondrial bioenergetics in avian muscle cells**

In mammals, orexin has been shown to induce differentiation of brown adipose tissue (BAT), subsequently leading to thermogenesis [58, 59]. One of the effects orexin has in this process is the regulation of genes involved in mitochondrial biogenesis. The treatment of mouse preadipocytes with ORX-A revealed a number of changes in

**47**

*Orexin System and Avian Muscle Mitochondria DOI: http://dx.doi.org/10.5772/intechopen.85177*

PGC-1β, and FoxO-1 with the high dose (**Figure 3e, f, h**).

Since mitochondria are the powerhouse of the cell with central importance for producing more than 90% of the ATP needed to carry out essential cellular functions, these organelles are important for proper growth and development of skeletal muscle tissue. The changes in expression of mitochondrial-related genes and their transcriptional regulators suggest that orexin may control mitochondrial respiratory function in avian muscle. To gain better insight into the physiological roles of orexins in avian muscle, mitochondrial bioenergetics in QM7 cells treated with 10 and 100 nM of rORX-A and rORX-B were assessed by monitoring basal oxygen consumption rate (OCR) followed by sequential treatment of cells with oligomycin, FCCP (carbonyl cyanide-4-phenylhydrazone), and antimycin A as shown in **Figure 4f**. As described previously [63], the decrease in OCR following oligomycin (which blocks ATP synthase) reveals OCR attributed to ATP synthesis activity. Maximal OCR is revealed in response to the uncoupling compound FCCP, and the difference between maximal OCR and basal OCR (prior to oligomycin) represents mitochondrial oxygen reserve capacity that cells can draw upon when increased energy production is needed. Oxygen consumption that remains following treatment with the electron transport inhibitor antimycin A is attributed to non-mitochondrial OCR (i.e., OCR due to activities other than non-mitochondrial c oxidase activity, such as mitochondrial reactive oxygen species production, oxidase activities, etc.). The amount of OCR attributed to proton leak is determined by the difference between oligomycin and antimycin A-inhibited OCR. When the non-mitochondrial component of cellular OCR was subtracted and by setting maximal OCR following FCCP at 100%, the effects of ORX-A and ORX-B on ATP synthesis, reserve capacity, and proton leak were determined. ATP synthesis was slightly elevated by both orexins, but the effect was not statistically discernable. Analysis of reserve capacity indicated no effect of both doses of rORX-A

mitochondrial dynamics, including up-regulation of the expression of a number of genes involved in mitochondrial biogenesis (i.e., PGC-1α, PGC-1β, PPARγ1, and UCP1) [58]. These findings were further supported when subsequent immunofluorescence staining revealed an increase in mitochondrial abundance of the treated cells. Studies using other cell types treated with ORX-A have also shown effects on mitochondrial function. Human neuroblastoma cells treated with ORX-A had increased mitochondrial membrane potential [60]. Additionally, in studies using human hepatoma cells [61] and human embryonic kidney cells [62], treatment with ORX-A resulted in increased ATP production that shifted from glycolysis in the cytoplasm to oxidative phosphorylation in the mitochondria. Taken together, these studies indicate that orexin is able to enhance mitochondrial function, biogenesis, and ATP production in mammalian vertebrates. Similar experiments were carried out by treating QM7 cells with recombinant human orexin, as previously described, in order to observe the differential effects on mitochondrial-related genes, transcriptional regulators, and bioenergetics. **Figure 3** illustrates how orexin causes differential expression of mitochondrial genes. rORX-A had no effect on av-UCP mRNA abundance (**Figure 3b**), but it downregulated the expression of av-ANT (mRNA and protein levels), Ski, and NRF-1 in a dose-dependent manner (**Figure 3a, c, d**). rORX-B, however, downregulated the expression of av-UCP and increased the expression of Ski and NRF-1 without altering the expression of av-ANT (**Figure 3a–d**). The mitochondrial transcriptional regulators related to these genes were also differentially regulated following treatment with recombinant orexins as seen in **Figure 3e–h**. Both doses of rORX-A caused a significant downregulation of PPARδ and FoxO-1 expression (**Figure 3g, h**). A high dose of rORX-A significantly downregulated the expression of PGC-1β, and both doses did not alter PGC-1α mRNA abundance (**Figure 3e, f**). rORX-B, however, induced the expression of these transcription factors in a dosedependent manner, but the effects were statistically significant only for PGC-1α,

### *Orexin System and Avian Muscle Mitochondria DOI: http://dx.doi.org/10.5772/intechopen.85177*

*Muscle Cells - Recent Advances and Future Perspectives*

**46**

**Figure 2.**

*means ± SE (n = 6). \**

**4.1 The orexin system differentially regulates mitochondrial-related genes and** 

*Significant difference between orexin-treated and control cells (P < 0.05).*

*Secretion of orexin by QM7 cells (a–c) and effect of orexin treatment on orexin system expression in QM7 cells (d–g). (a) Cell monolayers were incubated in serum-free medium with rORX-B (100 nM) for up to 72 h prior to Western blotting. (b) Percent change in ORX levels when compared to control (2 h post-treatment). (c) Cells were incubated in serum-free medium with or without brefeldin A (0.3 μg/ml) for 12 h. Medium and/or cell lysates were subjected to immunoblot analysis using an orexin antibody. (d–g) Cells were treated with 0 (control), 10, or 100 nM of recombinant orexin A and B for 24 h. Total protein and RNA were isolated and relative expression of ORX and ORXR1/2 was determined. Protein levels were measured by Western blot analysis (d, e). mRNA expression was measured by QPCR using 2−ΔΔCt method (f, g). Data are expressed as* 

In mammals, orexin has been shown to induce differentiation of brown adipose tissue (BAT), subsequently leading to thermogenesis [58, 59]. One of the effects orexin has in this process is the regulation of genes involved in mitochondrial biogenesis. The treatment of mouse preadipocytes with ORX-A revealed a number of changes in

**mitochondrial bioenergetics in avian muscle cells**

mitochondrial dynamics, including up-regulation of the expression of a number of genes involved in mitochondrial biogenesis (i.e., PGC-1α, PGC-1β, PPARγ1, and UCP1) [58]. These findings were further supported when subsequent immunofluorescence staining revealed an increase in mitochondrial abundance of the treated cells. Studies using other cell types treated with ORX-A have also shown effects on mitochondrial function. Human neuroblastoma cells treated with ORX-A had increased mitochondrial membrane potential [60]. Additionally, in studies using human hepatoma cells [61] and human embryonic kidney cells [62], treatment with ORX-A resulted in increased ATP production that shifted from glycolysis in the cytoplasm to oxidative phosphorylation in the mitochondria. Taken together, these studies indicate that orexin is able to enhance mitochondrial function, biogenesis, and ATP production in mammalian vertebrates.

Similar experiments were carried out by treating QM7 cells with recombinant human orexin, as previously described, in order to observe the differential effects on mitochondrial-related genes, transcriptional regulators, and bioenergetics. **Figure 3** illustrates how orexin causes differential expression of mitochondrial genes. rORX-A had no effect on av-UCP mRNA abundance (**Figure 3b**), but it downregulated the expression of av-ANT (mRNA and protein levels), Ski, and NRF-1 in a dose-dependent manner (**Figure 3a, c, d**). rORX-B, however, downregulated the expression of av-UCP and increased the expression of Ski and NRF-1 without altering the expression of av-ANT (**Figure 3a–d**). The mitochondrial transcriptional regulators related to these genes were also differentially regulated following treatment with recombinant orexins as seen in **Figure 3e–h**. Both doses of rORX-A caused a significant downregulation of PPARδ and FoxO-1 expression (**Figure 3g, h**). A high dose of rORX-A significantly downregulated the expression of PGC-1β, and both doses did not alter PGC-1α mRNA abundance (**Figure 3e, f**). rORX-B, however, induced the expression of these transcription factors in a dosedependent manner, but the effects were statistically significant only for PGC-1α, PGC-1β, and FoxO-1 with the high dose (**Figure 3e, f, h**).

Since mitochondria are the powerhouse of the cell with central importance for producing more than 90% of the ATP needed to carry out essential cellular functions, these organelles are important for proper growth and development of skeletal muscle tissue. The changes in expression of mitochondrial-related genes and their transcriptional regulators suggest that orexin may control mitochondrial respiratory function in avian muscle. To gain better insight into the physiological roles of orexins in avian muscle, mitochondrial bioenergetics in QM7 cells treated with 10 and 100 nM of rORX-A and rORX-B were assessed by monitoring basal oxygen consumption rate (OCR) followed by sequential treatment of cells with oligomycin, FCCP (carbonyl cyanide-4-phenylhydrazone), and antimycin A as shown in **Figure 4f**. As described previously [63], the decrease in OCR following oligomycin (which blocks ATP synthase) reveals OCR attributed to ATP synthesis activity. Maximal OCR is revealed in response to the uncoupling compound FCCP, and the difference between maximal OCR and basal OCR (prior to oligomycin) represents mitochondrial oxygen reserve capacity that cells can draw upon when increased energy production is needed. Oxygen consumption that remains following treatment with the electron transport inhibitor antimycin A is attributed to non-mitochondrial OCR (i.e., OCR due to activities other than non-mitochondrial c oxidase activity, such as mitochondrial reactive oxygen species production, oxidase activities, etc.). The amount of OCR attributed to proton leak is determined by the difference between oligomycin and antimycin A-inhibited OCR. When the non-mitochondrial component of cellular OCR was subtracted and by setting maximal OCR following FCCP at 100%, the effects of ORX-A and ORX-B on ATP synthesis, reserve capacity, and proton leak were determined. ATP synthesis was slightly elevated by both orexins, but the effect was not statistically discernable. Analysis of reserve capacity indicated no effect of both doses of rORX-A

### **Figure 3.**

*Effect of orexin treatment on mitochondrial-related genes (a–d) and mitochondrial-transcriptional regulators (e–h) in QM7 cells. Cells were treated with recombinant orexin A or B (10 and 100 nM) for 24 h and the relative abundance of avian (av)-adenosine nucleotide translocator (ANT; a), UCP (b), Ski (c), nuclear respiratory factor 1 (NRF-1; d), PGC-1α (e), PGC-1β (f), peroxisome proliferator-activated receptor δ (PPARδ; g), and FoxO-1 (h) were determined by QPCR. Untreated cells were used as control. Protein levels of av-ANT and PGC-1<sup>α</sup> were measured by Western blot analysis. Data are expressed as means ± SE (n = 6). \* Significant difference between orexin-treated and control cells (P < 0.05).*

and rORX-B; however, proton leak was significantly decreased by 10 nM of rORX-A, and by 100 nM of rORX-B (**Figure 4g**). The combined data from these experiments illustrate that orexins lead to the alteration of mitochondrial-related genes, their transcriptional regulators, and respiratory function in avian muscle cells. These changes suggest that orexin might also control mitochondrial dynamics (i.e., fusion/fission of mitochondria) in avian muscle.

**49**

**Figure 4.**

*\**

**avian muscle cells**

**4.2 Orexins differentially regulate mitochondrial biogenesis and dynamics in** 

*Significant difference between orexin-treated and control cells (P < 0.05).*

*Effect of orexin treatment on mitochondrial distribution (a), mitochondrial DNA and mass (b–e), and mitochondrial bioenergetics (f, g) in QM7 cells. a: Cells were cultured in chamber slides and treated with 100 nM of rORX-A or rORX-B for 24 h. Mitochondria were visualized with MitoTracker Red CMX Ros (75 nM) under a fluorescent microscope. Representative images acquired and deconvoluted are shown. (b–e) QM7 cells were treated with orexins (10 and 100 nM) for 24 h. The levels of mtDNA (b) and the relative expression of mtSSBP1 (c), mitochondrial markers CoxIV (d), and Cox5a (e) were determined by real-time PCR. (f, g) Oxygen consumption rate (OCR) (f) and the percentage of OCR due to proton leak (g) was determined using an XF24 flux Analyzer (Agilent Technologies). The values represent the means ±SE (n = 6).* 

Since mtDNA replication and quantitation are a necessary component of mitochondrial biogenesis, the expression of mtDNA and mtSSBP1 was measured in orexin-treated QM7 cells as shown in **Figure 4b, c**. In contrast to rORX-A, in which both doses significantly downregulated mtDNA and upregulated mtSSBP1 expression, rORX-B (high dose) significantly increased mtDNA expression without

*Orexin System and Avian Muscle Mitochondria DOI: http://dx.doi.org/10.5772/intechopen.85177* *Orexin System and Avian Muscle Mitochondria DOI: http://dx.doi.org/10.5772/intechopen.85177*

*Muscle Cells - Recent Advances and Future Perspectives*

**48**

**Figure 3.**

mitochondria) in avian muscle.

and rORX-B; however, proton leak was significantly decreased by 10 nM of rORX-A, and by 100 nM of rORX-B (**Figure 4g**). The combined data from these experiments illustrate that orexins lead to the alteration of mitochondrial-related genes, their transcriptional regulators, and respiratory function in avian muscle cells. These changes suggest that orexin might also control mitochondrial dynamics (i.e., fusion/fission of

*Significant difference between orexin-treated and control cells (P < 0.05).*

*Effect of orexin treatment on mitochondrial-related genes (a–d) and mitochondrial-transcriptional regulators (e–h) in QM7 cells. Cells were treated with recombinant orexin A or B (10 and 100 nM) for 24 h and the relative abundance of avian (av)-adenosine nucleotide translocator (ANT; a), UCP (b), Ski (c), nuclear respiratory factor 1 (NRF-1; d), PGC-1α (e), PGC-1β (f), peroxisome proliferator-activated receptor δ (PPARδ; g), and FoxO-1 (h) were determined by QPCR. Untreated cells were used as control. Protein levels of av-ANT and PGC-1<sup>α</sup> were measured by Western blot analysis. Data are expressed as means ± SE (n = 6). \**

### **Figure 4.**

*Effect of orexin treatment on mitochondrial distribution (a), mitochondrial DNA and mass (b–e), and mitochondrial bioenergetics (f, g) in QM7 cells. a: Cells were cultured in chamber slides and treated with 100 nM of rORX-A or rORX-B for 24 h. Mitochondria were visualized with MitoTracker Red CMX Ros (75 nM) under a fluorescent microscope. Representative images acquired and deconvoluted are shown. (b–e) QM7 cells were treated with orexins (10 and 100 nM) for 24 h. The levels of mtDNA (b) and the relative expression of mtSSBP1 (c), mitochondrial markers CoxIV (d), and Cox5a (e) were determined by real-time PCR. (f, g) Oxygen consumption rate (OCR) (f) and the percentage of OCR due to proton leak (g) was determined using an XF24 flux Analyzer (Agilent Technologies). The values represent the means ±SE (n = 6). \* Significant difference between orexin-treated and control cells (P < 0.05).*

### **4.2 Orexins differentially regulate mitochondrial biogenesis and dynamics in avian muscle cells**

Since mtDNA replication and quantitation are a necessary component of mitochondrial biogenesis, the expression of mtDNA and mtSSBP1 was measured in orexin-treated QM7 cells as shown in **Figure 4b, c**. In contrast to rORX-A, in which both doses significantly downregulated mtDNA and upregulated mtSSBP1 expression, rORX-B (high dose) significantly increased mtDNA expression without

### **Figure 5.**

*Effect of orexin treatment on mitochondrial dynamics-related genes in QM7 cells. QM7 cells were treated with orexins (10 and 100 nM) for 24 h. The relative expression of four genes involved in mitochondrial fusion, MFN1 (a), MFN2 (b), OPA1 (c), OMA1 (d) and three genes involved in mitochondrial fission, MTFP1 (f), DNM1 (g), and MTFR1 (h) was determined by real-time PCR. The protein levels of MFN1, MFN2, and OPA1 were determined by Western blot analysis (e). The values represent the means ±SE (n = 6). \* Significant difference between orexin-treated and control cells (P < 0.05).*

affecting mtSSBP1 levels. Consistent with these observations and in contrast to rORX-A, rORX-B increased mitochondrial content as visualized with MitoTracker Red probe staining (**Figure 4a**). Neither rORX-A nor rORX-B affected the expression of the mitochondrial transcription factor TFAM (data not shown). The expression of Cox IV and Cox 5a genes, commonly used markers for mitochondrial mass

**51**

*Orexin System and Avian Muscle Mitochondria DOI: http://dx.doi.org/10.5772/intechopen.85177*

growth and development.

