Therapeutic Approaches for Muscular Disorder

**3**

**Chapter 1**

Muscle

**Abstract**

combination therapy

**1. Introduction**

**1.1 Pathology of HF**

on the pathophysiology of skeletal muscle.

Exercise Therapy for Patients

with Heart Failure: Focusing on

the Pathophysiology of Skeletal

*Nobuo Morotomi, Kunihiro Sakuma and Kotomi Sakai*

In patients with heart failure (HF), it is important to perform exercise therapy with a focus on the pathophysiology of skeletal muscle. Patients with HF have multiple clinical symptoms due to cardiac dysfunction. Recent studies demonstrated the mechanism and treatment strategy for HF, and multiple signaling pathways involved in HF result in reduced exercise capacity and skeletal muscle mass. On the other hand, exercise therapy for HF is known to inhibit the inflammatory cytokines and neurohumoral factors, and increase muscle mass. Therefore, in this chapter, we discuss the importance of exercise therapy for HF, with a focus

**Keywords:** heart failure, skeletal muscle, muscle abnormality, exercise training,

HF is a condition characterized by cardiac decompensation due to organic or functional impaired pumping capacity. Reduced exercise capacity in patients with HF leads to numerous symptoms such as breath shortness, dyspnea, general fatigue, and edema in the foot [1]. One of the causes of reduced exercise capacity is the condition of HF. According to the JCS 2017/JHFS 2017 guidelines on the diagnosis and treatment of acute and chronic HF [1], HF is categorized into three groups by the left ventricular ejection fraction (LVEF). One is HF with LVEF <40%, termed heart failure with reduced ejection fraction (HFrEF). HFrEF develops due to left ventricular systolic dysfunction. HFrEF is a leading cause of ischemic heart disease and coronary sclerosis. Another group is HF with LVEF ≥50%, termed heart failure with preserved ejection fraction (HFpEF). HFpEF is known as diastolic dysfunction in HF. Diastolic dysfunction is mainly caused by hypertension. The last group is HR with LVEF from 40–49%, termed heart failure with mid-range ejection fraction (HFmrEF). HFmrEF can develop in the recovery process from HFrEF, but many other factors can cause HFmrEF. For example, arrhythmia with tachycardia, such

#### **Chapter 1**

## Exercise Therapy for Patients with Heart Failure: Focusing on the Pathophysiology of Skeletal Muscle

*Nobuo Morotomi, Kunihiro Sakuma and Kotomi Sakai*

### **Abstract**

In patients with heart failure (HF), it is important to perform exercise therapy with a focus on the pathophysiology of skeletal muscle. Patients with HF have multiple clinical symptoms due to cardiac dysfunction. Recent studies demonstrated the mechanism and treatment strategy for HF, and multiple signaling pathways involved in HF result in reduced exercise capacity and skeletal muscle mass. On the other hand, exercise therapy for HF is known to inhibit the inflammatory cytokines and neurohumoral factors, and increase muscle mass. Therefore, in this chapter, we discuss the importance of exercise therapy for HF, with a focus on the pathophysiology of skeletal muscle.

**Keywords:** heart failure, skeletal muscle, muscle abnormality, exercise training, combination therapy

#### **1. Introduction**

#### **1.1 Pathology of HF**

HF is a condition characterized by cardiac decompensation due to organic or functional impaired pumping capacity. Reduced exercise capacity in patients with HF leads to numerous symptoms such as breath shortness, dyspnea, general fatigue, and edema in the foot [1]. One of the causes of reduced exercise capacity is the condition of HF. According to the JCS 2017/JHFS 2017 guidelines on the diagnosis and treatment of acute and chronic HF [1], HF is categorized into three groups by the left ventricular ejection fraction (LVEF). One is HF with LVEF <40%, termed heart failure with reduced ejection fraction (HFrEF). HFrEF develops due to left ventricular systolic dysfunction. HFrEF is a leading cause of ischemic heart disease and coronary sclerosis. Another group is HF with LVEF ≥50%, termed heart failure with preserved ejection fraction (HFpEF). HFpEF is known as diastolic dysfunction in HF. Diastolic dysfunction is mainly caused by hypertension. The last group is HR with LVEF from 40–49%, termed heart failure with mid-range ejection fraction (HFmrEF). HFmrEF can develop in the recovery process from HFrEF, but many other factors can cause HFmrEF. For example, arrhythmia with tachycardia, such

as atrial fibrillation, places strain on the left heart and causes HFmrEF. The exact mechanism of HFmrEF remains unclear.

