**2.1. Basis of the regulation of cardiac mitochondrial function**

#### *2.1.1. Cardiac energy production and metabolism*

The heart relies mainly on mitochondrial metabolism to provide most of its energy. The heart has the largest demand for energy among all organs, since it beats continuously from its formation in the fetus until death, and thus cardiomyocytes contain the highest concentration of mitochondria in the body in order to meet its energy requirements [1]. Several interacting bioenergetic pathways contribute to energy metabolism of cardiac muscle including pyruvate oxidation, the tricarboxylic acid (TCA) cycle, the mitochondrial fatty acids oxidation (FAO), and oxidative phosphorylation (OXPHOS), which generates 80–90% of cellular ATP [2]. While the oxidation of pyruvate takes place in the cytosol, the other procedures occur in the mitochondria.

In the normal heart tissue, the supply of ATP from glycolytic mechanism is limited [2]. Fatty acids are the primary energy substrates used to produce ATP in cardiac muscle by OXPHOS, utilizing the carnitine shuttle to transport the fatty acids into the mitochondria. The heart also maintains stored high-energy phosphates, such as creatine phosphate (CP), that are produced from creatine by mitochondrial creatine kinase (mitoCK) using ATP from the closely associated adenine nucleotide translocase (ANT) and mitochondrial ATP synthase [2].

Additionally, the heart is a well vascularized organ, allowing for delivery of freshly oxygenated blood and quick removal of the waste products of metabolism. This constant supply of oxygen is important for OXPHOS to take place, as oxygen serves as the final electron acceptor in the ETC. Understanding the factors involved in the development and function of mitochondrial energy production pathways is increasingly important due to the many diseases associated with defects in this machinery.

Energy production within the cardiomyocytes of the heart is influenced by genetic factors as well as environmental factors. nDNA and mtDNA affect the enzymes and their cofactors as well as the availability of substrates to the mitochondria from their surroundings, which further influence OXPHOS. Cardiac tissue has specific gene regulations to meet its physiological and developmental needs. For example, the ATP synthase β-subunit is expressed at higher levels in cardiomyocyte-differentiated cells compared to control cells [3], and some isoforms of enzymes, e.g., cardiac specific isoforms of cytochrome c oxidase subunits VIa, VIIa, and VIII, are differentially expressed across tissues [4].

Besides the expression and function of the main proteins associated with the OXPHOS, the component of the ETC complexes I-IV and ATP synthase (complex V), many other molecules have been found to be involved in the regulation of the mitochondrial energy production through posttranslational modification. For example, proteins within the mitochondrial complexes can be nitrosylated (the addition of an NO group) or *O*-GlcNAcylated (the addition of *O*-linked β-*N*-acetylglucosamine (*O*-GlcNAc)) [5, 6]. These protein modifications modulate the activity of the complexes and hence change the efficiency of the mitochondria to meet the physiological function of the heart. In addition, our recent studies have also found a specific cell survival-promoting signaling that plays an important regulatory role in promoting ETC efficiency in cardiomyocytes, remarkably under the cardiac stress [7–9]. In particular, we found that this signaling pathway, which includes the heat shock protein 22(Hsp22), AKT, and valosin-containing protein (VCP), promotes ETC efficiency in cardiomyocyte through the increase of mitochondrial inducible nitric oxide synthase (iNOS) [7–9].

#### *2.1.2. Modulation of calcium signaling*

Mitochondrial abnormalities include impaired mitochondrial electron transport chain (ETC) activity, increased formation of ROS, shifted metabolic substrate utilization, aberrant mitochondrial dynamics, and altered ion homeostasis. Some of the mitochondrial abnormalities may have a genetic basis due to the changes of mitochondrial DNA (mtDNA) or the mutation of specific nuclear DNA (nDNA), while other abnormalities are due to environmental cardiotoxic insult or uncharacterized reasons. Although many specific mitochondrial targets have proven to be promising therapeutic strategies in experimental studies, most of them are pending for validation through clinical trials. Better understanding the molecular mechanism of mitochondria in cardiac pathology is important to provide diagnosis and treatment

In order to better understand the role that the mitochondrion plays in the heart, we provide in this chapter a brief background describing the regulation and function of mitochondria during normal cardiac development and aging as well as the pathological mechanisms involved in cardiac diseases. We also address the mitochondrial abnormalities–based diagnosis and

Mitochondria have long been described as the powerhouses of the cell. They are responsible for the generation of ATP, the main energy currency of the cell, while playing important roles in intracellular signaling, activation of apoptosis, and other mechanisms. Little information is currently available on mitochondrial function in the normal human heart as most of the studies on the role of mitochondria have relied on animal models, which may not be representative of the human. However, the development of new methods to study mitochondrial function provides an opportunity to use the small amount of tissue available from surgeries to understand mitochondrial function. In the near future, we expect more studies to be developed utilizing these techniques.