**5. Conclusions**

and biogenesis, was determined. The high dose (100 nM) of rORX-A decreased Cox IV gene expression; however, the high-dose of rORX-B significantly increased Cox

Mitochondria are dynamic organelles in the cell that constantly fuse and divide,

Orexins are originally identified as hypothalamic neuropeptides that have potent orexigenic effects on appetite and feeding behavior in mammals. In avian species, however, orexins seem to be myokines that regulate the expression of their own system as well as muscle mitochondrial function, biogenesis, bioenergetics, and dynamics. As intensive genetic selection for fast growth rate, driven by human nutritional needs and economic demands, have resulted in dramatic increase in chicken body weight arising mainly from increased muscle mass, these finding open new vistas on the role of orexin system in muscle development and energy metabolism. Further in depth investigations are warranted to understand the relationship between orexin system, mitochondrial network, and muscle growth and development which, in turn, will be beneficial to the poultry industry as it has the potential

to lead to increased production efficiency and reduced economic costs.

IV and Cox 5a mRNA levels compared with untreated cells (**Figure 4d, e**).

forming constantly changing tubular networks, according to the needs of the organism, thus leading to alterations in their morphology and function [64]. Since orexin is shown to alter expression of mitochondrial-related genes and transcriptional regulators, it may also control avian muscle mitochondrial dynamics. The molecular mechanisms that control mitochondrial dynamics are complex and require participation and coordination of both the nuclear and mitochondrial genomes. In mammals this network has been partially unraveled after the identification of some of the genes responsible for mitochondrial fusion [mitofusins (MFN1 and MFN2), and optic atrophy 1 (OPA1)] and fission [dyanamin-related protein 1 (Drp1 or DNM1), fission 1 (FIS1), and mitochondrial protein 18 kDa]. Up until the current study, the integration of such a mitochondrial network is unknown in avian species. Following orexin treatment, the expression of four genes related to mitochondrial fusion and three genes related to mitochondrial fission were measured as shown in **Figure 5**. Recombinant ORX-B at high dose significantly induced the expression of MFN2, OPA1, and OMA1, but decreased the mRNA levels of MFN1 (**Figure 5a–d**). The same effect was observed at the protein levels (**Figure 5e**). However, rORX-A significantly downregulated the expression of MFN1 and OMA1 with both doses, and OPA1 with the high dose, but did not affect that of MFN2 (**Figure 5a–d**). Interestingly, and in contrast to rORX-B, where no significant effects were observed, rORX-A upregulated the expression of mitofission-related genes MTFP1, DNM1, and MTFR1 (**Figure 5f–h**). These orexin-induced changes in the expression of dynamics-related genes may serve in regulating mitochondrial metabolism in muscle cells in response to the needs of the animal during stages of

### *Orexin System and Avian Muscle Mitochondria DOI: http://dx.doi.org/10.5772/intechopen.85177*

*Muscle Cells - Recent Advances and Future Perspectives*

**50**

**Figure 5.**

affecting mtSSBP1 levels. Consistent with these observations and in contrast to rORX-A, rORX-B increased mitochondrial content as visualized with MitoTracker Red probe staining (**Figure 4a**). Neither rORX-A nor rORX-B affected the expression of the mitochondrial transcription factor TFAM (data not shown). The expression of Cox IV and Cox 5a genes, commonly used markers for mitochondrial mass

*difference between orexin-treated and control cells (P < 0.05).*

*Significant* 

*Effect of orexin treatment on mitochondrial dynamics-related genes in QM7 cells. QM7 cells were treated with orexins (10 and 100 nM) for 24 h. The relative expression of four genes involved in mitochondrial fusion, MFN1 (a), MFN2 (b), OPA1 (c), OMA1 (d) and three genes involved in mitochondrial fission, MTFP1 (f), DNM1 (g), and MTFR1 (h) was determined by real-time PCR. The protein levels of MFN1, MFN2, and OPA1 were determined by Western blot analysis (e). The values represent the means ±SE (n = 6). \**

and biogenesis, was determined. The high dose (100 nM) of rORX-A decreased Cox IV gene expression; however, the high-dose of rORX-B significantly increased Cox IV and Cox 5a mRNA levels compared with untreated cells (**Figure 4d, e**).

Mitochondria are dynamic organelles in the cell that constantly fuse and divide, forming constantly changing tubular networks, according to the needs of the organism, thus leading to alterations in their morphology and function [64]. Since orexin is shown to alter expression of mitochondrial-related genes and transcriptional regulators, it may also control avian muscle mitochondrial dynamics. The molecular mechanisms that control mitochondrial dynamics are complex and require participation and coordination of both the nuclear and mitochondrial genomes. In mammals this network has been partially unraveled after the identification of some of the genes responsible for mitochondrial fusion [mitofusins (MFN1 and MFN2), and optic atrophy 1 (OPA1)] and fission [dyanamin-related protein 1 (Drp1 or DNM1), fission 1 (FIS1), and mitochondrial protein 18 kDa]. Up until the current study, the integration of such a mitochondrial network is unknown in avian species. Following orexin treatment, the expression of four genes related to mitochondrial fusion and three genes related to mitochondrial fission were measured as shown in **Figure 5**. Recombinant ORX-B at high dose significantly induced the expression of MFN2, OPA1, and OMA1, but decreased the mRNA levels of MFN1 (**Figure 5a–d**). The same effect was observed at the protein levels (**Figure 5e**). However, rORX-A significantly downregulated the expression of MFN1 and OMA1 with both doses, and OPA1 with the high dose, but did not affect that of MFN2 (**Figure 5a–d**). Interestingly, and in contrast to rORX-B, where no significant effects were observed, rORX-A upregulated the expression of mitofission-related genes MTFP1, DNM1, and MTFR1 (**Figure 5f–h**). These orexin-induced changes in the expression of dynamics-related genes may serve in regulating mitochondrial metabolism in muscle cells in response to the needs of the animal during stages of growth and development.

### **5. Conclusions**

Orexins are originally identified as hypothalamic neuropeptides that have potent orexigenic effects on appetite and feeding behavior in mammals. In avian species, however, orexins seem to be myokines that regulate the expression of their own system as well as muscle mitochondrial function, biogenesis, bioenergetics, and dynamics. As intensive genetic selection for fast growth rate, driven by human nutritional needs and economic demands, have resulted in dramatic increase in chicken body weight arising mainly from increased muscle mass, these finding open new vistas on the role of orexin system in muscle development and energy metabolism. Further in depth investigations are warranted to understand the relationship between orexin system, mitochondrial network, and muscle growth and development which, in turn, will be beneficial to the poultry industry as it has the potential to lead to increased production efficiency and reduced economic costs.

*Muscle Cells - Recent Advances and Future Perspectives*

### **Author details**

Kentu Lassiter\* and Sami Dridi Department of Poultry Science, Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, Arkansas

\*Address all correspondence to: klassit@uark.edu

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*Orexin System and Avian Muscle Mitochondria DOI: http://dx.doi.org/10.5772/intechopen.85177*

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**52**

**Author details**

Kentu Lassiter\* and Sami Dridi

provided the original work is properly cited.

University of Arkansas, Fayetteville, Arkansas

\*Address all correspondence to: klassit@uark.edu

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Department of Poultry Science, Center of Excellence for Poultry Science,

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expression in the chicken hypothalamus.

[38] Ohkubo T, Tsukada A, Shamoto K. cDNA cloning of chicken orexin receptor and tissue distribution: Sexually dimorphic expression in chicken gonads. Journal of Molecular Endocrinology. 2003;**31**:499-508. DOI:

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2013;**1513**:34-40. DOI: 10.1016/j.

Migues PV, Pompeiano M. Early expression of hypocretin/orexin in the chick embryo brain. PLoS One. 2014;**9**(9):e106977. DOI: 10.1371/

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complex. Cellular Signalling. 1998;**10**:447-455. DOI: 10.1016/ S0898-6568(98)00006-0

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Patent No. WO9634877

jcem.86.9.7849

jcem.86.10.7921

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*Muscle Cells - Recent Advances and Future Perspectives*

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[22] Hood RL. The cellular basis for growth of the abdominal fat pad in broiler-type chickens. Poultry Science. 1982;**61**:117-121. DOI: 10.3382/ps.0610117

[23] Furuse M, Ando R, Bungo T, Ao R,

Intracerebroventricular injection of orexins does not stimulate food intake in neonatal chicks. British Poultry Science. 1999;**40**:698-700. DOI: 10.1080/00071669987115

[24] Stadel JM, Wilson S, Bergsma D. Orphan g protein-coupled receptors: A neglected opportunity for pioneer drug discovery. Trends in Pharmacological Sciences. 1997;**18**:430-437. DOI: 10.1016/

[25] Shibahara M, Sakurai T, Nambu T, Takenouchi T, Iwaasa H, Egashira SI, et al. Structure, tissue distribution, and pharmacological characterization

[26] Alvarez CE, Sutcliffe JG. Hypocretin is an early member of the incretin gene family. Neuroscience Letters. 2002;**324**:169-172. DOI: 10.1016/

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Shimojo M, Masuda Y.

S0165-6147(97)01117-6

of xenopus orexins. Peptides. 1999;**20**:1169-1176. DOI: 10.1016/

S0196-9781(99)00120-5

S0304-3940(02)00195-7

regpep.2004.08.006

pr.109.001321

php?startid=9#/1 [Accessed: 7

[15] Havenstein GB, Ferket PR, Qureshi MA. Growth, livability, and feed conversion of 1957 versus 2001 broilers when fed representative 1957 and 2001 broiler diets. Poultry Science. 2003;**82**:1500-1508. DOI: 10.1093/

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[17] Guernec A, Berri C, Chevalier B, Wacrenier-Cere N, Le Bihan-Duval E, Duclos MJ. Muscle development, insulin-like growth factor-I and myostatin mRNA levels in chickens selected for increased breast muscle yield. Growth Hormone & IGF Research. 2003;**13**:8-18. DOI: 10.1016/

[18] Krzysik-Walker SM, Ocon-Grove OM, Maddineni SR, Hendricks GL 3rd, Ramachandran R. Is visfatin an adipokine or myokine? Evidence for greater visfatin expression in skeletal muscle than visceral fat in chickens. Endocrinology 2008;**149**: 1543-1550.

December 2018]

ps/82.10.1500

meatsci.2007.07.031

S1096-6374(02)00136-3

DOI: 10.1210/en.2007-1301

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[30] Karteris E, Randeva HS, Grammatopoulos DK, Jaffe RB, Hillhouse EW. Expression and coupling characteristics of the CRH and orexin type 2 receptors in human fetal adrenals. The Journal of Clinical Endocrinology and Metabolism. 2001;**86**:4512-4519. DOI: 10.1210/ jcem.86.9.7849

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[32] Karteris E, Machado RJ, Chen J, Zervou S, Hillhouse EW, Randeva HS. Food deprivation differentially modulates orexin receptor expression and signaling in the rat hypothalamus and adrenal cortex. American Journal of Physiology. Endocrinology and Metabolism. 2005;**288**:E1089-E1100. DOI: 10.1152/ajpendo.00351.2004

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[36] Kukkonen JP, Leonard CS. Orexin/ hypocretin receptor signaling cascades. British Journal of Pharmacology. 2014;**171**(2):314-331. DOI: 10.1111/ bph.12324

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[39] Miranda B, Esposito V, de Girolamo P, Sharp PJ, Wilson PW, Dunn IC. Orexin in the chicken hypothalamus: Immunocytochemical localization and comparison of mRNA concentrations during the day and night, and after chronic food restriction. Brain Research. 2013;**1513**:34-40. DOI: 10.1016/j. brainres.2013.03.036

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feeding-associated glucose utilization in skeletal muscle via sympathetic nervous system. Cell Metabolism. 2009;**10**: 466-480. DOI: 10.1016/j.cmet.

[52] Wong KK, Ng SY, Lee LT, Ng HK, Chow BK. Orexins and their receptors from fish to mammals: A comparative approach. General and Comparative Endocrinology. 2011;**171**:124-130. DOI:

[53] Kaslin J, Nystedt JM, Ostergard M, Peitsaro N, Panula P. The orexin/ hypocretin system in zebrafish is connected to the aminergic and cholinergic systems. The Journal of Neuroscience. 2004;**24**:2678-2689. DOI: 10.1523/JNEUROSCI.4908-03.2004

[54] Matsuda K, Azuma M, Kang KS. Orexin system in teleost fish. In: Litwack G, editor. Vitamins and Hormones. 1st ed. San Diego: Elsevier; 2012. pp. 341-361. DOI: 10.1016/ B978-0-12-394623-2.00018-4

[55] Lassiter K, Greene E, Piekarski A, Faulkner OB, Hargis BM, Bottje W,

10.1016/j.ygcen.2011.01.001

microinjections into the medial medulla. Journal of Neurophysiology. 2002;**87**:2480-2489. DOI: 10.1152/

10.1016/j.ygcen.2013.05.006

jn.2001.85.5.2008

jn.2002.87.5.2480

2009.09.013

neuropeptide Y, melanin-concentrating hormone and galanin. The Journal of Endocrinology. 1999;**160**:R7-R12. DOI:

[43] Katayama S, Hamasu K, Shigemi K,

[44] da Silva ES, dos Santos TV, Hoeller AA,

Meneghelli C, et al. Behavioral and metabolic effects of central injections or orexins/hypocretins in pigeons (*Columba livia*). Regulatory Peptides.

[45] Phillips-Singh D, Li Q, Takeuchi S, Ohkubo T, Sharp PJ, Boswell T. Fasting differentially regulates expression of agouti-related peptide, pro-

opiomelanocortin, prepro-orexin, and vasoactive intestinal polypeptide mRNAs in the hypothalamus of Japanese quail. Cell and Tissue Research. 2003;**313**: 217-225. DOI: 10.1007/s00441-003-0755-8

[46] Song Z, Liu L, Yue Y, Jiao H, Lin H, Sheikhahmadi A, et al. Fasting alters protein expression of AMP-activated protein kinase in the hypothalamus of broiler chicks (*Gallus gallus* 

*domesticus*). General and Comparative Endocrinology. 2012;**178**:546-555. DOI:

[47] Katayama S, Shigemi K, Cline MA, Furuse M. Clorgyline inhibits orexin-A-induced arousal in layer-type chicks. The Journal of Veterinary Medical Science. 2011;**73**:471-474. DOI: 10.1292/

[48] Song Z, Everaert N, Wang Y, Decuypere E, Buyse J. The endocrine control of energy homeostasis in chickens. General and Comparative

10.1016/j.ygcen.2012.06.026

Intracerebroventricular injection of orexin-A, but not orexin-B, induces arousal of layer-type neonatal chicks. Comparative Biochemistry and Physiology A. 2010;**157**:132-135. DOI:

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10.1016/j.cbpa.2010.05.018

dos Santos TS, Pereira GV,

2008;**147**:9-18. DOI: 10.1016/j.

regpep.2007.12.003

Cline MA, Furuse M.