Reduced exercise capacity in patients with HF can also cause organic and functional abnormality of skeletal muscle not only due to abnormal LVEF. Patients with HF can lose weight due to these abnormalities of skeletal muscle. A previous study reported that weight loss of 7.5% over six months was an independent predictor of long-term mortality in patients with HF [2]. In addition to the condition of HR and its related skeletal muscle abnormalities, reduced exercise capacity develops slowly with age. On the other hand, in 1998, Rosenberg proposed the term "sarcopenia" to describe age-related muscle decrease. Elderly patients with HF are included in "secondary sarcopenia" and advanced HF patients are defined as having "cardiac cachexia". The Asian definition of sarcopenia was established by the Asian Working Group for Sarcopenia in 2014 [3]. Sarcopenia is diagnosed by a low muscle mass and low muscle strength or low physical performance. Sarcopenia develops due to aging, undernutrition, sedentary lifestyle, and progression of inflammatory diseases, cancer, and chronic diseases such as HF and COPD. The reported prevalence of sarcopenia is between 4.1% and 11.5% in Asian older people. On the other hand, in 2013, Fülster reported a prevalence of 19.4% among 200 ambulatory patients with stable chronic HF in Germany [4].

The exact mechanism of the development of sarcopenia related to HF is unclear. However, previous studies noted specific changes in skeletal muscle in patients with HF. Kinugawa et al. reported that patients with HF have skeletal muscle abnormalities, including energy dysbolisms, fiber transformation from type 1 (*slow*-twitch muscle fibers) to type 2 (*fast*-twitch muscle fibers), and mitochondrial dysfunction [5]. Brown et al. demonstrated decreases in mitochondrial density, gene expression, and oxygenated capacity in skeletal muscle in patients with HF [6]. Using animal studies, Takada et al. found that chronic *heart failure (*CHF) model mice have a low level of protein expression of phosphorylated AMPKα, sirtuin-1, PGC-1, and mitochondrial transcription factor A (Tfam) in skeletal muscle [7].

In this review, we describe the molecular mechanisms in skeletal muscle and the treatment for HF.

#### **2. Molecular mechanisms of HF**

This section presents three representative skeletal muscle mechanisms unique to HF that cause organic and functional physical abnormalities.

#### **2.1 Inflammatory cytokines**

Inflammatory cytokines are biologically active substances that exert a variety of functions by signaling through specific receptors on the cell surface. Inflammatory cytokines include interleukin (IL)-6, interferon, tumor necrotic factor (TNF) family, and colony-stimulating factor. The excess secretion of inflammatory cytokines due to HF disrupts the anabolic catabolic balance of skeletal muscle in HF patients. Impaired cardiac cell and vascular endothelial cells with high stress result in the secretion of inflammatory cytokines and myocardial infarction. Levine et al. reported that HF patients have a high circulating concentration of TNF-α [8].

In 2001, Reid et al. reported that secretion of TNF-α increased the production of reactive oxygen species through the mitochondrial electron transport system, resulting in the activated ubiquitin-proteasome system through the NF-κB signaling pathway [9]. This study suggested progressive skeletal muscle wasting through these systems in HF patients. In addition, Chojkier et al. reported that

**5**

of TGF-β.