The heart relies mainly on mitochondrial metabolism to provide most of its energy. The heart has the largest demand for energy among all organs, since it beats continuously from its formation in the fetus until death, and thus cardiomyocytes contain the highest concentration of mitochondria in the body in order to meet its energy requirements [1]. Several interacting bioenergetic pathways contribute to energy metabolism of cardiac muscle including pyruvate oxidation, the tricarboxylic acid (TCA) cycle, the mitochondrial fatty acids oxidation (FAO), and oxidative phosphorylation (OXPHOS), which generates 80–90% of cellular ATP [2]. While the oxidation of pyruvate takes place in the cytosol, the other procedures occur in the mitochondria. In the normal heart tissue, the supply of ATP from glycolytic mechanism is limited [2]. Fatty acids are the primary energy substrates used to produce ATP in cardiac muscle by OXPHOS, utilizing the carnitine shuttle to transport the fatty acids into the mitochondria. The heart also maintains stored high-energy phosphates, such as creatine phosphate (CP), that are produced

of mitochondrial-based cardiac diseases.

32 Mitochondrial Diseases

therapeutic options available in heart disease.

**2. The role of mitochondria in the normal heart**

**2.1. Basis of the regulation of cardiac mitochondrial function**

*2.1.1. Cardiac energy production and metabolism*

Ca2+ concentration is highly regulated in the myocardium and is responsible for the induction and intensity of contraction in the myocytes [10]. Mitochondria are able to modulate the Ca2+ concentration in the cardiomyocyte, which plays an important role in the cardiac function [11].

Mitochondria can directly decrease the Ca2+ concentration in the cytosol of the cell by importing Ca2+ via the mitochondrial Ca2+ uniporter. Reciprocally, they can also increase the Ca2+ concentration in the cytosol by expelling calcium stored within the mitochondria through Na+ /Ca2+ or H+ /Ca2+ exchangers [12]. This elaborate system of channels and transporters allows for physiological responses to cytosolic calcium signals and the loading of Ca2+ in the mitochondrial matrix. Mitochondria partake in the cardiac excitation-contraction coupling (ECC) by storing Ca2+, responding to cytosolic calcium signals and generating the ATP required for cardiac contraction. Ca2+ influx via L-type Ca2+ channels triggers further release of Ca2+ from the sarcoplasmic reticulum (SR), which binds to troponin C, and allows for the myosin and actin filaments to interact [10]. During diastole, the Ca2+ either goes back into the SR or is exported out of the cell via the Na+ /Ca2+ exchanger [13]. An increase in workload, as triggered by β-adrenergic stimulation, increases the number of Ca2+ transients as well as the size of the transients, leading to stronger cardiac contractions [14]. Additionally, mitochondria can also indirectly contribute to Ca2+ regulation by inducing changes in the concentration of ATP, NAD(P)H, pyruvate, and ROS, which in turn regulate other Ca2+ signaling machinery components [15]. This associated Ca2+ signaling is involved in the Ca2+ buffering, the Ca2+ release from internal stores and the influx from the extracellular solution, the Ca2+ uptake into cellular organelles, and the extrusion by plasma membrane Ca2+ pumps [16].

three superoxide dismutase (SOD) isoforms with precise subcellular compartmentalization: the Cu,Zn-dependent isoform (Cu,Zn SOD, SOD1) is found in the cytosol; the Mn-dependent isoform (Mn SOD, SOD2) is located in the mitochondrial matrix; and Cu,Zn SOD is located in the extracellular space (ecSOD, SOD3) [22]. Mitochondrial ROS have emerged as an impor-

·− is the proximal mitochondrial ROS and is produced by the one-electron reduction of oxygen

ical conditions, the balance between ROS generation and ROS scavenging is highly controlled. ROS generation can initiate diverse cellular responses, which include triggering signaling pathways involved in cell protection, initiating coordinated activation of mitochondrial fission and autophagy to optimize removal of abnormal mitochondria and cells, and ensuring that the damage does not spread to neighboring mitochondria and cells [21]. Both high levels of ROS (oxidative stress) and excessively low levels of ROS (reductive stress) are harmful and may play causative roles in the pathologies related to the dramatic change of redox environment [21]. Excess ROS production in the heart under pathophysiological conditions leads to mitochondrial dysfunction and bioenergetic decline and contributes to a number of cell pathologies in the heart. For example, ROS is favored by high membrane potential, low ATP formation, and hampering the flow of electrons through the complexes in cardiomyocytes. In addition, ROS formation is the result of the uncoupling of respiration as seen during the opening of the mPTP [21].

reliable methods that can be used to measure the mitochondrial ROS production *in vivo* [24].

occur on antimycin A binding may be responsible for the production of ROS [21].

ing is a key essential mechanism for cardiomyocyte survival and function.