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jvms.10-0358

et al. Orexin system is expressed in avian muscle cells and regulates mitochondrial dynamics. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 2015;**308**:R173-R187. DOI: 10.1152/ ajpregu.00394.2014

[56] Nguyen PH, Greene E, Kong BW, Bottje W, Anthony N, Dridi S. Acute heat stress alters the expression of orexin system in quail muscle. Frontiers in Physiology. 2017;**8**:1079. DOI: 10.3389/fphys.2017.01079

[57] Arihara Z, Takahashi K, Murakami O, Totsune K, Sone M, Satoh F, et al. Immunoreactive orexin-A in human plasma. Peptides. 2001;**22**:139-142. DOI: 10.1016/S0196-9781(00)00369-7

[58] Sellayah D, Bharaj P, Sikder D. Orexin is required for brown adipose tissue development, differentiation, and function. Cell Metabolism. 2011;**14**: 478-490. DOI: 10.1016/j. cmet.2011.08.010

[59] Swami M. Metabolism: Orexin acts on brown fat. Nature Medicine. 2011;**17**:1356. DOI: 10.1038/nm.2563

[60] Pasban-Aliabadi H, Esmaeili-Mahani S, Abbasnejad M. Orexin-A protects human neuroblastoma SH-SY5Y cells against 6-hydroxydopamine-induced neurotoxicity: Involvement of PKC and PI3K signaling pathways. Rejuvenation Research. 2017;**20**:125-133. DOI: 10.1089/rej.2016.1836

[61] Wan X, Liu Y, Zhao Y, Sun X, Fan D, Guo L. Orexin A affects HepG2 human hepatocellular carcinoma cells glucose metabolism via HIF-1α-dependent and -independent mechanism. PLoS One. 2017;**12**(9):e0184213. DOI: 10.1371/ journal.pone.0184213

[62] Sikder D, Kodadek T. The neurohormone orexin stimulates hypoxia-inducible factor-1 activity. Genes & Development. 2007;**21**: 2995-3005. DOI: 10.1101/gad.1584307

[63] Hill BG, Awe SO, Vladykovskaya E, Ahmed Y, Liu SQ, Bhatnagar A, et al. Myocardial ischaemia inhibits mitochondrial metabolism of 4-hydroxy-trans-2-nonenal. The Biochemical Journal. 2009;**417**:513-524. DOI: 10.1042/BJ20081615

[64] Chen H, Chan DC. Emerging functions of mammalian mitochondrial fusion and, fission. Human Molecular Genetics. 2005;**14**(2):R283-R289. DOI: 10.1093/hmg/ddi270

**59**

**Chapter 4**

**Abstract**

**1. Introduction**

Noncoding RNAs in the

Training Effects

*and Tiago Fernandes*

Cardiovascular System: Exercise

*Noemy Pereira, Camila Gatto, Edilamar Menezes de Oliveira* 

Exercise training (ET) represents a non-pharmacological treatment that can attenuate or even reverse the process of cardiovascular diseases (CVD), by stimulating protein synthesis, angiogenesis, mitochondrial biogenesis, anti-inflammatory, and anti-oxidative effects that are involved to enhance the performance and improved quality of life. Despite the benefits of exercise, the intricacies of their underlying molecular mechanisms remain largely unknown. Noncoding RNAs (ncRNAs) have been recognized as a major regulatory network governing gene expression in several physiological processes and appeared as pivotal modulators in a myriad of cardiovascular processes under physiological and pathological conditions. However, little is known about ncRNA expression and role in response to exercise. Here we review the current understanding of the ncRNA role in exerciseinduced adaptations focused on the cardiovascular system and address their

**Keywords:** microRNA, lncRNAs, heart, vessels, exercise and cardiovascular diseases

Exercise training (ET) has been shown to undergo beneficial changes in both cardiac structure and function, protecting the heart and vessels against the development of cardiovascular diseases (CVD) [1–4]. CVD are a growing cause of morbidity and mortality throughout the world and often develop after a period of abnormal heart growth termed pathological cardiac hypertrophy (CH). The occurrence of risk factors, such as diabetes, hypertension, obesity, and advanced age leads to substantial complications of CVD. Loss of cardiomyocytes and capillaries accompanied by fibrosis contributes to decreased cardiac function ultimately leading to heart failure (HF). Although current management has improved survival in patients with CVD, such therapies do not fully address the underlying cause and, as a result, HF progresses. Thus, understanding the pathways that rescue cardiovascular remodeling (CR) and function could have important clinical implications [3–6]. Genome-wide analyses and ribonucleic acids (RNA) sequencing revealed that a large part of the human genome (~99%) do not encode proteins but are transcriptionally active and give rise to a broad spectrum of noncoding RNAs (ncRNAs).

potential role in clinical applications for cardiovascular diseases.

### **Chapter 4**

## Noncoding RNAs in the Cardiovascular System: Exercise Training Effects

*Noemy Pereira, Camila Gatto, Edilamar Menezes de Oliveira and Tiago Fernandes*

### **Abstract**

Exercise training (ET) represents a non-pharmacological treatment that can attenuate or even reverse the process of cardiovascular diseases (CVD), by stimulating protein synthesis, angiogenesis, mitochondrial biogenesis, anti-inflammatory, and anti-oxidative effects that are involved to enhance the performance and improved quality of life. Despite the benefits of exercise, the intricacies of their underlying molecular mechanisms remain largely unknown. Noncoding RNAs (ncRNAs) have been recognized as a major regulatory network governing gene expression in several physiological processes and appeared as pivotal modulators in a myriad of cardiovascular processes under physiological and pathological conditions. However, little is known about ncRNA expression and role in response to exercise. Here we review the current understanding of the ncRNA role in exerciseinduced adaptations focused on the cardiovascular system and address their potential role in clinical applications for cardiovascular diseases.

**Keywords:** microRNA, lncRNAs, heart, vessels, exercise and cardiovascular diseases

### **1. Introduction**

Exercise training (ET) has been shown to undergo beneficial changes in both cardiac structure and function, protecting the heart and vessels against the development of cardiovascular diseases (CVD) [1–4]. CVD are a growing cause of morbidity and mortality throughout the world and often develop after a period of abnormal heart growth termed pathological cardiac hypertrophy (CH). The occurrence of risk factors, such as diabetes, hypertension, obesity, and advanced age leads to substantial complications of CVD. Loss of cardiomyocytes and capillaries accompanied by fibrosis contributes to decreased cardiac function ultimately leading to heart failure (HF). Although current management has improved survival in patients with CVD, such therapies do not fully address the underlying cause and, as a result, HF progresses. Thus, understanding the pathways that rescue cardiovascular remodeling (CR) and function could have important clinical implications [3–6].

Genome-wide analyses and ribonucleic acids (RNA) sequencing revealed that a large part of the human genome (~99%) do not encode proteins but are transcriptionally active and give rise to a broad spectrum of noncoding RNAs (ncRNAs).

The ENCODE Project in 2000 (see https://www.encodeproject.org/), that aimed to characterize all the functional elements in the human genome, helped researchers to understand the eukaryotic genome complexity. Although ncRNAs have been considered as junk in the human genome, currently it represent as one of the newest study targets of CVD by potential role in controlling gene expression [7, 8].

Bartolomei et al. [9] discovered the first ncRNA (H19) in mice and it was one of the first maternally imprinted long ncRNAs (lncRNAs) to be identified [9]. Since then, thousands of ncRNAs have been described and are classified into two large groups: small ncRNAs, which are up to 200 nucleotides (nt), and lncRNAs, which are longer than 200 nucleotides. Small ncRNAs comprise microRNAs (miRNAs), PIWI interacting RNAs (piRNAs), transfer RNAs (tRNAs), and small nucleolar RNAs (snoRNAs). Long ncRNAs mainly refers to natural antisense transcripts and other lncRNAs. The discovery of the new class of RNAs increased our knowledge about epigenetic, transcriptional, and translational regulation of gene expression under physiological and pathological conditions. Thus, the possibility of ncRNAs as a potential tool for the treatment of CVD is of great interest [6, 8, 10–12].

ncRNAs such as miRNAs and lncRNAs are regulators of cell development, differentiation, and growth, exert their functions in both nuclear and cytoplasmic compartments. In the cardiovascular system, ncRNAs regulate cardiac development, inflammation, hypertrophy, fibrosis, and regeneration [6, 10–12]. Beyond this, altered miRNA expression can be found in the blood of patients with various CVD [10, 13]. ET is well known to promote cardiovascular profit in which it can vary according to type, intensity, and duration of exercise. However, little is known about the regulatory interaction networks among the multiple classes of RNAs or the mechanisms regulated by exercise-induced ncRNAs on heart and vessel [1, 3, 5, 14]. The contribution of ncRNAs to these adaptations has the potential not only to reveal novel aspects of cardiovascular system but also to identify new targets for prevention and treatment of the CVD.

In this review, we provide an overview of the current knowledge of the cardiovascular effects of ET on ncRNAs, with a specific focus on miRNAs, their role in regulating cardiovascular remodeling (CR) and their potential application for the treatment of CVD. These findings will provide insights that can aid in the development of new therapeutic interventions for these pathologies.

### **2. miRNAs, a family of small ncRNAs**

miRNAs, the most popular class of small ncRNAs, are a class of single-stranded, endogenous, conserved, RNAs (∼21–23 nucleotides) that regulate gene expression at the posttranscriptional level. They bind to messenger RNA (mRNA) by base pairing to complementary sequences within the 3′ untranslated region (3′UTR), and cause gene silencing through the inhibition of translation and/or degradation of mRNA [15–17]. However, recent studies have suggested that miRNA-binding sites are also located in 5′ UTRs or open reading frames (ORFs), and the mechanism of miRNA-mediated regulation from these sites has not been identified [18]. miRNA sequences are in diverse regions of the genome. Most canonical miRNAs in humans are encoded within introns of coding or noncoding genes. However, some miRNAs may be encoded by exons or may be associated in the same loci, organized as a polycistronic transcription unit. Small sequences and imperfect complementary, a single miRNA can target several to hundreds of distinct mRNAs. Conversely, an mRNA can be targeted by diverse miRNAs simultaneously, thus miRNAs are estimated to regulate the expression of more than a third of human protein-coding genes [15–17].

**61**

**Figure 1.**

*Noncoding RNAs in the Cardiovascular System: Exercise Training Effects*

The first miRNA, lin-4, was discovered in *C. elegans* in 1993. Lin-4 regulates *C. elegans* development by binding to the lin-14 mRNA to inhibit lin-14 protein expression [19]. Since then, it has been recognized that miRNAs are a highly conserved class of small RNAs present in most organisms and a variety of miRNAs have been discovered suggesting that there are in excess of 2650 mature sequences in human (see http://www.mirbase.org). Studies have demonstrated that miRNAs influence several biological events, including embryogenesis, cell death, differentiation, proliferation, and cell growth. Furthermore, developments in miRNA-related technologies, such as miRNA expression profiling and synthetic oligoRNA, have contributed to the identification of miRNAs involved in a number of physiological

The biogenesis of the miRNA starts in the nucleus; RNA polymerase II initially transcribes miRNAs into long segments of coding or noncoding RNA, known as pri-miRNAs, which are usually capped and polyadenylated. The pri-miRNAs harbor a local hairpin structure that is then cropped by a nuclear enzyme Drosha and their cofactor Pasha (also known as DGCR8) into pre-miRNAs (∼70 nucleotides). After nuclear processing, the pre-miRNA is exported into the cytoplasm by Exportin-5 in a RanGTP-dependent manner. Subsequently, the enzyme Dicer removes the terminal loop of the pre-miRNAs to generate the miRNA:miRNA∗ duplex (dsRNA) of ∼20–25 nucleotides. The dsRNA is loaded into the miRNA-associated multiprotein RNA-induced silencing complex (RISC), which includes the Argonaute proteins. One strand of the miRNA is preferentially retained in this complex and becomes

*miRNA biogenesis. miRNA genes are transcribed by RNA polymerase II (Pol II) to generate the primary transcripts (pri-miRNAs). The initial processing of the primary transcript is mediated by the Drosha-DiGeorge syndrome critical region gene 8 (DGCR8) complex (also known as the microprocessor complex) that generates ~70 nucleotide (nt) pre-miRNAs. Pre-miRNA has a short stem plus, which is recognized by the nuclear export factor exportin 5. Once exported from the nucleus, the cytoplasmic RNase III Dicer catalyzes the production of miRNA duplexes. Dicer, TRBP (TAR RNA-binding protein; also known as TARBP2), and Argonaute (AGO) 1–4 mediate the processing of pre-miRNA and the assembly of the RISC (RNA-induced silencing complex). Within this complex, one strand of the miRNA duplex is removed resulting in a singlestranded miRNA, partially complementary to target mRNA, which remains in the complex. miRNA complex interaction with mRNA induces posttranscriptional silencing through as both mRNA destabilization and* 

*translational repression (https://smart.servier.com/image-set-download/).*

*DOI: http://dx.doi.org/10.5772/intechopen.86054*

and pathological conditions [15, 16, 20].

### *Noncoding RNAs in the Cardiovascular System: Exercise Training Effects DOI: http://dx.doi.org/10.5772/intechopen.86054*

The first miRNA, lin-4, was discovered in *C. elegans* in 1993. Lin-4 regulates *C. elegans* development by binding to the lin-14 mRNA to inhibit lin-14 protein expression [19]. Since then, it has been recognized that miRNAs are a highly conserved class of small RNAs present in most organisms and a variety of miRNAs have been discovered suggesting that there are in excess of 2650 mature sequences in human (see http://www.mirbase.org). Studies have demonstrated that miRNAs influence several biological events, including embryogenesis, cell death, differentiation, proliferation, and cell growth. Furthermore, developments in miRNA-related technologies, such as miRNA expression profiling and synthetic oligoRNA, have contributed to the identification of miRNAs involved in a number of physiological and pathological conditions [15, 16, 20].

The biogenesis of the miRNA starts in the nucleus; RNA polymerase II initially transcribes miRNAs into long segments of coding or noncoding RNA, known as pri-miRNAs, which are usually capped and polyadenylated. The pri-miRNAs harbor a local hairpin structure that is then cropped by a nuclear enzyme Drosha and their cofactor Pasha (also known as DGCR8) into pre-miRNAs (∼70 nucleotides). After nuclear processing, the pre-miRNA is exported into the cytoplasm by Exportin-5 in a RanGTP-dependent manner. Subsequently, the enzyme Dicer removes the terminal loop of the pre-miRNAs to generate the miRNA:miRNA∗ duplex (dsRNA) of ∼20–25 nucleotides. The dsRNA is loaded into the miRNA-associated multiprotein RNA-induced silencing complex (RISC), which includes the Argonaute proteins. One strand of the miRNA is preferentially retained in this complex and becomes

### **Figure 1.**

*Muscle Cells - Recent Advances and Future Perspectives*

prevention and treatment of the CVD.

**2. miRNAs, a family of small ncRNAs**

ment of new therapeutic interventions for these pathologies.

The ENCODE Project in 2000 (see https://www.encodeproject.org/), that aimed to characterize all the functional elements in the human genome, helped researchers to understand the eukaryotic genome complexity. Although ncRNAs have been considered as junk in the human genome, currently it represent as one of the newest

Bartolomei et al. [9] discovered the first ncRNA (H19) in mice and it was one of the first maternally imprinted long ncRNAs (lncRNAs) to be identified [9]. Since then, thousands of ncRNAs have been described and are classified into two large groups: small ncRNAs, which are up to 200 nucleotides (nt), and lncRNAs, which are longer than 200 nucleotides. Small ncRNAs comprise microRNAs (miRNAs), PIWI interacting RNAs (piRNAs), transfer RNAs (tRNAs), and small nucleolar RNAs (snoRNAs). Long ncRNAs mainly refers to natural antisense transcripts and other lncRNAs. The discovery of the new class of RNAs increased our knowledge about epigenetic, transcriptional, and translational regulation of gene expression under physiological and pathological conditions. Thus, the possibility of ncRNAs as

study targets of CVD by potential role in controlling gene expression [7, 8].

a potential tool for the treatment of CVD is of great interest [6, 8, 10–12].

ncRNAs such as miRNAs and lncRNAs are regulators of cell development, differentiation, and growth, exert their functions in both nuclear and cytoplasmic compartments. In the cardiovascular system, ncRNAs regulate cardiac development, inflammation, hypertrophy, fibrosis, and regeneration [6, 10–12]. Beyond this, altered miRNA expression can be found in the blood of patients with various CVD [10, 13]. ET is well known to promote cardiovascular profit in which it can vary according to type, intensity, and duration of exercise. However, little is known about the regulatory interaction networks among the multiple classes of RNAs or the mechanisms regulated by exercise-induced ncRNAs on heart and vessel [1, 3, 5, 14]. The contribution of ncRNAs to these adaptations has the potential not only to reveal novel aspects of cardiovascular system but also to identify new targets for

In this review, we provide an overview of the current knowledge of the cardiovascular effects of ET on ncRNAs, with a specific focus on miRNAs, their role in regulating cardiovascular remodeling (CR) and their potential application for the treatment of CVD. These findings will provide insights that can aid in the develop-

miRNAs, the most popular class of small ncRNAs, are a class of single-stranded, endogenous, conserved, RNAs (∼21–23 nucleotides) that regulate gene expression at the posttranscriptional level. They bind to messenger RNA (mRNA) by base pairing to complementary sequences within the 3′ untranslated region (3′UTR), and cause gene silencing through the inhibition of translation and/or degradation of mRNA [15–17]. However, recent studies have suggested that miRNA-binding sites are also located in 5′ UTRs or open reading frames (ORFs), and the mechanism of miRNA-mediated regulation from these sites has not been identified [18]. miRNA sequences are in diverse regions of the genome. Most canonical miRNAs in humans are encoded within introns of coding or noncoding genes. However, some miRNAs may be encoded by exons or may be associated in the same loci, organized as a polycistronic transcription unit. Small sequences and imperfect complementary, a single miRNA can target several to hundreds of distinct mRNAs. Conversely, an mRNA can be targeted by diverse miRNAs simultaneously, thus miRNAs are estimated to regulate the expression of more than a third of human protein-coding

**60**

genes [15–17].