**2.3 Autophagy**

*Exercise Therapy for Patients with Heart Failure: Focusing on the Pathophysiology of Skeletal…*

TNF-α-injected mice had increased expression of nitric oxide synthase activity and the expression of their albumin synthesis genes was downregulated [10]. Schaap et al. reported that *high* expression of TNF-α and IL-6 led to a reduction of the quadriceps muscle area and grip strength in older people [11]. Langhaus et al. reported that the secretion of TNF-α induced appetite loss [12]. Saitoh et al. found that appetite loss caused malnutrition and resulted in the inhibition of protein anabolic action. Moreover, appetite loss was associated with a poor prognosis in HF patients in their study [13]. On the other hand, Hollriegel et al. reported that HF patients did not have high expression of atrogin-1 mRNA or protein in skeletal muscle [14]. Of note, there are few studies on this topic in human subjects [15]. Although TNF-α and IL-6 activity affect muscle abnormalities in HF patients, it remains unclear how

The accelerated renin*-*angiotensin*-*aldosterone (RAA) system leads to skeletal muscle dysfunction in HF patients. Angiotensinogen is produced from the liver and is the first substrate of the RAA system. Thereafter, renin from the juxtaglomerular apparatus of the kidney is released into the blood. Angiotensin І is produced from angiotensinogen by activated renin. Then, angiotensin ІІ is produced from angiotensin І by angiotensin-converting enzyme. Angiotensin ІІ causes vasoconstriction and stimulates aldosterone secretion through *angiotensin* ІІ *type 1 receptor.* Angiotensin ІІ causes an accumulation of fibrosis in skeletal muscle through transforming growth factor-β (TGF-β)-dependent signaling [16]. Activation of the RAA system in HF patients causes the overproduction of angiotensin ІІ. Their RAA system is activated regardless of the severity of HF [17]. This study also revealed that their renin activity is significantly higher than that in those without HF

(3.0 ± 3.7 and 1.2 ± 1.2 ng/mL). In 2007, Cohn et al. investigated the effects of TGF-β neutralizing antibodies and angiotensin-receptor blockers (ARB) on skeletal muscle function. In this study, the intervention group using ARB had significantly reduced TGF-β signaling through angiotensin ІІ and improved skeletal muscle function [18]. TGF-β is also known as a factor that elicits apoptosis of muscle satellite cells during the process of repairing damaged skeletal muscles. The author suggested that inhibition of the angiotensin ІІ type1 receptor by ARB led to the low expression

Fukushima et al. demonstrated that the phosphorylation of Akt (p-Akt) decreased in skeletal muscle from mice with HF after myocardial infarction and this decrease was caused by an increase in plasma angiotensin ІІ [19]. Another study using HF model mice found that angiotensin ІІ directly induced skeletal muscle abnormalities [20]. This study reported a significant decrease in the amount of p-Akt protein in angiotensin ІІ-treated mice. In addition, angiotensin ІІ induced fiber transformation from type І to type ІІ, skeletal muscle atrophy, and weight loss. Based on these involved factors, the RAA system is considered to elicit skeletal muscle dysfunction in HF. In the future, studies on increased protein catabolism by

Autophagy is an intracellular system that delivers cytoplasmic substrates to lysosomes for subsequent degradation and removal. Autophagy is divided into three types depending on the mechanisms; macroautophagy, microautophagy, and chaperone-mediated autophagy. The research area of macroautophagy is the most advanced of these three types. Fasting induces the autophagy system and

inflammatory cytokine families influence each other in humans.

*DOI: http://dx.doi.org/10.5772/intechopen.97291*

**2.2 Renin***–***angiotensin***–***aldosterone system**

the RAA system need to be conducted in humans.