The molecular mechanisms of ROS generation in the cardiac mitochondrion remain unclear. It has been showed that complex I (NADH-ubiquinone oxidoreductase) is the main source of ROS in the mitochondrion. However, the ROS production at complex I is high under pathological conditions, not physiological condition [21]. Further mechanistic studies suggest that the major site of ROS production in complex I is either upstream of a rotenone-binding site or tightly coupled to the increased level of NAD(P)H after rotenone supplementation [21]. ROS production at complex II is low at physiological concentrations of succinate, suggesting that complex II is not a key contributor to the mitochondrial ROS. ROS production at complex III only occurs after the binding of antimycin A, suggesting that conformational changes that

Mitochondria can mediate cell death through the opening or activation of the mPTP [25]. The mPTP is a high conductance channel that generates a sudden increase in inner mitochondrial membrane (IMM) permeability to ions and small solutes when opened [26, 27]. The pore is regulated by the concentration of Ca2+, ADP, NADH, and ROS. Regulation of the mPTP open-

Intense research efforts have been focused on elucidating the molecular components of the mPTP. The original mPTP model hypothesized that the channel comprised these principal proteins: cyclophilin D (CyPD), located in the mitochondrial matrix; the ANT, found in the inner membrane; the voltage-dependent anion channel (VDAC) in the outer membrane [28];

·− production takes place at redox-active prosthetic groups within pro-

·− formation [23]. Under physiolog-

Mitochondria and Heart Disease

35

http://dx.doi.org/10.5772/intechopen.72611

·− produced in isolated mitochondria, there are few

tant mechanism of disease and redox signaling in the cardiovascular system.

teins where the kinetic factors are key to the production of O2

Although many studies have detected O2

O2

[23]. Mitochondrial O2

*2.1.4. mPTP opening*

Calcium signaling in the mitochondria also contributes to the regulation of cellular energy metabolism. ATP is hydrolyzed to ADP in order to power energy-requiring processes and is shuttled into the mitochondria to be reconverted into ATP as a final step in respiration. This enhances the electron flux within the ETC, resulting in the oxidation of NADH to NAD<sup>+</sup> . Concurrently, Ca2+ is transferred into the mitochondria through the mitochondrial Ca2+ uniporter (MCU), activating the enzymes of the Krebs cycle to adjust NADH regeneration to match its oxidation [14]. In addition, excessive mitochondrial Ca2+ uptake and Ca2+ accumulation, irreversible ΔΨ collapse, ATP depletion, and oxidative stress contribute to the opening of the mPTP [17].

Type 2 ryanodine receptors (RyR2s) and type 2 inositol 1,4,5-trisphosphate receptors (IP3R2s) are Ca2+ release channels found on cardiac SR. Recent studies have demonstrated that leaky RyR2 channels, but not IP3R2, contribute to mitochondrial Ca2+ overload and dysfunction in heart failure (HF) [11]. NO signaling and its downstream effectors such as *S*-nitrosylation have also been shown to be key processes in regulating calcium signaling. The neuronal nitric oxide synthase (nNOS or NOS1) has been linked to the reduction of calcium influx through the L-type Ca2+ channel [5, 18]. This decrease in Ca2+ influx may be responsible for the cardioprotection induced by NO. Furthermore, decreased *S*-nitrosylation of key SR Ca2+ handling proteins such as the RyR2s due to impaired NOS1 can result in increased Ca2+ mediated ventricular arrhythmia in the setting of elevated myocardia [Ca2+] i [19]. Inhibition of *S*-nitrosylation of the SR Ca2+ ATPase (SERCA) has been associated with lower Ca2+ uptake in the SR and impaired myocardial relaxation [20].

While substantial efforts were undertaken to characterize the kinetic properties of mitochondrial calcium cycling, the experimental approaches and techniques have not been able to reach explicit conclusions on cardiac mitochondrial responses to cytosolic Ca2+ oscillations during each heartbeat. However, it is widely accepted that Ca2+ is a second messenger for the regulation of mitochondrial tasks and represents a crucial link for the role of mitochondria for excitation-metabolism and excitation-contraction coupling in the heart.