*miRNA biogenesis. miRNA genes are transcribed by RNA polymerase II (Pol II) to generate the primary transcripts (pri-miRNAs). The initial processing of the primary transcript is mediated by the Drosha-DiGeorge syndrome critical region gene 8 (DGCR8) complex (also known as the microprocessor complex) that generates ~70 nucleotide (nt) pre-miRNAs. Pre-miRNA has a short stem plus, which is recognized by the nuclear export factor exportin 5. Once exported from the nucleus, the cytoplasmic RNase III Dicer catalyzes the production of miRNA duplexes. Dicer, TRBP (TAR RNA-binding protein; also known as TARBP2), and Argonaute (AGO) 1–4 mediate the processing of pre-miRNA and the assembly of the RISC (RNA-induced silencing complex). Within this complex, one strand of the miRNA duplex is removed resulting in a singlestranded miRNA, partially complementary to target mRNA, which remains in the complex. miRNA complex interaction with mRNA induces posttranscriptional silencing through as both mRNA destabilization and translational repression (https://smart.servier.com/image-set-download/).*

the mature miRNA depending on its thermodynamic stability; the opposite strand, known as the passenger strand, is eliminated from the complex. The RISC acts as a guide by base pairing of miRNA with its target mRNAs, resulting in mRNA cleavage, mRNA deadenylation, and translation repression (**Figure 1**). It is well recognized that pairing in the 'seed' region of the miRNA (nucleotides 2–7 or 8) appears critical for target recognition. Thus, miRNAs that bind to target mRNAs with imperfect complementarity repress target gene expression via translational silencing. In contrast, miRNAs that bind to their target mRNAs with perfect complementarily induce mRNA degradation [15–17, 20].

### **3. miRNAs regulation by long noncoding RNAs**

Emerging evidences suggest that miRNAs may be affected by long noncoding RNAs (lncRNAs) [14, 21]. While there is plenty evidence of miRNAs involvement in exercise adaptations, investigations on lncRNAs are still lacking [14]. LncRNAs are a heterogeneous group of transcripts modulating gene expression at multiple and more complex levels than miRNAs. One way to classify lncRNAs is according to their mechanism of action: signal, decoy, guide, scaffold, enhancer, or sponge lncRNAs (see the review by [22, 23]).

Similar to miRNAs, lncRNAs have been shown to be involved in CVD with several reports about their specific expression in different cardiac diseases [21, 24–30]. In this context, it was recently showed that H19, which is a maternally imprinted lncRNA, is implicated in CVD [24, 31]. H19 is highly expressed during embryogenesis but is strongly repressed after birth, with a significant re-expression of lncRNA H19 in CVD. Liu et al. [24] showed that miRNA-675 mediates the anti-hypertrophic effect of H19 on cardiomyocytes acting as a negative regulator of cardiac hypertrophy. In addition, it was demonstrated that H19 exon1 encodes miR-675-3p and miR-675-5p, which are up-regulated in pathological CH. Prediction algorithms identified a pro-hypertrophic factor, Ca/calmodulin-dependent protein kinase IIδ (CaMKIIδ), as potential targets for miR-675-3p and miR-675-5p, which was confirmed by luciferase reporter gene assays [24]. Moreover, high H19 expression has been linked to hyperhomocysteinemia, a known risk factor for coronary artery disease [32]. Furthermore, H19 was reported to sponge let-7 family miRNAs, which are believed

### **Figure 2.**

*Cardiac disease-induced cardiac remodeling (CR) is characterized by an aberrant profile of myocardium growth, accompanied by fibrosis, apoptosis, and cardiac dysfunction. lncRNAs H19, CHRF, Myheart, Chast, and Chaer have been described to regulate pathological cardiac hypertrophy; cardiac fibroblast-enriched lncRNAs Meg3 and Wisper controlling cardiac fibrosis; and lncRNAs CHRF, Myheart, and Chaer associated with heart failure in response to cardiac injury.*

**63**

HF [41–43].

*Noncoding RNAs in the Cardiovascular System: Exercise Training Effects*

**4.1 Cardiac remodeling and dysfunction: exercise training effects**

according to environmental stimuli or external stimuli.

to have atheroprotective role [21, 33]. Thus, lncRNA-miRNA-mRNA axis reveals a

Cardiac hypertrophy-related factor (CHRF) [26], myosin heavy-chain-associated RNA transcripts (Myheart) [25], cardiac hypertrophy-associated epigenetic regulator (Chaer) [27], cardiac hypertrophy-associated transcript (Chast) [28], cardiac fibroblast-enriched lncRNA maternally expressed 3 (Meg3) [29] and Wisp2 super-enhancer-associated RNA (Wisper) [30] are lncRNAs linked to CR (**Figure 2**). Additionally, recent gain- and loss-of-function studies, both *in vitro* in cell lines and *in vivo* in mouse models, have demonstrated that lncRNAs play critical

The mammalian heart is a muscle and is the first organ to form in the embryo. The development of heart is highly complex and involves the integration of multiple cell types as well as a connection between vascular system and blood vessels. However, its uninterrupted development and function are determinant to organism survival [34, 35]. The primary function of heart is to contract and pump blood throughout the circulatory system to deliver oxygen and nutrients to tissues and to transport carbon dioxide back to the lungs [36, 37]. This organ can be remodeled

Cardiac remodeling (CR) is an adaptation to increased cardiac overload including a variety of mechanical, hemodynamic, and hormonal factors that promotes changes in structure, dimensions, mass, shape, and functions of the heart as well as cardiac cells [34, 38], and has been generally defined as either physiological (i.e., normal) or pathological (i.e., detrimental) [34]. Physiological growth occurs in response to exercise, pregnancy, or postnatal growth [34] causing heart hypertrophy, which is reversible and characterized by normal cardiac morphology and function [39]. On the other hand, in pathological hypertrophy, contractile performance, for example, can be perturbed or reduced in response to diverse pathophysiological stimuli, which also causes the typically heart remodeling, in association with increases in myocyte cell volume [36, 39]. However, it can slowly progressives to become maladaptive, leading to a decompensation in

Cardiac dysfunction is the main consequence of pathological CR, a process that involves a complex series of transcriptional, signaling, structural, electrophysiological, and functional events, occurring within the cardiac myocyte and in the other cellular elements within the ventricle (fibroblasts, endothelial cells, T-cells, etc.). Thus, it starts with genetic changes in response to cardiac insults, with re-expression of fetal genes (atrial and β-type natriuretic peptide, β-myosin heavy chain, and α-skeletal muscle actin), fibrosis, endothelial dysfunction, and inflammation. Consequently, cellular and molecular changes occur, resulting in progressive loss of ventricular function, asymptomatic at first, that evolves to signs and symptoms of

ET is the most effective non-pharmacological intervention to prevent or reduce cardiovascular disturbances [2, 44]. ET can be classified as static or dynamic and leads to two different types of intermittent chronic cardiac overload, which induces morphological changes in the heart [45]. This changes will depend on factors such as the type of exercise used (aerobic or resistance), volume (training time), intensity (degree of training load), and frequency of ET (number of training session

*DOI: http://dx.doi.org/10.5772/intechopen.86054*

promising therapeutic strategy for CVD.

roles in cardiovascular biology and diseases.

**4. Heart, exercise, and miRNAs**

cardiac function [40].

to have atheroprotective role [21, 33]. Thus, lncRNA-miRNA-mRNA axis reveals a promising therapeutic strategy for CVD.

Cardiac hypertrophy-related factor (CHRF) [26], myosin heavy-chain-associated RNA transcripts (Myheart) [25], cardiac hypertrophy-associated epigenetic regulator (Chaer) [27], cardiac hypertrophy-associated transcript (Chast) [28], cardiac fibroblast-enriched lncRNA maternally expressed 3 (Meg3) [29] and Wisp2 super-enhancer-associated RNA (Wisper) [30] are lncRNAs linked to CR (**Figure 2**). Additionally, recent gain- and loss-of-function studies, both *in vitro* in cell lines and *in vivo* in mouse models, have demonstrated that lncRNAs play critical roles in cardiovascular biology and diseases.

### **4. Heart, exercise, and miRNAs**

*Muscle Cells - Recent Advances and Future Perspectives*

tarily induce mRNA degradation [15–17, 20].

lncRNAs (see the review by [22, 23]).

**3. miRNAs regulation by long noncoding RNAs**

the mature miRNA depending on its thermodynamic stability; the opposite strand, known as the passenger strand, is eliminated from the complex. The RISC acts as a guide by base pairing of miRNA with its target mRNAs, resulting in mRNA cleavage, mRNA deadenylation, and translation repression (**Figure 1**). It is well recognized that pairing in the 'seed' region of the miRNA (nucleotides 2–7 or 8) appears critical for target recognition. Thus, miRNAs that bind to target mRNAs with imperfect complementarity repress target gene expression via translational silencing. In contrast, miRNAs that bind to their target mRNAs with perfect complemen-

Emerging evidences suggest that miRNAs may be affected by long noncoding RNAs (lncRNAs) [14, 21]. While there is plenty evidence of miRNAs involvement in exercise adaptations, investigations on lncRNAs are still lacking [14]. LncRNAs are a heterogeneous group of transcripts modulating gene expression at multiple and more complex levels than miRNAs. One way to classify lncRNAs is according to their mechanism of action: signal, decoy, guide, scaffold, enhancer, or sponge

Similar to miRNAs, lncRNAs have been shown to be involved in CVD with several reports about their specific expression in different cardiac diseases [21, 24–30]. In this context, it was recently showed that H19, which is a maternally imprinted lncRNA, is implicated in CVD [24, 31]. H19 is highly expressed during embryogenesis but is strongly repressed after birth, with a significant re-expression of lncRNA H19 in CVD. Liu et al. [24] showed that miRNA-675 mediates the anti-hypertrophic effect of H19 on cardiomyocytes acting as a negative regulator of cardiac hypertrophy. In addition, it was demonstrated that H19 exon1 encodes miR-675-3p and miR-675-5p, which are up-regulated in pathological CH. Prediction algorithms identified a pro-hypertrophic factor, Ca/calmodulin-dependent protein kinase IIδ (CaMKIIδ), as potential targets for miR-675-3p and miR-675-5p, which was confirmed by luciferase reporter gene assays [24]. Moreover, high H19 expression has been linked to hyperhomocysteinemia, a known risk factor for coronary artery disease [32]. Furthermore, H19 was reported to sponge let-7 family miRNAs, which are believed

*Cardiac disease-induced cardiac remodeling (CR) is characterized by an aberrant profile of myocardium growth, accompanied by fibrosis, apoptosis, and cardiac dysfunction. lncRNAs H19, CHRF, Myheart, Chast, and Chaer have been described to regulate pathological cardiac hypertrophy; cardiac fibroblast-enriched lncRNAs Meg3 and Wisper controlling cardiac fibrosis; and lncRNAs CHRF, Myheart, and Chaer associated* 

**62**

**Figure 2.**

*with heart failure in response to cardiac injury.*

### **4.1 Cardiac remodeling and dysfunction: exercise training effects**

The mammalian heart is a muscle and is the first organ to form in the embryo. The development of heart is highly complex and involves the integration of multiple cell types as well as a connection between vascular system and blood vessels. However, its uninterrupted development and function are determinant to organism survival [34, 35]. The primary function of heart is to contract and pump blood throughout the circulatory system to deliver oxygen and nutrients to tissues and to transport carbon dioxide back to the lungs [36, 37]. This organ can be remodeled according to environmental stimuli or external stimuli.

Cardiac remodeling (CR) is an adaptation to increased cardiac overload including a variety of mechanical, hemodynamic, and hormonal factors that promotes changes in structure, dimensions, mass, shape, and functions of the heart as well as cardiac cells [34, 38], and has been generally defined as either physiological (i.e., normal) or pathological (i.e., detrimental) [34]. Physiological growth occurs in response to exercise, pregnancy, or postnatal growth [34] causing heart hypertrophy, which is reversible and characterized by normal cardiac morphology and function [39]. On the other hand, in pathological hypertrophy, contractile performance, for example, can be perturbed or reduced in response to diverse pathophysiological stimuli, which also causes the typically heart remodeling, in association with increases in myocyte cell volume [36, 39]. However, it can slowly progressives to become maladaptive, leading to a decompensation in cardiac function [40].

Cardiac dysfunction is the main consequence of pathological CR, a process that involves a complex series of transcriptional, signaling, structural, electrophysiological, and functional events, occurring within the cardiac myocyte and in the other cellular elements within the ventricle (fibroblasts, endothelial cells, T-cells, etc.). Thus, it starts with genetic changes in response to cardiac insults, with re-expression of fetal genes (atrial and β-type natriuretic peptide, β-myosin heavy chain, and α-skeletal muscle actin), fibrosis, endothelial dysfunction, and inflammation. Consequently, cellular and molecular changes occur, resulting in progressive loss of ventricular function, asymptomatic at first, that evolves to signs and symptoms of HF [41–43].

ET is the most effective non-pharmacological intervention to prevent or reduce cardiovascular disturbances [2, 44]. ET can be classified as static or dynamic and leads to two different types of intermittent chronic cardiac overload, which induces morphological changes in the heart [45]. This changes will depend on factors such as the type of exercise used (aerobic or resistance), volume (training time), intensity (degree of training load), and frequency of ET (number of training session

at any given time) [2, 45], and is directly related to maximal aerobic capacity or VO2 max [42]. It is known that ET is one of major stimuli for physiological cardiac hypertrophy (CH) [46]. The main physiological responses attributed to CR are the increase in contractility, the improvement of the transient Ca2+ intracellular, increase in expansion with significant improvements in myocardial oxygenation, and additional endothelium-dependent functions that prevent ischemic events [42].

In contrast to pathological CH that occurs in response to continuous stimulus on heart (arterial hypertension, valve disease, or aortic stenosis, for example), CH induced by ET consists of a frequent, but intermittent stimulus cardiac hemodynamic overload [2]. Thus, a pressure overload in response to resistance training, for example, causes an increase in the width of the cardiac myocytes without dilation of the chamber and causes a concentric left ventricle (LV) hypertrophy [39]. On the other hand, the increase in size of cardiomyocytes by aerobic ET is mainly associated with an eccentric LV hypertrophy phenotype, induced by volume overload, with predominant myocyte growth in series, promoting dilation of the cardiac chamber. Either concentric or eccentric CH by ET is not followed by fibrosis and re-expression of fetal genes, which is linked to preserved or improved cardiac function [39, 44].

Medeiros et al. [47] showed that CH induced by moderate aerobic ET causes resting bradycardia in rats by an increase in cardiac vagal activity, which is associated to increased VO2 peak [44, 47, 48] and an increase in nitric oxide (NO) and angiogenesis levels in heart of experimental animals. The increased cross-sectional area in the heart contributes to the increase in ventricular systolic volume and cardiac output, which improves aerobic capacity [49]. Thus, ET has been widely used as a preventive therapeutic strategy for prevention or treatment of diseases in cardiovascular system. However, the underlying molecular mechanisms that regulate ET responses have not been fully elucidated, especially with regard to CH [50]. In this sense, emerging evidences have shown that miRNAs may play an important role in both heart development and CR [44, 51] as in the pathogenesis of HF through their ability to regulate the expression levels of genes that govern the process of adaptive and maladaptive CR [52]. Besides, miRNAs can mediate the beneficial effects of ET in heart in both physiological and pathological CR well as in cardiac disorders [2, 53, 54].