*Exercise Therapy for Patients with Heart Failure: Focusing on the Pathophysiology of Skeletal… DOI: http://dx.doi.org/10.5772/intechopen.97291*

TNF-α-injected mice had increased expression of nitric oxide synthase activity and the expression of their albumin synthesis genes was downregulated [10]. Schaap et al. reported that *high* expression of TNF-α and IL-6 led to a reduction of the quadriceps muscle area and grip strength in older people [11]. Langhaus et al. reported that the secretion of TNF-α induced appetite loss [12]. Saitoh et al. found that appetite loss caused malnutrition and resulted in the inhibition of protein anabolic action. Moreover, appetite loss was associated with a poor prognosis in HF patients in their study [13]. On the other hand, Hollriegel et al. reported that HF patients did not have high expression of atrogin-1 mRNA or protein in skeletal muscle [14]. Of note, there are few studies on this topic in human subjects [15]. Although TNF-α and IL-6 activity affect muscle abnormalities in HF patients, it remains unclear how inflammatory cytokine families influence each other in humans.

#### **2.2 Renin***–***angiotensin***–***aldosterone system**

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

mechanism of HFmrEF remains unclear.

stable chronic HF in Germany [4].

**2. Molecular mechanisms of HF**

**2.1 Inflammatory cytokines**

treatment for HF.

as atrial fibrillation, places strain on the left heart and causes HFmrEF. The exact

Reduced exercise capacity in patients with HF can also cause organic and functional abnormality of skeletal muscle not only due to abnormal LVEF. Patients with HF can lose weight due to these abnormalities of skeletal muscle. A previous study reported that weight loss of 7.5% over six months was an independent predictor of long-term mortality in patients with HF [2]. In addition to the condition of HR and its related skeletal muscle abnormalities, reduced exercise capacity develops slowly with age. On the other hand, in 1998, Rosenberg proposed the term "sarcopenia" to describe age-related muscle decrease. Elderly patients with HF are included in "secondary sarcopenia" and advanced HF patients are defined as having "cardiac cachexia". The Asian definition of sarcopenia was established by the Asian Working Group for Sarcopenia in 2014 [3]. Sarcopenia is diagnosed by a low muscle mass and low muscle strength or low physical performance. Sarcopenia develops due to aging, undernutrition, sedentary lifestyle, and progression of inflammatory diseases, cancer, and chronic diseases such as HF and COPD. The reported prevalence of sarcopenia is between 4.1% and 11.5% in Asian older people. On the other hand, in 2013, Fülster reported a prevalence of 19.4% among 200 ambulatory patients with

The exact mechanism of the development of sarcopenia related to HF is unclear. However, previous studies noted specific changes in skeletal muscle in patients with HF. Kinugawa et al. reported that patients with HF have skeletal muscle abnormalities, including energy dysbolisms, fiber transformation from type 1 (*slow*-twitch muscle fibers) to type 2 (*fast*-twitch muscle fibers), and mitochondrial dysfunction [5]. Brown et al. demonstrated decreases in mitochondrial density, gene expression, and oxygenated capacity in skeletal muscle in patients with HF [6]. Using animal studies, Takada et al. found that chronic *heart failure (*CHF) model mice have a low level of protein expression of phosphorylated AMPKα, sirtuin-1, PGC-1, and

In this review, we describe the molecular mechanisms in skeletal muscle and the

This section presents three representative skeletal muscle mechanisms unique to

Inflammatory cytokines are biologically active substances that exert a variety of functions by signaling through specific receptors on the cell surface. Inflammatory cytokines include interleukin (IL)-6, interferon, tumor necrotic factor (TNF) family, and colony-stimulating factor. The excess secretion of inflammatory

cytokines due to HF disrupts the anabolic catabolic balance of skeletal muscle in HF patients. Impaired cardiac cell and vascular endothelial cells with high stress result in the secretion of inflammatory cytokines and myocardial infarction. Levine et al. reported that HF patients have a high circulating concentration of TNF-α [8]. In 2001, Reid et al. reported that secretion of TNF-α increased the production of reactive oxygen species through the mitochondrial electron transport system, resulting in the activated ubiquitin-proteasome system through the NF-κB signaling pathway [9]. This study suggested progressive skeletal muscle wasting through these systems in HF patients. In addition, Chojkier et al. reported that

mitochondrial transcription factor A (Tfam) in skeletal muscle [7].