Therefore, in the below section we will highlight the role of miRNAs in both physiological CR well as diseased heart. In another section, we will highlight what is known until now about the effects of ET in the regulation of miRNA expression in heart in these different scenarios.

### **4.2 miRNAs in cardiac remodeling and heart diseases**

The miRNAs may be essential regulators in the development and normal physiology. Dysregulation of miRNA expression has been found to correlate with CR and disease in both human and mouse [55, 56]. Therefore, identification of a signature of miRNAs is an essential and promising prerequisite for the study of the biological functions of this class of molecules in the cardiovascular system [57, 58].

A single miRNA can be highly expressed in one tissue and may not have, or have a low expression in another [58]. In this sense, miRNA-1, -133, -206, and -208 are muscle specific and are primarily expressed in the cardiac and skeletal muscle [57, 59], with miRNA-208 being solely expressed in heart [57]. During cardiac development, the expression of miRNA-1 increases, which promotes a decrease in levels of the heart and neural crest derivatives expressed 2 levels (Hand2), a genetarget, reaching it at the levels found in mature cardiac myocytes [57]. miRNA-1 also promotes myogenesis, targeting the histone deacetylase 4 (HDAC4). The

**65**

*Noncoding RNAs in the Cardiovascular System: Exercise Training Effects*

miRNA-1 family represents more than 40% of all the miRNAs expressed in the heart [60], which consists of a subfamily composed of miRNA-1-1, miRNA-1-2 and

The miRNA-133 family consists of the subfamily miRNA-133a-1, 133a-2, and 133b. miRNA-133, like miRNA-1, also plays an important role in cardiac development. In fact, the deletion of miRNA-133 results in severe cardiac malformations, together with embryonic and post-natal lethality, as a consequence of an insuffi-

The miRNA-208a, -208b, and -499 are specifically encoded by introns of Myh6 (α-MHC), Myh7 (β-MHC), and Myh7b, respectively. In mice, Myh7/miRNA-208b is expressed in the embryonic heart and Myh6/miR-208a is expressed predominantly in the postnatal stages. Myh7 is re-expressed in the adult's heart only after cardiac stress. Myh7b/miRNA-499 is expressed in the embryonic heart and in the adult heart [61]. However, the targeted deletion of miRNA-208a in mice results in only a mild cardiac phenotype, such as the ectopic expression of rapid muscle markers of the skeleton and the impaired ability to respond to postnatal stress. Therefore, this scenario suggests a not so essential role of these three miRNAs during cardiac

Considering the importance of miRNAs even in heart development, it is understandable that dysregulation in its expression is implicated in a variety of pathological disorders, including diabetes, various cancers, and CVD [51]. Pathological CH, unlike the physiological CH promoted by ET, is a common response to various cardiac disorders, which includes not only hypertension, but also cardiac ischemic disease, valvular diseases, and endocrine disorders, and is therefore, considered the major determinant of mortality and morbidity in CVD [58, 63]. Currently, existent literature suggest that mainly miRNA-1, -18b, -21, -133, -195, and -208 play impor-

In this sense, Carè et al. [64] found a decrease in expression of both miRNA-133 and miRNA-1 in three different animal models of CH, including hypertrophy induced by pressure overload, hypertrophy induced by Akt overexpression, and adaptive CH in mice and humans [64]. However, *in vitro* overexpression of these miRNAs inhibited hypertrophy, while *in vivo* inhibition of miRNA-133 promoted a pronounced and sustained CH. In this same study, three new specific targets for miRNA-133 have been identified, which include RhoA and CH regulatory GDP-GTP exchange protein; the Cdc42, a signal transduction kinase implicated in hypertrophy; and Nelf-A/WHSC2, a nuclear factor involved in cardiogenesis [64]. miRNA-1, in turn, controls cardiac myocyte growth by negatively regulating the expression of calcium-calmodulin signaling components, Mef2a and Gata4, which are key transcription factors that mediate calcium-dependent changes in gene

Cheng et al. [55] showed that miRNAs are aberrantly expressed in culture of neonatal cardiomyocytes stimulated with angiotensin II or phenylephrine, and that miRNA-21 knockdown exerts a negative effect on cardiomyocyte hypertrophy *in vitro*. The mitogen-activated protein kinase (MAPK), FasLigand (FasL), and homolog of deleted phosphatase on chromosome 10 (PTEN) are targets that have already been validated for miRNA-21, which stimulate the Akt pathway [66, 67]. In addition, there is evidence that depletion of miRNA-21 exerts anti-apoptotic and pro-proliferative effects on rat proliferative vascular smooth muscle cells (VSMCs) [68]. On the other hand, overexpression of miRNA-1 (which was aberrantly downregulated in induced-hypertrophy model in mouse), prevented the hypertrophic

Cardiac fibrosis is an important contributor for the development of cardiac dysfunction in diverse pathological conditions, such as myocardial infarction (MI),

growth of cardiac myocytes that were stimulated by endothelin-1 [67].

*DOI: http://dx.doi.org/10.5772/intechopen.86054*

cient number of cardioblasts [57].

development [51, 62].

expression [65].

tant roles in modulating CH [52].

miRNA-206.

*Noncoding RNAs in the Cardiovascular System: Exercise Training Effects DOI: http://dx.doi.org/10.5772/intechopen.86054*

*Muscle Cells - Recent Advances and Future Perspectives*

function [39, 44].

cardiac disorders [2, 53, 54].

heart in these different scenarios.

**4.2 miRNAs in cardiac remodeling and heart diseases**

at any given time) [2, 45], and is directly related to maximal aerobic capacity or VO2 max [42]. It is known that ET is one of major stimuli for physiological cardiac hypertrophy (CH) [46]. The main physiological responses attributed to CR are the increase in contractility, the improvement of the transient Ca2+ intracellular, increase in expansion with significant improvements in myocardial oxygenation, and additional endothelium-dependent functions that prevent ischemic events [42]. In contrast to pathological CH that occurs in response to continuous stimulus on heart (arterial hypertension, valve disease, or aortic stenosis, for example), CH induced by ET consists of a frequent, but intermittent stimulus cardiac hemodynamic overload [2]. Thus, a pressure overload in response to resistance training, for example, causes an increase in the width of the cardiac myocytes without dilation of the chamber and causes a concentric left ventricle (LV) hypertrophy [39]. On the other hand, the increase in size of cardiomyocytes by aerobic ET is mainly associated with an eccentric LV hypertrophy phenotype, induced by volume overload, with predominant myocyte growth in series, promoting dilation of the cardiac chamber. Either concentric or eccentric CH by ET is not followed by fibrosis and re-expression of fetal genes, which is linked to preserved or improved cardiac

Medeiros et al. [47] showed that CH induced by moderate aerobic ET causes resting bradycardia in rats by an increase in cardiac vagal activity, which is associated to increased VO2 peak [44, 47, 48] and an increase in nitric oxide (NO) and angiogenesis levels in heart of experimental animals. The increased cross-sectional area in the heart contributes to the increase in ventricular systolic volume and cardiac output, which improves aerobic capacity [49]. Thus, ET has been widely used as a preventive therapeutic strategy for prevention or treatment of diseases in cardiovascular system. However, the underlying molecular mechanisms that regulate ET responses have not been fully elucidated, especially with regard to CH [50]. In this sense, emerging evidences have shown that miRNAs may play an important role in both heart development and CR [44, 51] as in the pathogenesis of HF through their ability to regulate the expression levels of genes that govern the process of adaptive and maladaptive CR [52]. Besides, miRNAs can mediate the beneficial effects of ET in heart in both physiological and pathological CR well as in

Therefore, in the below section we will highlight the role of miRNAs in both physiological CR well as diseased heart. In another section, we will highlight what is known until now about the effects of ET in the regulation of miRNA expression in

The miRNAs may be essential regulators in the development and normal physiology. Dysregulation of miRNA expression has been found to correlate with CR and disease in both human and mouse [55, 56]. Therefore, identification of a signature of miRNAs is an essential and promising prerequisite for the study of the biological

A single miRNA can be highly expressed in one tissue and may not have, or have a low expression in another [58]. In this sense, miRNA-1, -133, -206, and -208 are muscle specific and are primarily expressed in the cardiac and skeletal muscle [57, 59], with miRNA-208 being solely expressed in heart [57]. During cardiac development, the expression of miRNA-1 increases, which promotes a decrease in levels of the heart and neural crest derivatives expressed 2 levels (Hand2), a genetarget, reaching it at the levels found in mature cardiac myocytes [57]. miRNA-1 also promotes myogenesis, targeting the histone deacetylase 4 (HDAC4). The

functions of this class of molecules in the cardiovascular system [57, 58].

**64**

miRNA-1 family represents more than 40% of all the miRNAs expressed in the heart [60], which consists of a subfamily composed of miRNA-1-1, miRNA-1-2 and miRNA-206.

The miRNA-133 family consists of the subfamily miRNA-133a-1, 133a-2, and 133b. miRNA-133, like miRNA-1, also plays an important role in cardiac development. In fact, the deletion of miRNA-133 results in severe cardiac malformations, together with embryonic and post-natal lethality, as a consequence of an insufficient number of cardioblasts [57].

The miRNA-208a, -208b, and -499 are specifically encoded by introns of Myh6 (α-MHC), Myh7 (β-MHC), and Myh7b, respectively. In mice, Myh7/miRNA-208b is expressed in the embryonic heart and Myh6/miR-208a is expressed predominantly in the postnatal stages. Myh7 is re-expressed in the adult's heart only after cardiac stress. Myh7b/miRNA-499 is expressed in the embryonic heart and in the adult heart [61]. However, the targeted deletion of miRNA-208a in mice results in only a mild cardiac phenotype, such as the ectopic expression of rapid muscle markers of the skeleton and the impaired ability to respond to postnatal stress. Therefore, this scenario suggests a not so essential role of these three miRNAs during cardiac development [51, 62].

Considering the importance of miRNAs even in heart development, it is understandable that dysregulation in its expression is implicated in a variety of pathological disorders, including diabetes, various cancers, and CVD [51]. Pathological CH, unlike the physiological CH promoted by ET, is a common response to various cardiac disorders, which includes not only hypertension, but also cardiac ischemic disease, valvular diseases, and endocrine disorders, and is therefore, considered the major determinant of mortality and morbidity in CVD [58, 63]. Currently, existent literature suggest that mainly miRNA-1, -18b, -21, -133, -195, and -208 play important roles in modulating CH [52].

In this sense, Carè et al. [64] found a decrease in expression of both miRNA-133 and miRNA-1 in three different animal models of CH, including hypertrophy induced by pressure overload, hypertrophy induced by Akt overexpression, and adaptive CH in mice and humans [64]. However, *in vitro* overexpression of these miRNAs inhibited hypertrophy, while *in vivo* inhibition of miRNA-133 promoted a pronounced and sustained CH. In this same study, three new specific targets for miRNA-133 have been identified, which include RhoA and CH regulatory GDP-GTP exchange protein; the Cdc42, a signal transduction kinase implicated in hypertrophy; and Nelf-A/WHSC2, a nuclear factor involved in cardiogenesis [64]. miRNA-1, in turn, controls cardiac myocyte growth by negatively regulating the expression of calcium-calmodulin signaling components, Mef2a and Gata4, which are key transcription factors that mediate calcium-dependent changes in gene expression [65].

Cheng et al. [55] showed that miRNAs are aberrantly expressed in culture of neonatal cardiomyocytes stimulated with angiotensin II or phenylephrine, and that miRNA-21 knockdown exerts a negative effect on cardiomyocyte hypertrophy *in vitro*. The mitogen-activated protein kinase (MAPK), FasLigand (FasL), and homolog of deleted phosphatase on chromosome 10 (PTEN) are targets that have already been validated for miRNA-21, which stimulate the Akt pathway [66, 67]. In addition, there is evidence that depletion of miRNA-21 exerts anti-apoptotic and pro-proliferative effects on rat proliferative vascular smooth muscle cells (VSMCs) [68]. On the other hand, overexpression of miRNA-1 (which was aberrantly downregulated in induced-hypertrophy model in mouse), prevented the hypertrophic growth of cardiac myocytes that were stimulated by endothelin-1 [67].

Cardiac fibrosis is an important contributor for the development of cardiac dysfunction in diverse pathological conditions, such as myocardial infarction (MI),

ischemic, dilated, hypertrophic cardiomyopathies, and HF and can be defined as an inappropriate accumulation of extracellular matrix proteins in the heart [69]. The exacerbated expression of extracellular matrix proteins is determinant in the differentiation between physiological and pathological CH [2]. In this sense, the negative regulation of miRNA-29 with anti-miRNAs *in vitro* and *in vivo* in a MI model induces the expression of collagens, whereas miRNA-29 overexpression in fibroblasts reduces this expression, suggesting this miRNA as a regulator of cardiac fibrosis [46]. Currently, the miRNA-29 family has been validated as a regulator of the gene expression of collagen I, III, fibrillin I, and elastin and is involved in pathological CH and the HF process [46, 51, 66]. In addition, decreased expression of the miRNA-29 family, miRNA-24, and miRNA-320 occurs after MI, and the decrease in miRNA-29 expression promotes fibrosis and scar formation in the heart [51].

Through microarray analysis, van Rooij et al. [56] related the miRNAs found both in response to hypertrophy and HF and identified more than 12 miRNAs dysregulated in the heart of mice. Many of these miRNAs were similarly regulated in failing human hearts. Interestingly, overexpression of miRNA-23a, miRNA-23b, miRNA-24, miRNA-195, or miRNA-214 induced hypertrophic growth in cardiomyocyte culture, whereas overexpression of miRNA-150 or miRNA-181b decreased cell size of the cardiomyocyte. The miRNA-23a knockdown attenuated hypertrophy [56] and the overexpression of miRNA-195 *in vivo* alone (transgenic mouse), was sufficient to lead to hypertrophy and HF [56]. The miRNA-23a is transcriptionally regulated by the nuclear factor of activated T cells, cytoplasmic 3 (NFATc3), which mediates the signaling pathway of cardiac stress. Thus, miRNA-23a overexpression can triggers a hypertrophic response by means of its target-gene muRF1 (muscle specific ring finger protein 1), an anti-hypertrophic protein, while negative regulation of miRNA-23a abolishes CH induced by isoproterenol in mice [70]. Other miRNAs exhibited pro-hypertrophic profile, including miRNA-208, -21, -18b, -27, and -9 [56].

Transgenic overexpression of miRNA-208a in the adult heart of mice is enough to induce hypertrophic growth, resulting in pronounced repression of regulatory target-genes, such as THRAP1 and myostatin (negative regulators of muscle growth and hypertrophy). In contrast, the deletion of cardiac miRNA-208a protects these animals from CH in response to hemodynamic cardiac stress by canceling the re-expression of fetal gene β-MHC [71]. In addition, therapeutic inhibition of miRNA-208 (by subcutaneous delivery of anti-miRNA-208a) reduces deleterious CR, improves cardiac function, and survival during HF in hypertensive rats [72]. Curiously, inhibition of cardiac-specific miR-208a may have therapeutic usefulness in a variety of metabolic disorders, such as obesity and type-2 diabetes in the setting of cardiac dysfunction by targeting and increasing MED13 in heart [73].

miRNA-199a is predominantly expressed in cardiomyocytes, where it maintains cell size and may play a role in the regulation of CH [56]. miRNA-199 is downregulated under hypoxic conditions in cardiac myocytes (SIRT1) and hypoxia-1α inducible factor (HIF-α), an essential transcription factor for induction of the gene network of response to hypoxia. Thus, miRNA-24, miRNA-29, miRNA-199, and miRNA-320 represent potential therapeutic targets for ischemic injury in cardiac myocytes [51].

An overexpression of miRNA-22 was sufficient to induce pathological CH [74], showing itself as an essential miRNA for this process. On the other hand, the silencing of miRNA-22 in the heart of mice abolished CH and remodeling in response to two independent stressors: the infusion of isoproterenol and the activated calcineurin transgene. In addition, SIRT1 and HDAC4 were validated as target genes for miRNA-22 in the heart [74]. miRNA-155 is also required for the development

**67**

*Noncoding RNAs in the Cardiovascular System: Exercise Training Effects*

pathological CR, in addition to preventing the progression of HF [75].

of CH in response to stress. In fact, the knockout of miRNA-155 in mice suppresses

In addition, Bernardo et al. [76] showed that only the inhibition of the whole miRNA-34 family, but not the inhibition of miRNA-34 alone, improves cardiac function in mice with preexisting hypertrophy induced by TAC and also attenuates the pathological remodeling of the LV after MI [76]. In this same work, four targets for miRNA-34 (vinculin, Sema4b, Pofut1, and Bcl6) were validated, which were positively regulated and associated with the cardiac protection found in both TAC and/or MI mice treated with the antimiR-34 LNA. Thus, the authors believe that, since LNA-based therapies have already entered clinical trials, inhibition of the

Therefore, it is clear that miRNAs play pivotal roles in cardiovascular development and disease [51, 56]. Recent evidences show that miRNAs are dynamically regulated by exercise [1, 54, 81]. Thus, the elucidation of the relationship between ET and miRNAs in heart in physiological and pathological (i.e., hypertension, HF, spinal cord injury, etc.) conditions is fundamental to understand how exercise regulates the cardiovascular system at the molecular level, which may be promising

**4.3 miRNAs regulation by exercise training in cardiac remodeling and heart** 

The miRNA-1 and miRNA-133 are decreased in both aerobic treadmill-induced physiological hypertrophy and pressure overload-induced pathologic hypertrophy [64]. Additionally, swimming training is recognized for its efficiency in inducing myocardial hypertrophy and a significant increase in LV end-diastolic volume in rats [82]. In study conducted by Soci et al. [66], female rats were submitted to two swimming protocols (one moderate and other with high volume training to mimic an active individual and an elite athlete, respectively) for 10 weeks and developed physiological CH in a proportional way to the training volume. It was associated to a down regulation of miRNAs-1, -133a, and -133b in LV of trained rats also compared to sedentary group, which is inversely proportional to the physiological CH [66]. These results suggest that these myomiRs can be regulated by exercise, regardless of type (running or swimming) or volume (moderate and high) training, maintaining a similar expression profile [1]. Besides, Ma et al. [49] found that CH in rats induced by swimming exercise correlates with the modulation of other miRNAs, including miRNA-21, miRNA-124, miRNA-144, and miRNA-145 [49]. These miRNAs have as

entire miRNA-34 family has great therapeutic potential.

for the development of new drugs [2].

**diseases**

The miRNA-34 family (miRNA-34a, -34b, and -34c) expression levels are elevated in both hearts of mice with cardiac stress and in aging process, as well as in patients with HF [76, 77]. In this way, inhibition with antimiR-34a/antimiR-34 has emerged as a promising therapeutic strategy [78]. miRNA-34a appears to be an essential regulator in cardiac repair and regeneration after MI in the heart. In fact, in a study by Yang et al., miRNA-34a mimic delivery limited cardiomyocyte proliferation and subsequent MI recovery in mice, whereas antimiR-34a treatment improved remodeling post infarction. Furthermore, in isolated cardiomyocytes, miRNA-34a directly regulated cell cycle activity and death, via modulation of its targets, which include Bcl2, Cyclin D1, and SIRT1 [79]. Thus, at first, modulation of miRNA-34a appears to be important in the process of repairing adult myocardium after injury. The study conducted by Baker et al. showed that miRNA-34a is induced by oxidative stress (OE) in patients with chronic obstructive pulmonary disease, reducing the gene expression of SIRT1 and SIRT6 in bronchial epithelial cells [80]. Thus, miRNA-34a antigens may have therapeutic potential in several pathological

*DOI: http://dx.doi.org/10.5772/intechopen.86054*

conditions, including CH.

### *Noncoding RNAs in the Cardiovascular System: Exercise Training Effects DOI: http://dx.doi.org/10.5772/intechopen.86054*

*Muscle Cells - Recent Advances and Future Perspectives*

heart [51].

and -9 [56].

ischemic, dilated, hypertrophic cardiomyopathies, and HF and can be defined as an inappropriate accumulation of extracellular matrix proteins in the heart [69]. The exacerbated expression of extracellular matrix proteins is determinant in the differentiation between physiological and pathological CH [2]. In this sense, the negative regulation of miRNA-29 with anti-miRNAs *in vitro* and *in vivo* in a MI model induces the expression of collagens, whereas miRNA-29 overexpression in fibroblasts reduces this expression, suggesting this miRNA as a regulator of cardiac fibrosis [46]. Currently, the miRNA-29 family has been validated as a regulator of the gene expression of collagen I, III, fibrillin I, and elastin and is involved in pathological CH and the HF process [46, 51, 66]. In addition, decreased expression of the miRNA-29 family, miRNA-24, and miRNA-320 occurs after MI, and the decrease in miRNA-29 expression promotes fibrosis and scar formation in the

Through microarray analysis, van Rooij et al. [56] related the miRNAs found both in response to hypertrophy and HF and identified more than 12 miRNAs dysregulated in the heart of mice. Many of these miRNAs were similarly regulated in failing human hearts. Interestingly, overexpression of miRNA-23a, miRNA-23b, miRNA-24, miRNA-195, or miRNA-214 induced hypertrophic growth in cardiomyocyte culture, whereas overexpression of miRNA-150 or miRNA-181b decreased cell size of the cardiomyocyte. The miRNA-23a knockdown attenuated hypertrophy [56] and the overexpression of miRNA-195 *in vivo* alone (transgenic mouse), was sufficient to lead to hypertrophy and HF [56]. The miRNA-23a is transcriptionally regulated by the nuclear factor of activated T cells, cytoplasmic 3 (NFATc3), which mediates the signaling pathway of cardiac stress. Thus, miRNA-23a overexpression can triggers a hypertrophic response by means of its target-gene muRF1 (muscle specific ring finger protein 1), an anti-hypertrophic protein, while negative regulation of miRNA-23a abolishes CH induced by isoproterenol in mice [70]. Other miRNAs exhibited pro-hypertrophic profile, including miRNA-208, -21, -18b, -27,

Transgenic overexpression of miRNA-208a in the adult heart of mice is enough

miRNA-199a is predominantly expressed in cardiomyocytes, where it maintains

An overexpression of miRNA-22 was sufficient to induce pathological CH [74], showing itself as an essential miRNA for this process. On the other hand, the silencing of miRNA-22 in the heart of mice abolished CH and remodeling in response to two independent stressors: the infusion of isoproterenol and the activated calcineurin transgene. In addition, SIRT1 and HDAC4 were validated as target genes for miRNA-22 in the heart [74]. miRNA-155 is also required for the development

to induce hypertrophic growth, resulting in pronounced repression of regulatory target-genes, such as THRAP1 and myostatin (negative regulators of muscle growth and hypertrophy). In contrast, the deletion of cardiac miRNA-208a protects these animals from CH in response to hemodynamic cardiac stress by canceling the re-expression of fetal gene β-MHC [71]. In addition, therapeutic inhibition of miRNA-208 (by subcutaneous delivery of anti-miRNA-208a) reduces deleterious CR, improves cardiac function, and survival during HF in hypertensive rats [72]. Curiously, inhibition of cardiac-specific miR-208a may have therapeutic usefulness in a variety of metabolic disorders, such as obesity and type-2 diabetes in the setting

of cardiac dysfunction by targeting and increasing MED13 in heart [73].

cell size and may play a role in the regulation of CH [56]. miRNA-199 is downregulated under hypoxic conditions in cardiac myocytes (SIRT1) and hypoxia-1α inducible factor (HIF-α), an essential transcription factor for induction of the gene network of response to hypoxia. Thus, miRNA-24, miRNA-29, miRNA-199, and miRNA-320 represent potential therapeutic targets for ischemic injury in cardiac

**66**

myocytes [51].

of CH in response to stress. In fact, the knockout of miRNA-155 in mice suppresses pathological CR, in addition to preventing the progression of HF [75].

The miRNA-34 family (miRNA-34a, -34b, and -34c) expression levels are elevated in both hearts of mice with cardiac stress and in aging process, as well as in patients with HF [76, 77]. In this way, inhibition with antimiR-34a/antimiR-34 has emerged as a promising therapeutic strategy [78]. miRNA-34a appears to be an essential regulator in cardiac repair and regeneration after MI in the heart. In fact, in a study by Yang et al., miRNA-34a mimic delivery limited cardiomyocyte proliferation and subsequent MI recovery in mice, whereas antimiR-34a treatment improved remodeling post infarction. Furthermore, in isolated cardiomyocytes, miRNA-34a directly regulated cell cycle activity and death, via modulation of its targets, which include Bcl2, Cyclin D1, and SIRT1 [79]. Thus, at first, modulation of miRNA-34a appears to be important in the process of repairing adult myocardium after injury. The study conducted by Baker et al. showed that miRNA-34a is induced by oxidative stress (OE) in patients with chronic obstructive pulmonary disease, reducing the gene expression of SIRT1 and SIRT6 in bronchial epithelial cells [80]. Thus, miRNA-34a antigens may have therapeutic potential in several pathological conditions, including CH.

In addition, Bernardo et al. [76] showed that only the inhibition of the whole miRNA-34 family, but not the inhibition of miRNA-34 alone, improves cardiac function in mice with preexisting hypertrophy induced by TAC and also attenuates the pathological remodeling of the LV after MI [76]. In this same work, four targets for miRNA-34 (vinculin, Sema4b, Pofut1, and Bcl6) were validated, which were positively regulated and associated with the cardiac protection found in both TAC and/or MI mice treated with the antimiR-34 LNA. Thus, the authors believe that, since LNA-based therapies have already entered clinical trials, inhibition of the entire miRNA-34 family has great therapeutic potential.

Therefore, it is clear that miRNAs play pivotal roles in cardiovascular development and disease [51, 56]. Recent evidences show that miRNAs are dynamically regulated by exercise [1, 54, 81]. Thus, the elucidation of the relationship between ET and miRNAs in heart in physiological and pathological (i.e., hypertension, HF, spinal cord injury, etc.) conditions is fundamental to understand how exercise regulates the cardiovascular system at the molecular level, which may be promising for the development of new drugs [2].

### **4.3 miRNAs regulation by exercise training in cardiac remodeling and heart diseases**

The miRNA-1 and miRNA-133 are decreased in both aerobic treadmill-induced physiological hypertrophy and pressure overload-induced pathologic hypertrophy [64]. Additionally, swimming training is recognized for its efficiency in inducing myocardial hypertrophy and a significant increase in LV end-diastolic volume in rats [82]. In study conducted by Soci et al. [66], female rats were submitted to two swimming protocols (one moderate and other with high volume training to mimic an active individual and an elite athlete, respectively) for 10 weeks and developed physiological CH in a proportional way to the training volume. It was associated to a down regulation of miRNAs-1, -133a, and -133b in LV of trained rats also compared to sedentary group, which is inversely proportional to the physiological CH [66]. These results suggest that these myomiRs can be regulated by exercise, regardless of type (running or swimming) or volume (moderate and high) training, maintaining a similar expression profile [1]. Besides, Ma et al. [49] found that CH in rats induced by swimming exercise correlates with the modulation of other miRNAs, including miRNA-21, miRNA-124, miRNA-144, and miRNA-145 [49]. These miRNAs have as

validated targets Pik3a, PTEN, and TSC2, which are negative regulators of the PI3K/ AKT/mTOR signaling pathway. Importantly, Liu et al. [5] showed that the inhibition of miRNA-222 *in vivo* completely blocked cardiac and cardiomyocyte growth in response to exercise while reducing markers of cardiomyocyte proliferation. Indeed, the authors revealed that miRNA-222 delivery was sufficient to protect the heart against dysfunction and adverse remodeling after ischemic injury [5].

In the same work cited before, Soci et al. [66] found an increase in miRNA-29c expression in LV in response to swimming training, which was inversely correlated with a decrease in both collagen I and III expression as well as in OH-proline concentration, being essential for the improvement of LV compliance observed in rats [66]. These findings exhibit an important integrative and regulatory role for miRNAs in the fibrotic response of trained heart.

Other work from our group showed that swimming training also causes an increase in miRNA-126 in the heart of rats, targeting and decreasing in expression of Spred-1, which contributes to angiogenesis in this tissue [83]. Although Pi3kr2 is also a well-validated target for miRNA-126, it was not altered by the swimming protocol. Physiological CH also involves the regulation of some miRNAs related to increased expression of angiotensin receptor-1 (AT1R), regardless of the participation of Ang-II. In parallel, increased angiotensin-converting enzyme 2 (ACE2), angiotensin (1–7), and Ang-II receptor in heart, suggest that miRNAs are involved in the regulation of the non-classical renin-angiotensin-aldosterone system (RAAS), counteracting the classic cardiac RAAS in CH [2, 54]. Fernandes et al. [54] evaluated that swimming alter the expression of cardiac miRNAs targeting the components of the RAAS, which were associated with the development of ventricular CH in rats [54]. In fact, swimming training increased the expression of miRNAs-27a and 27b by targeting the angiotensin-converting enzyme (ACE) and decreased miRNA-143 expression by targeting ACE2 in the heart of healthy rats.

Differently from which is found in pathological CH [61], high volume ET decreases the expression of cardiac miRNA-208a in healthy, induces the upregulation of targets such as THRAP-1, Purβ, and Sox6, and improves the balance between βMHC and αMHC gene expression [2, 84]. In addition, in recent study, Fernandes et al. [85] showed that aerobic ET prevents weight gain and pathological CH in obese Zucker rats, via increase of cardiac MED13 by regulation of miRNA-208. Thus, miRNA-208 represents a potential potent therapeutic target for modulation of cardiac function and remodeling during progression of heart disease.

In addition, the study conducted by Fernandes et al. [86] showed that swimming training reestablishes the peripheral levels of the -16, -21, and -126 miRNAs associated with revascularization in hypertension. This alteration was accompanied by normalization of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), and phosphatidylinositol-3 kinase regulatory subunit (PI3KR2) in parallel to a normalization of pro-apoptotic (Bad) and anti-apoptotic mediators (Bcl-2, Bcl-x, and p-Badser112: Bad ratio) [86].

Swimming ET restores the miRNA-29a and miRNA-29c levels of the heart and prevents the deposition of type I and III collagen in the border zone as well as in remote regions of the infarcted myocardium of rats, which can contribute to the improvement of the ventricular function induced by the aerobic training [87]. Cardiac fibrosis, which impairs cardiac contractility, is a major aspect of the remodeling process after MI. Impaired cardiomyocyte contractility and calcium transient are hallmarks of LV contractile dysfunction [88]. In this sense, Melo et al. [81] showed that moderate aerobic ET also restores cardiac Ca2+ transient after MI, regulating miRNA-1 and miRNA-214 levels [81]. Additionally, resistance training has been shown to induce CH, with improved cardiac function of isolated cardiomyocytes, which is partially explained by the decrease of miRNA-214 and

**69**

**5.1 miRNA-126**

*Noncoding RNAs in the Cardiovascular System: Exercise Training Effects*

the increase of gene SERCA2a expression [81]. There are few studies that demonstrate the protective role of resistance training in heart through changes in miRNA expression pattern. Thus, there is a potential role of these miRNAs in promoting

The vascular disease in its various forms is one of the leading causes of death worldwide, and in accordance with the projection of the World Health Organization, this number only increases in the next 30 years [89]. ET prevents the progression of vascular diseases and reduces cardiovascular morbidity and mortality. ET also ameliorates vascular changes including endothelial dysfunction and arterial remodeling and stiffness, usually present in type 2 diabetes, obesity,

Although there are major advances in the surgical area, the great challenge that anyone who presents a framework of CVD remains the prevention of this disease. Currently, there are some ways to alleviate this disease such as the use of beta-blockers, ACE inhibitors, statins; all these strategies are for mitigate the risk factors such as hypertension and dyslipidemia. Already in more severe cases, surgical procedures such as coronary artery bypass grafting, angioplasty (in severe cases of atheroscle-

Despite these strategies of mitigate the risk factors, this is still not enough since the rate of individuals affected and morbimortality has been increasing in relation to this class of diseases. So, the search for new therapies and approaches to prevent the onset and reduce the progression of vascular disease is desperately needed. Therefore, the processes time consuming since the mechanisms involved in this

Atherosclerosis is the main cause of CVD. This is a chronic inflammatory disease that affects the blood vessels and arteries of medium or large size, usually in the areas of bifurcation, due to the non-laminar flow, leading to an endothelial dysfunction due to chronic exposure to pro-inflammatory molecules, and also to an inhibition of anti-inflammatory molecules, such as NO, this causes a narrowing of

In this disease, the blood flow changes from laminar to turbulent, due to the narrowing of the vascular lumen, the endothelial cells (EC) respond to this flow, that is, to the shear stress, this response is due to mechanosensors genes present on the cell surface and also the activation of the adhesion molecules and other inflammatory cytokines that are activated to alleviate the excess of cholesterol low-density

Another important factor that has been widely studied in vascular diseases and in atherosclerosis are the miRNAs, which can regulate the cellular phenotype of both VSMCs and EC, such as the proliferation, migration, and cell growth, and can

miRNA-126, for example, can regulate both inflammation and angiogenesis (the process of new vessel formation). This miRNA is basically expressed in platelets and EC, which in turn play an important role in the blood circulation, as well, regulating vascular tone, recruitment of other cells, and even vascular homeostasis [102, 103], and the transcription of this miRNA is regulated by the transcription factor Ets1, which is known for their role in specific gene expression in EC [104]. Harris et al.

the arterial lumen due to atheroma plaque, impeding blood flow [94, 95].

lipoprotein (LDL) that is encompassed in the vascular intima [96–98].

also regulate the handling of lipids and inflammatory molecules [99–101].