HF that cause organic and functional physical abnormalities.

**4**

The accelerated renin*-*angiotensin*-*aldosterone (RAA) system leads to skeletal muscle dysfunction in HF patients. Angiotensinogen is produced from the liver and is the first substrate of the RAA system. Thereafter, renin from the juxtaglomerular apparatus of the kidney is released into the blood. Angiotensin І is produced from angiotensinogen by activated renin. Then, angiotensin ІІ is produced from angiotensin І by angiotensin-converting enzyme. Angiotensin ІІ causes vasoconstriction and stimulates aldosterone secretion through *angiotensin* ІІ *type 1 receptor.* Angiotensin ІІ causes an accumulation of fibrosis in skeletal muscle through transforming growth factor-β (TGF-β)-dependent signaling [16]. Activation of the RAA system in HF patients causes the overproduction of angiotensin ІІ. Their RAA system is activated regardless of the severity of HF [17]. This study also revealed that their renin activity is significantly higher than that in those without HF (3.0 ± 3.7 and 1.2 ± 1.2 ng/mL). In 2007, Cohn et al. investigated the effects of TGF-β neutralizing antibodies and angiotensin-receptor blockers (ARB) on skeletal muscle function. In this study, the intervention group using ARB had significantly reduced TGF-β signaling through angiotensin ІІ and improved skeletal muscle function [18]. TGF-β is also known as a factor that elicits apoptosis of muscle satellite cells during the process of repairing damaged skeletal muscles. The author suggested that inhibition of the angiotensin ІІ type1 receptor by ARB led to the low expression of TGF-β.

Fukushima et al. demonstrated that the phosphorylation of Akt (p-Akt) decreased in skeletal muscle from mice with HF after myocardial infarction and this decrease was caused by an increase in plasma angiotensin ІІ [19]. Another study using HF model mice found that angiotensin ІІ directly induced skeletal muscle abnormalities [20]. This study reported a significant decrease in the amount of p-Akt protein in angiotensin ІІ-treated mice. In addition, angiotensin ІІ induced fiber transformation from type І to type ІІ, skeletal muscle atrophy, and weight loss. Based on these involved factors, the RAA system is considered to elicit skeletal muscle dysfunction in HF. In the future, studies on increased protein catabolism by the RAA system need to be conducted in humans.

#### **2.3 Autophagy**

Autophagy is an intracellular system that delivers cytoplasmic substrates to lysosomes for subsequent degradation and removal. Autophagy is divided into three types depending on the mechanisms; macroautophagy, microautophagy, and chaperone-mediated autophagy. The research area of macroautophagy is the most advanced of these three types. Fasting induces the autophagy system and

the isolation membrane is formed. The isolation membrane elongates, engulfing protein aggregates and organelles within the cytoplasm, and finally forms doublemembraned structures called autophagosomes. Autophagosomes subsequently fuse with lysosomes to degrade their cargo by lysosomal enzymes.

Autophagy influences the muscle abnormalities in HF. There are two mechanisms. First, a maladaptive response for autophagy exacerbates the condition of HF, resulting in reduced muscle function. For example, the decrease in Beclin 1 expression weakens the macroautophagy system in patients with ischemic cardiac myopathy [21]. In contrast, Zhu et al. reported that pressure overload in mice markedly increased cardiac autophagy and load-induced autophagic activity remained significantly high for at least 3 weeks [22]. This study reported that Beclin 1 overexpression increased autophagic activity and promoted pathological remodeling. Using mice with pressure-overload heart failure, this study revealed that lysosome abundance calculated by measuring the lysosomal markers LAMP-1 and cathepsin D increased in wild-type hearts and to a greater extent in Beclin 1 transgenic hearts. Another study demonstrated that cardiac-specific deficiency of autophagy-related 5, a protein required for autophagy, leads to cardiac hypertrophy in adult mice [23]. Therefore, HF is exacerbated by dysfunctional autophagy and results in skeletal muscle abnormality.

Second, cardiac autophagy may directly cause skeletal muscle atrophy regardless of the progression of HF. Janning et al. investigated the autophagy pathway using myocardial infarction model mice [24]. Their study revealed that although myoatrophy in the soleus muscle and plantaris muscle progressed, the expression levels of autophagic markers, such as GABARAPL-1 and AtG7, increased in the plantaris but not in the soleus muscle. This study provides evidence of autophagy signaling regulation in HF-induced muscle atrophy. In addition, the selective degradation of mitochondria is termed mitophagy. Oka et al. reported that cardiac cells are abundant in mitochondria and dysfunctional mitophagy leads to reduced cardiac function through the inflammation inside the cells [25]. It is also possible that mitophagy causes skeletal abnormalities.

There is increasing evidence supporting a role of autophagy in age-related disease states of the cardiovascular system. Sasaki et al. reviewed autophagy in cardiovascular disease [26]. Their report states that autophagy is related to age-associated cardiovascular diseases, HF, ischemic heart disease, cardiomyopathy, hypertension, and atherosclerosis. However, the mechanisms of skeletal muscle dysfunction caused by autophagy in HF remain unclear.

#### **3. Exercise training as treatment for HF**

Exercise training improved the reduced exercise capacity and skeletal muscle power due to HF in several studies. There are two types of exercise; aerobic exercise and resistance training (RT). Aerobic exercise induced peroxisome proliferatoractivated receptor gamma coactivator 1-alpha (PGC1-α) expression and improved insulin resistance [27]. High-intensity aerobic exercise increases the ratio of type 1 muscle fibers. Gielen et al. investigated how the expression of cathepsin-L, E3 ligases MuRF-1, and MaFbx changed after exercise training among HF patients, and compared them with healthy subjects [28]. As a result, the expression of MuRF-1 in HF patients was significantly higher than that in healthy subjects. In addition, after four weeks of exercise training, the expression of MuRF-1 mRNA in HF patients was reduced by 32.8% and 37.0% in people aged ≤55 years and ≥ 65 years, respectively. In another study, they investigated the expression of inflammatory cytokines (TNF-α, IL-6, and IL-1-β) before and after exercise training in HF patients [29].

**7**

patients [34].

*Exercise Therapy for Patients with Heart Failure: Focusing on the Pathophysiology of Skeletal…*

Exercise training did not affect the serum levels of TNF-α, IL-6, or IL-1-β, but it significantly reduced the expression levels of these cytokines and iNOS (by 52%) in skeletal muscle. Thus, exercise training may reduce the expression of inflammatory

Aerobic exercise increases the exercise capacity, and RT improves skeletal muscle mass and strength. Pu et al. demonstrated the effects of resistance training on muscle function in HF patients [32]. In their study, the improvement of knee extensor muscle power was 43% higher and the six-minute walking distance was 49 m greater in HF patients than those in the control group. Another study also found that RT improved skeletal muscle mass and power more than aerobic exercise in dialysis patients [33]. Multimodal exercise programs, including aerobic exercise, RT, and respiratory muscle training, were reported to significantly improve dyspnea and the quality of life, in addition to quadriceps power and exercise time, in HF

Saitoh et al. suggested that combination therapy of exercise training with standard drugs, such as angiotensin-converting enzyme inhibitors, beta-receptor blockers, ghrelin agonists, and myostatin inhibitors, is better than exercise training and nutritional supplements for treating cardiac sarcopenia [35]. The following section explains the possibility of treatment for HF using combinations of exercise

Sufficient nutritional supplements improve skeletal muscle dysfunction accompanying HF. Aquilani et al. investigated the effects of exercise training with 8 g of daily essential amino acids (EAA) in 21 HF patients [36]. Based on the cardiopulmonary exercise test, the EAA group had no change in oxygen consumption but increased their exercise load. Although there was no significant change in the 6-minute walking distance in the control group, the EAA group increased their walked distance by 74 m on average. Rozentryt et al. reported that the intake of nutritional supplements with a high-calorie (600 kcal) and high-protein (20 g) diet increased the body weight by 2.0 kg at the 6-week follow-up and 2.3 kg at the 18-week follow-up [37]. In this study, oral nutritional supplementation did not affect the albumin concentration or peak oxygen consumption, but reduced the