*DOI: http://dx.doi.org/10.5772/intechopen.86054*

**5. Vascular, exercise, and miRNAs**

hypertension, and metabolic syndrome [90].

rosis), and others are performed [91–93].

disease are not yet fully understood.

cardioprotective effects on CR.

the increase of gene SERCA2a expression [81]. There are few studies that demonstrate the protective role of resistance training in heart through changes in miRNA expression pattern. Thus, there is a potential role of these miRNAs in promoting cardioprotective effects on CR.

### **5. Vascular, exercise, and miRNAs**

*Muscle Cells - Recent Advances and Future Perspectives*

miRNAs in the fibrotic response of trained heart.

validated targets Pik3a, PTEN, and TSC2, which are negative regulators of the PI3K/ AKT/mTOR signaling pathway. Importantly, Liu et al. [5] showed that the inhibition of miRNA-222 *in vivo* completely blocked cardiac and cardiomyocyte growth in response to exercise while reducing markers of cardiomyocyte proliferation. Indeed, the authors revealed that miRNA-222 delivery was sufficient to protect the heart against dysfunction and adverse remodeling after ischemic injury [5].

In the same work cited before, Soci et al. [66] found an increase in miRNA-29c expression in LV in response to swimming training, which was inversely correlated with a decrease in both collagen I and III expression as well as in OH-proline concentration, being essential for the improvement of LV compliance observed in rats [66]. These findings exhibit an important integrative and regulatory role for

Other work from our group showed that swimming training also causes an increase in miRNA-126 in the heart of rats, targeting and decreasing in expression of Spred-1, which contributes to angiogenesis in this tissue [83]. Although Pi3kr2 is also a well-validated target for miRNA-126, it was not altered by the swimming protocol. Physiological CH also involves the regulation of some miRNAs related to increased expression of angiotensin receptor-1 (AT1R), regardless of the participation of Ang-II. In parallel, increased angiotensin-converting enzyme 2 (ACE2), angiotensin (1–7), and Ang-II receptor in heart, suggest that miRNAs are involved in the regulation of the non-classical renin-angiotensin-aldosterone system (RAAS), counteracting the classic cardiac RAAS in CH [2, 54]. Fernandes et al. [54] evaluated that swimming alter the expression of cardiac miRNAs targeting the components of the RAAS, which were associated with the development of ventricular CH in rats [54]. In fact, swimming training increased the expression of miRNAs-27a and 27b by targeting the angiotensin-converting enzyme (ACE) and decreased

miRNA-143 expression by targeting ACE2 in the heart of healthy rats.

of cardiac function and remodeling during progression of heart disease.

mediators (Bcl-2, Bcl-x, and p-Badser112: Bad ratio) [86].

Differently from which is found in pathological CH [61], high volume ET decreases the expression of cardiac miRNA-208a in healthy, induces the upregulation of targets such as THRAP-1, Purβ, and Sox6, and improves the balance between βMHC and αMHC gene expression [2, 84]. In addition, in recent study, Fernandes et al. [85] showed that aerobic ET prevents weight gain and pathological CH in obese Zucker rats, via increase of cardiac MED13 by regulation of miRNA-208. Thus, miRNA-208 represents a potential potent therapeutic target for modulation

In addition, the study conducted by Fernandes et al. [86] showed that swimming training reestablishes the peripheral levels of the -16, -21, and -126 miRNAs associated with revascularization in hypertension. This alteration was accompanied by normalization of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), and phosphatidylinositol-3 kinase regulatory subunit (PI3KR2) in parallel to a normalization of pro-apoptotic (Bad) and anti-apoptotic

Swimming ET restores the miRNA-29a and miRNA-29c levels of the heart and prevents the deposition of type I and III collagen in the border zone as well as in remote regions of the infarcted myocardium of rats, which can contribute to the improvement of the ventricular function induced by the aerobic training [87]. Cardiac fibrosis, which impairs cardiac contractility, is a major aspect of the remodeling process after MI. Impaired cardiomyocyte contractility and calcium transient are hallmarks of LV contractile dysfunction [88]. In this sense, Melo et al. [81] showed that moderate aerobic ET also restores cardiac Ca2+ transient after MI, regulating miRNA-1 and miRNA-214 levels [81]. Additionally, resistance training has been shown to induce CH, with improved cardiac function of isolated cardiomyocytes, which is partially explained by the decrease of miRNA-214 and

**68**

The vascular disease in its various forms is one of the leading causes of death worldwide, and in accordance with the projection of the World Health Organization, this number only increases in the next 30 years [89]. ET prevents the progression of vascular diseases and reduces cardiovascular morbidity and mortality. ET also ameliorates vascular changes including endothelial dysfunction and arterial remodeling and stiffness, usually present in type 2 diabetes, obesity, hypertension, and metabolic syndrome [90].

Although there are major advances in the surgical area, the great challenge that anyone who presents a framework of CVD remains the prevention of this disease. Currently, there are some ways to alleviate this disease such as the use of beta-blockers, ACE inhibitors, statins; all these strategies are for mitigate the risk factors such as hypertension and dyslipidemia. Already in more severe cases, surgical procedures such as coronary artery bypass grafting, angioplasty (in severe cases of atherosclerosis), and others are performed [91–93].

Despite these strategies of mitigate the risk factors, this is still not enough since the rate of individuals affected and morbimortality has been increasing in relation to this class of diseases. So, the search for new therapies and approaches to prevent the onset and reduce the progression of vascular disease is desperately needed. Therefore, the processes time consuming since the mechanisms involved in this disease are not yet fully understood.

Atherosclerosis is the main cause of CVD. This is a chronic inflammatory disease that affects the blood vessels and arteries of medium or large size, usually in the areas of bifurcation, due to the non-laminar flow, leading to an endothelial dysfunction due to chronic exposure to pro-inflammatory molecules, and also to an inhibition of anti-inflammatory molecules, such as NO, this causes a narrowing of the arterial lumen due to atheroma plaque, impeding blood flow [94, 95].

In this disease, the blood flow changes from laminar to turbulent, due to the narrowing of the vascular lumen, the endothelial cells (EC) respond to this flow, that is, to the shear stress, this response is due to mechanosensors genes present on the cell surface and also the activation of the adhesion molecules and other inflammatory cytokines that are activated to alleviate the excess of cholesterol low-density lipoprotein (LDL) that is encompassed in the vascular intima [96–98].

Another important factor that has been widely studied in vascular diseases and in atherosclerosis are the miRNAs, which can regulate the cellular phenotype of both VSMCs and EC, such as the proliferation, migration, and cell growth, and can also regulate the handling of lipids and inflammatory molecules [99–101].

### **5.1 miRNA-126**

miRNA-126, for example, can regulate both inflammation and angiogenesis (the process of new vessel formation). This miRNA is basically expressed in platelets and EC, which in turn play an important role in the blood circulation, as well, regulating vascular tone, recruitment of other cells, and even vascular homeostasis [102, 103], and the transcription of this miRNA is regulated by the transcription factor Ets1, which is known for their role in specific gene expression in EC [104]. Harris et al.

[104] demonstrated that miRNA-126 negatively regulates the vascular cell adhesion molecule (VCAM-1). This protein mediates the adhesion of lymphocytes, monocytes, eosinophils, and basophils of the vascular endothelium, thus interfering with the adhesion of leukocytes induced by tumor necrosis factor (TNF) in the endothelium, which may decrease leukocyte infiltration in the vascular wall and also in the interaction of leukocytes with EC [104].

This miRNA can influence the reduction of apoptotic cells contained in the vascular thrombus, softening the inflammatory reaction characteristic of atherosclerosis; so the miRNA-126 has an anti-atherosclerotic effect, which is also related to the G 16 protein signaling regulator, which results in recruitment of circulating progenitor cells, leading to stabilization of atherosclerotic plaque [105, 106]. Studies show that on deletion of this miRNA, vascular integrity decreases; in the case of infarcted individuals, a defect in cardiac neovascularization occurs [107].

This miRNA may be pro-angiogenic because of its binding to the PI3K pathway, which is important in regulating mitogenesis, cell differentiation, and insulin-stimulated glucose transport, and mitogen-activated protein kinases (MAPKs) that respond to extracellular stimuli and regulate various cellular activities such as gene expression, mitosis, differentiation, cell survival, and apoptosis. PI3K pathway promotes the suppression of the sprouty-related EVH1 domain containing protein-1 protein (Spred-1) that generally inhibits the signaling pathway [108–111].

Recent studies showed that the miRNA-126 levels in the circulatory system increase to varying degrees after different ET protocols [1]. It is already known that regular aerobic exercise has a positive effect on arterial endothelial function, mainly by increasing circulating NO and reducing endothelin-1. Aerobic exercise can also improve vascular endothelial and mitochondrial function to protect blood vessels, thus reducing the incidence of CVD [83, 112, 113].

Recent study demonstrated that the plasma levels of miRNA-126 were increased in obese subjects after an acute aerobic exercise session [114] Already noted that improvement of vascular endothelial function in adolescents after 6 weeks of aerobic exercise combined with dietary control may be related to changes in serum level miRNA-126, this improved peripheral vasodilator capacity, indirectly reflecting the improvement of vascular endothelial function in obese adolescents [115]. This increase of serum levels of the miRNA-126 through ET represents the beneficial aspects for obese individuals and associated diseases such as CVD. This may be related to the increase of NO provided by physical training, which is already well established in the literature, that aerobic exercise increases the expression and phosphorylation of endothelial nitric oxide synthase (eNOS) and with that the levels of oxidative stress decreased, leading to an increase in the use of NO and an increase in vascular function [90, 102, 116–118].

### **5.2 miRNA-21**

miRNA-21 regulates cell proliferation through your gene target PTEN, which is a protein that acts as phosphatase and operates primarily on dephosphorylation in the PI3K/Akt signaling pathway. PTEN suppresses Akt signaling, which decreases the activity of eNOS and PTEN also inhibits the expression of VCAM-1 in EC stimulated by TNF-α [119]. A pathological condition that increases the expression of the miRNA-21 is shear stress in your state turbulent flow [120]. miRNA-21 also contributes to phenotypic change in EC, which is induced by transforming growth factor β (TGF-β) [121]. These data indicate that miRNA-21 modulates vascular homeostasis through PTEN and Akt.

**71**

*Noncoding RNAs in the Cardiovascular System: Exercise Training Effects*

Although miRNA-21 has been involved in promoting the proliferation of VSMCs in response to a series of models of vascular mechanical injury, its role remains to be defined in the formation of atherosclerotic lesion [68]. miRNA-21 also mediates VSMC differentiation from the contractile phenotype to the synthetic through TGF-β and BMP signaling in a single posttranscriptional processing step, implying that Smad proteins can control the maturation of

It was demonstrated by [86] that aerobic ET has a beneficial effect on the regression of arterial hypertension, restoring the normal expression of miRNA-16, -21, and -126 of skeletal muscle microcirculation, normalization of levels of VEGF, eNOS, and PI3KR2, as well as proapoptotic (bad) and antiapoptotic mediators (Bcl-2, Bcl-x, and p-Badser112: Bad ratio), indicating that the balance between angiogenesis and apoptotic factors that can prevent microvascular abnormalities in rats hypertensive, demonstrating once again the protective effect of aerobic physi-

The miRNA-221/222 is expressed in EC, VSMCs, and hematopoietic cells. It has been shown that the endothelial progenitor cells of patients with coronary artery disease have an increased expression of these miRNAs, these cells have a factor important in endothelial integrity and vascular repair [123]. One of the target genes of these miRNAs is signal transducer and activator of transcription 5A (Stat5A), a transcription factor involved in the regulation of cell proliferation and migration, it has been identified that Stat5A is negatively regulated by miR-222 and it has been observed in EC of atherosclerotic lesions that Stat5A is low expression, so it is possible that Stat5A may be a new therapeutic target for

The study by Liu et al. [123] also demonstrated that miRNA-221/222 has opposite biological functions depending on the cell; the study observed that inhibition of miRNA-221/222 is able to increase reendothelization, whereas in VSMC neointimal formation was significantly reduced. Thus, miRNA-221/222 could be an ideal therapeutic target in patients with atherosclerosis, angioplasty, stent implantation,

The increased expression of the miRNA-146a has been observed in vascular injury after balloon angioplasty. This miRNA can mediate cell-to-cell communication between VSMCs and CE and also other cell types in the vessel wall [125]. A study by Sun et al. [126] attested that transcription of miRNA-146a is regulated by the transcriptional factors KLF-4 (Krüppel-like factor 4) and KLF-5 (Krüppel-like factor 5) and they play an important role in promoting proliferation of VSMCs (*in vitro*) and when the carotid artery is injured with a balloon catheter there occurs a hyperplasia of the neointima (*in vivo*) [126]. Ji et al. [68] also found an increase in miRNA-146a in injured arteries, resulting in a remarkable formation of neointima. In this study, the expression of several miRNAs in the wall of the vessel after angioplasty was observed for the first time [68]. In the study by Chen et al. [127], several miRNAs have been shown to play roles such as proliferation, cell differentiation, and apoptosis in the inflammatory responses of monocytes and macrophages with stimulation of oxidized LDL that are primordial in the develop-

*DOI: http://dx.doi.org/10.5772/intechopen.86054*

miRNAs [122].

cal training.

**5.3 miRNA-221/222**

atherosclerosis [103, 123, 124].

ment of atherosclerosis [127].

**5.4 miRNA-146a**

or myocardial revascularization surgery [123].

*Noncoding RNAs in the Cardiovascular System: Exercise Training Effects DOI: http://dx.doi.org/10.5772/intechopen.86054*

Although miRNA-21 has been involved in promoting the proliferation of VSMCs in response to a series of models of vascular mechanical injury, its role remains to be defined in the formation of atherosclerotic lesion [68]. miRNA-21 also mediates VSMC differentiation from the contractile phenotype to the synthetic through TGF-β and BMP signaling in a single posttranscriptional processing step, implying that Smad proteins can control the maturation of miRNAs [122].

It was demonstrated by [86] that aerobic ET has a beneficial effect on the regression of arterial hypertension, restoring the normal expression of miRNA-16, -21, and -126 of skeletal muscle microcirculation, normalization of levels of VEGF, eNOS, and PI3KR2, as well as proapoptotic (bad) and antiapoptotic mediators (Bcl-2, Bcl-x, and p-Badser112: Bad ratio), indicating that the balance between angiogenesis and apoptotic factors that can prevent microvascular abnormalities in rats hypertensive, demonstrating once again the protective effect of aerobic physical training.

### **5.3 miRNA-221/222**

*Muscle Cells - Recent Advances and Future Perspectives*

interaction of leukocytes with EC [104].

ing pathway [108–111].

individuals, a defect in cardiac neovascularization occurs [107].

thus reducing the incidence of CVD [83, 112, 113].

increase in vascular function [90, 102, 116–118].

[104] demonstrated that miRNA-126 negatively regulates the vascular cell adhesion molecule (VCAM-1). This protein mediates the adhesion of lymphocytes, monocytes, eosinophils, and basophils of the vascular endothelium, thus interfering with the adhesion of leukocytes induced by tumor necrosis factor (TNF) in the endothelium, which may decrease leukocyte infiltration in the vascular wall and also in the

This miRNA can influence the reduction of apoptotic cells contained in the vascular thrombus, softening the inflammatory reaction characteristic of atherosclerosis; so the miRNA-126 has an anti-atherosclerotic effect, which is also related to the G 16 protein signaling regulator, which results in recruitment of circulating progenitor cells, leading to stabilization of atherosclerotic plaque [105, 106]. Studies show that on deletion of this miRNA, vascular integrity decreases; in the case of infarcted

This miRNA may be pro-angiogenic because of its binding to the PI3K pathway, which is important in regulating mitogenesis, cell differentiation, and insulin-stimulated glucose transport, and mitogen-activated protein kinases (MAPKs) that respond to extracellular stimuli and regulate various cellular activities such as gene expression, mitosis, differentiation, cell survival, and apoptosis. PI3K pathway promotes the suppression of the sprouty-related EVH1 domain containing protein-1 protein (Spred-1) that generally inhibits the signal-

Recent studies showed that the miRNA-126 levels in the circulatory system increase to varying degrees after different ET protocols [1]. It is already known that regular aerobic exercise has a positive effect on arterial endothelial function, mainly by increasing circulating NO and reducing endothelin-1. Aerobic exercise can also improve vascular endothelial and mitochondrial function to protect blood vessels,

Recent study demonstrated that the plasma levels of miRNA-126 were increased

miRNA-21 regulates cell proliferation through your gene target PTEN, which is a protein that acts as phosphatase and operates primarily on dephosphorylation in the PI3K/Akt signaling pathway. PTEN suppresses Akt signaling, which decreases the activity of eNOS and PTEN also inhibits the expression of VCAM-1 in EC stimulated by TNF-α [119]. A pathological condition that increases the expression of the miRNA-21 is shear stress in your state turbulent flow [120]. miRNA-21 also contributes to phenotypic change in EC, which is induced by transforming growth factor β (TGF-β) [121]. These data indicate that miRNA-21 modulates vascular homeostasis

in obese subjects after an acute aerobic exercise session [114] Already noted that improvement of vascular endothelial function in adolescents after 6 weeks of aerobic exercise combined with dietary control may be related to changes in serum level miRNA-126, this improved peripheral vasodilator capacity, indirectly reflecting the improvement of vascular endothelial function in obese adolescents [115]. This increase of serum levels of the miRNA-126 through ET represents the beneficial aspects for obese individuals and associated diseases such as CVD. This may be related to the increase of NO provided by physical training, which is already well established in the literature, that aerobic exercise increases the expression and phosphorylation of endothelial nitric oxide synthase (eNOS) and with that the levels of oxidative stress decreased, leading to an increase in the use of NO and an

**70**

**5.2 miRNA-21**

through PTEN and Akt.

The miRNA-221/222 is expressed in EC, VSMCs, and hematopoietic cells. It has been shown that the endothelial progenitor cells of patients with coronary artery disease have an increased expression of these miRNAs, these cells have a factor important in endothelial integrity and vascular repair [123]. One of the target genes of these miRNAs is signal transducer and activator of transcription 5A (Stat5A), a transcription factor involved in the regulation of cell proliferation and migration, it has been identified that Stat5A is negatively regulated by miR-222 and it has been observed in EC of atherosclerotic lesions that Stat5A is low expression, so it is possible that Stat5A may be a new therapeutic target for atherosclerosis [103, 123, 124].

The study by Liu et al. [123] also demonstrated that miRNA-221/222 has opposite biological functions depending on the cell; the study observed that inhibition of miRNA-221/222 is able to increase reendothelization, whereas in VSMC neointimal formation was significantly reduced. Thus, miRNA-221/222 could be an ideal therapeutic target in patients with atherosclerosis, angioplasty, stent implantation, or myocardial revascularization surgery [123].

### **5.4 miRNA-146a**

The increased expression of the miRNA-146a has been observed in vascular injury after balloon angioplasty. This miRNA can mediate cell-to-cell communication between VSMCs and CE and also other cell types in the vessel wall [125]. A study by Sun et al. [126] attested that transcription of miRNA-146a is regulated by the transcriptional factors KLF-4 (Krüppel-like factor 4) and KLF-5 (Krüppel-like factor 5) and they play an important role in promoting proliferation of VSMCs (*in vitro*) and when the carotid artery is injured with a balloon catheter there occurs a hyperplasia of the neointima (*in vivo*) [126]. Ji et al. [68] also found an increase in miRNA-146a in injured arteries, resulting in a remarkable formation of neointima. In this study, the expression of several miRNAs in the wall of the vessel after angioplasty was observed for the first time [68]. In the study by Chen et al. [127], several miRNAs have been shown to play roles such as proliferation, cell differentiation, and apoptosis in the inflammatory responses of monocytes and macrophages with stimulation of oxidized LDL that are primordial in the development of atherosclerosis [127].

Cao et al. [128] observed that levels of miRNA-146a and miRNA-21 were increased in atherosclerotic plaques and that these miRNAs suppress the expression of Notch2 (neurogenic locus notch homolog protein) which is a membrane protein, and Jag1 (Jagged1) is a cell surface protein [128]. These provide important negative feedback on the proliferation of VSMCs in response to vascular injury, so inhibitions of these miRNAs may present a new strategy for the prevention of fibroproliferative vascular diseases.

### **5.5 miRNAs-143/145**

The miRNAs-143/145 have an important role in the differentiation of VSMC as well as miRNA-146a, with a critical role in cell phenotype change [129, 130]. Boettger et al. [130] demonstrated that the detection of miRNA-143 and miRNA-145 altered the VSMC phenotype from contractile (physiological) to synthetic (pathological), in which this led to neointimal formation in elderly mice [130]. Another study highlighted the suppression of these miRNAs in human aneurysms [131]. Several targets of miRNA-143 and miRNA-145 have been reported in which they regulate the functions of VSMCs, for example, the transcription factors KLF-4 (Krüppel-like factor 4), myocardin, Elk-1 (ELK1, ETS member of the family of oncogenes), and KLF5. In addition, the ACE has been shown to contribute to vascular tonus dysregulation and to reduce blood pressure in mice with high expression of miRNA-143/145 [129, 130]. Thus, these miRNAs through their targets are antiatherosclerotic and have the function of regulating the phenotype of the VSMCs and inhibit the formation of neointima.

### **5.6 Others miRNAs**

Other miRNAs are also involved with EC and VSMCs like miRNA-27b and let-7. Kuehbacher et al. [103] demonstrated that inhibition of miRNA-27b and let-7f in the EC significantly reduced the onset of the endothelium, therefore when these miRNAs are with increased expression, it is suggested that it has a pro-angiogenic action [103]. It has also been observed that shear stress increases the expression of miRNA-27b supporting an activity endothelial protection [132]. Other miRNA vascular protector is the miRNA-296 which regulates directly the substrate of tyrosine kinase regulated by the hepatocyte growth factor (HGS), a protein responsible for the degradation of pro-angiogenic receptors VEGFR2 and PDGFRβ, promoting angiogenesis [133, 134]. The miRNA-130a was also identified as angiogenic, targeting the homeobox anti-angiogenic GAX (homeoboxstop-growth), and HoxA5 proteins. It has been observed that miRNA-130a antagonizes the inhibitory effects of GAX on the proliferation, migration, and formation of EC [135].

That said, miRNAs are important regulators of vascular cell functions and contribute to many vascular diseases, such as coronary artery disease and, in general, atherosclerosis. It is supposed that the human genome encodes several miRNA genes, but only a few vascular-specific miRNAs have been identified so far. In addition, there is only some information about its specific functions of the cell type and target genes. Many studies have revealed an aberrant miRNA expression profile under pathological conditions. Thus, miRNAs can be used as new biomarkers and, on the other hand, may represent powerful new therapeutic targets. Several miRNA-based therapeutic strategies are used to modify miRNA expression as therapeutic approaches; one of miRNA modulators is physical training, as already mentioned in miRNAs-126 and 21.

**73**

*Noncoding RNAs in the Cardiovascular System: Exercise Training Effects*

ET, especially aerobic exercise, plays a protective role in the cardiovascular system and has been used as a non-pharmacological therapeutic tool for the prevention and treatment of CVD. Although much knowledge already exists, there is insufficient evidence to establish a direct link between epigenetic modulations and changes, caused by exercise, in the heart and blood vessels [42]. There is currently evidence that the protective effects of aerobic ET involve changes in pattern expression of specific miRNAs by positively regulating cardiovascular remodeling. In fact, miRNAs play important roles in the regulation of distinct processes in mammals, and although they exhibit limited complementarity with their target RNAs, it is still sufficient for them to regulate various physiological processes, including cell proliferation, differentiation, migration, angiogenesis, apoptosis, tissue development, and remodeling [14, 136, 137]. However, new studies to understand the pathways and mechanisms in which the ET acts on the miRNAs are required. Thus, studies with miRNAs hope to broaden the perspective of its use in the correction of pathological processes from miRNAs therapy, contributing as a therapeutic intervention in cardiovascular damages as well as for the survival and quality of life of patients.

The researchers were supported by Sao Paulo Research Foundation (FAPESP: 2016/26156-5, 2015/22814-5 and 2015/17275-8), USP/PRP-NAPmiR, National Council for Scientific and Technological Development (CNPq: 307591/2009-3 and 159827/2011-6), and Coordination for the Improvement of Higher Education

Thanks to the support of data basis Smart Servier Medical Art in the design of

the figures by authors (https://smart.servier.com/image-set-download/).

*DOI: http://dx.doi.org/10.5772/intechopen.86054*

**6. Conclusions**

**Acknowledgements**

Personnel (CAPES-Proex).

There are no conflicts of interest.

**Notes/Thanks/Other declarations**

**Conflict of interest**

### **6. Conclusions**

*Muscle Cells - Recent Advances and Future Perspectives*

and inhibit the formation of neointima.

vascular diseases.

**5.5 miRNAs-143/145**

**5.6 Others miRNAs**

formation of EC [135].

mentioned in miRNAs-126 and 21.

Cao et al. [128] observed that levels of miRNA-146a and miRNA-21 were increased in atherosclerotic plaques and that these miRNAs suppress the expression of Notch2 (neurogenic locus notch homolog protein) which is a membrane protein, and Jag1 (Jagged1) is a cell surface protein [128]. These provide important negative feedback on the proliferation of VSMCs in response to vascular injury, so inhibitions of these miRNAs may present a new strategy for the prevention of fibroproliferative

The miRNAs-143/145 have an important role in the differentiation of VSMC as well as miRNA-146a, with a critical role in cell phenotype change [129, 130]. Boettger et al. [130] demonstrated that the detection of miRNA-143 and miRNA-145 altered the VSMC phenotype from contractile (physiological) to synthetic (pathological), in which this led to neointimal formation in elderly mice [130]. Another study highlighted the suppression of these miRNAs in human aneurysms [131]. Several targets of miRNA-143 and miRNA-145 have been reported in which they regulate the functions of VSMCs, for example, the transcription factors KLF-4 (Krüppel-like factor 4), myocardin, Elk-1 (ELK1, ETS member of the family of oncogenes), and KLF5. In addition, the ACE has been shown to contribute to vascular tonus dysregulation and to reduce blood pressure in mice with high expression of miRNA-143/145 [129, 130]. Thus, these miRNAs through their targets are antiatherosclerotic and have the function of regulating the phenotype of the VSMCs

Other miRNAs are also involved with EC and VSMCs like miRNA-27b and let-7. Kuehbacher et al. [103] demonstrated that inhibition of miRNA-27b and let-7f in the EC significantly reduced the onset of the endothelium, therefore when these miRNAs are with increased expression, it is suggested that it has a pro-angiogenic action [103]. It has also been observed that shear stress increases the expression of miRNA-27b supporting an activity endothelial protection [132]. Other miRNA vascular protector is the miRNA-296 which regulates directly the substrate of tyrosine kinase regulated by the hepatocyte growth factor (HGS), a protein responsible for the degradation of pro-angiogenic receptors VEGFR2 and PDGFRβ, promoting angiogenesis [133, 134]. The miRNA-130a was also identified as angiogenic, targeting the homeobox anti-angiogenic GAX (homeoboxstop-growth), and HoxA5 proteins. It has been observed that miRNA-130a antagonizes the inhibitory effects of GAX on the proliferation, migration, and

That said, miRNAs are important regulators of vascular cell functions and contribute to many vascular diseases, such as coronary artery disease and, in general, atherosclerosis. It is supposed that the human genome encodes several miRNA genes, but only a few vascular-specific miRNAs have been identified so far. In addition, there is only some information about its specific functions of the cell type and target genes. Many studies have revealed an aberrant miRNA expression profile under pathological conditions. Thus, miRNAs can be used as new biomarkers and, on the other hand, may represent powerful new therapeutic targets. Several miRNA-based therapeutic strategies are used to modify miRNA expression as therapeutic approaches; one of miRNA modulators is physical training, as already

**72**

ET, especially aerobic exercise, plays a protective role in the cardiovascular system and has been used as a non-pharmacological therapeutic tool for the prevention and treatment of CVD. Although much knowledge already exists, there is insufficient evidence to establish a direct link between epigenetic modulations and changes, caused by exercise, in the heart and blood vessels [42]. There is currently evidence that the protective effects of aerobic ET involve changes in pattern expression of specific miRNAs by positively regulating cardiovascular remodeling. In fact, miRNAs play important roles in the regulation of distinct processes in mammals, and although they exhibit limited complementarity with their target RNAs, it is still sufficient for them to regulate various physiological processes, including cell proliferation, differentiation, migration, angiogenesis, apoptosis, tissue development, and remodeling [14, 136, 137]. However, new studies to understand the pathways and mechanisms in which the ET acts on the miRNAs are required. Thus, studies with miRNAs hope to broaden the perspective of its use in the correction of pathological processes from miRNAs therapy, contributing as a therapeutic intervention in cardiovascular damages as well as for the survival and quality of life of patients.

### **Acknowledgements**

The researchers were supported by Sao Paulo Research Foundation (FAPESP: 2016/26156-5, 2015/22814-5 and 2015/17275-8), USP/PRP-NAPmiR, National Council for Scientific and Technological Development (CNPq: 307591/2009-3 and 159827/2011-6), and Coordination for the Improvement of Higher Education Personnel (CAPES-Proex).

### **Conflict of interest**

There are no conflicts of interest.

### **Notes/Thanks/Other declarations**

Thanks to the support of data basis Smart Servier Medical Art in the design of the figures by authors (https://smart.servier.com/image-set-download/).

*Muscle Cells - Recent Advances and Future Perspectives*

### **Author details**

Noemy Pereira, Camila Gatto, Edilamar Menezes de Oliveira and Tiago Fernandes\* Laboratory of Biochemistry and Molecular Biology of the Exercise, Physical Education and Sports School, University of Sao Paulo, Sao Paulo, Brazil

\*Address all correspondence to: tifernandes@usp.br

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**75**

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S2468054017300483

s00109-011-0840-5

CS20171463

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**Author details**

Noemy Pereira, Camila Gatto, Edilamar Menezes de Oliveira and Tiago Fernandes\* Laboratory of Biochemistry and Molecular Biology of the Exercise, Physical Education and Sports School, University of Sao Paulo, Sao Paulo, Brazil

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: tifernandes@usp.br

provided the original work is properly cited.

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Section 2

Muscle Disorder:

Inflammation-Induced

Section 2