β-Hydroxy-β-methylbutyrate (HMB) is a metabolite of the amino acid leucine and has a positive effect on muscle protein anabolism. A study using rats reported that HMB supplementation resulted in greater expression of Akt, mTOR, and S6K1 than leucine [38]. Berk et al. investigated the effects of a mixture of HMB, glutamine, and arginine in advanced cancer patients. However, there were no significant differences in the 8-week lean body mass between the placebo and the HMB/Arg/Gln groups [39]. Another study examining chronic pulmonary patients

**3.1 Combination of nutritional supplements and exercise training**

St-Jean-Pelletier et al. investigated myofiber changes and mitochondrial density in the vastus lateralis in healthy subjects [30]. They found an increased ratio of type 2a myofibers and decreased mitochondrial density in people aged ≥65 years with low physical activity. Campos et al. investigated the effects of exercise training on mitochondrial dysfunction using myocardial infarction model mice [31]. This study suggested that the improvement in mitochondrial number, density, and oxygenation by exercise training aid in recovery from cardiac dysfunction. Although exercise training improved mitochondrial dysfunction in HF mice model, the exact

*DOI: http://dx.doi.org/10.5772/intechopen.97291*

cytokines and maintain high-level muscle function.

mechanism in HF patients remains to be elucidated.

training and other therapies (**Figure 1**).

*3.1.1 Amino acids and micronutrients*

serum level of TNF-α.

#### *Exercise Therapy for Patients with Heart Failure: Focusing on the Pathophysiology of Skeletal… DOI: http://dx.doi.org/10.5772/intechopen.97291*

Exercise training did not affect the serum levels of TNF-α, IL-6, or IL-1-β, but it significantly reduced the expression levels of these cytokines and iNOS (by 52%) in skeletal muscle. Thus, exercise training may reduce the expression of inflammatory cytokines and maintain high-level muscle function.

St-Jean-Pelletier et al. investigated myofiber changes and mitochondrial density in the vastus lateralis in healthy subjects [30]. They found an increased ratio of type 2a myofibers and decreased mitochondrial density in people aged ≥65 years with low physical activity. Campos et al. investigated the effects of exercise training on mitochondrial dysfunction using myocardial infarction model mice [31]. This study suggested that the improvement in mitochondrial number, density, and oxygenation by exercise training aid in recovery from cardiac dysfunction. Although exercise training improved mitochondrial dysfunction in HF mice model, the exact mechanism in HF patients remains to be elucidated.

Aerobic exercise increases the exercise capacity, and RT improves skeletal muscle mass and strength. Pu et al. demonstrated the effects of resistance training on muscle function in HF patients [32]. In their study, the improvement of knee extensor muscle power was 43% higher and the six-minute walking distance was 49 m greater in HF patients than those in the control group. Another study also found that RT improved skeletal muscle mass and power more than aerobic exercise in dialysis patients [33]. Multimodal exercise programs, including aerobic exercise, RT, and respiratory muscle training, were reported to significantly improve dyspnea and the quality of life, in addition to quadriceps power and exercise time, in HF patients [34].

Saitoh et al. suggested that combination therapy of exercise training with standard drugs, such as angiotensin-converting enzyme inhibitors, beta-receptor blockers, ghrelin agonists, and myostatin inhibitors, is better than exercise training and nutritional supplements for treating cardiac sarcopenia [35]. The following section explains the possibility of treatment for HF using combinations of exercise training and other therapies (**Figure 1**).

#### **3.1 Combination of nutritional supplements and exercise training**

#### *3.1.1 Amino acids and micronutrients*

Sufficient nutritional supplements improve skeletal muscle dysfunction accompanying HF. Aquilani et al. investigated the effects of exercise training with 8 g of daily essential amino acids (EAA) in 21 HF patients [36]. Based on the cardiopulmonary exercise test, the EAA group had no change in oxygen consumption but increased their exercise load. Although there was no significant change in the 6-minute walking distance in the control group, the EAA group increased their walked distance by 74 m on average. Rozentryt et al. reported that the intake of nutritional supplements with a high-calorie (600 kcal) and high-protein (20 g) diet increased the body weight by 2.0 kg at the 6-week follow-up and 2.3 kg at the 18-week follow-up [37]. In this study, oral nutritional supplementation did not affect the albumin concentration or peak oxygen consumption, but reduced the serum level of TNF-α.

β-Hydroxy-β-methylbutyrate (HMB) is a metabolite of the amino acid leucine and has a positive effect on muscle protein anabolism. A study using rats reported that HMB supplementation resulted in greater expression of Akt, mTOR, and S6K1 than leucine [38]. Berk et al. investigated the effects of a mixture of HMB, glutamine, and arginine in advanced cancer patients. However, there were no significant differences in the 8-week lean body mass between the placebo and the HMB/Arg/Gln groups [39]. Another study examining chronic pulmonary patients

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

with lysosomes to degrade their cargo by lysosomal enzymes.

muscle abnormality.

mitophagy causes skeletal abnormalities.

caused by autophagy in HF remain unclear.

**3. Exercise training as treatment for HF**

the isolation membrane is formed. The isolation membrane elongates, engulfing protein aggregates and organelles within the cytoplasm, and finally forms doublemembraned structures called autophagosomes. Autophagosomes subsequently fuse

Autophagy influences the muscle abnormalities in HF. There are two mechanisms. First, a maladaptive response for autophagy exacerbates the condition of HF, resulting in reduced muscle function. For example, the decrease in Beclin 1 expression weakens the macroautophagy system in patients with ischemic cardiac myopathy [21]. In contrast, Zhu et al. reported that pressure overload in mice markedly increased cardiac autophagy and load-induced autophagic activity remained significantly high for at least 3 weeks [22]. This study reported that Beclin 1 overexpression increased autophagic activity and promoted pathological remodeling. Using mice with pressure-overload heart failure, this study revealed that lysosome abundance calculated by measuring the lysosomal markers LAMP-1 and cathepsin D increased in wild-type hearts and to a greater extent in Beclin 1 transgenic hearts. Another study demonstrated that cardiac-specific deficiency of autophagy-related 5, a protein required for autophagy, leads to cardiac hypertrophy in adult mice [23]. Therefore, HF is exacerbated by dysfunctional autophagy and results in skeletal

Second, cardiac autophagy may directly cause skeletal muscle atrophy regardless of the progression of HF. Janning et al. investigated the autophagy pathway using myocardial infarction model mice [24]. Their study revealed that although myoatrophy in the soleus muscle and plantaris muscle progressed, the expression levels of autophagic markers, such as GABARAPL-1 and AtG7, increased in the plantaris but not in the soleus muscle. This study provides evidence of autophagy signaling regulation in HF-induced muscle atrophy. In addition, the selective degradation of mitochondria is termed mitophagy. Oka et al. reported that cardiac cells are abundant in mitochondria and dysfunctional mitophagy leads to reduced cardiac function through the inflammation inside the cells [25]. It is also possible that

There is increasing evidence supporting a role of autophagy in age-related disease states of the cardiovascular system. Sasaki et al. reviewed autophagy in cardiovascular disease [26]. Their report states that autophagy is related to age-associated cardiovascular diseases, HF, ischemic heart disease, cardiomyopathy, hypertension, and atherosclerosis. However, the mechanisms of skeletal muscle dysfunction

Exercise training improved the reduced exercise capacity and skeletal muscle power due to HF in several studies. There are two types of exercise; aerobic exercise and resistance training (RT). Aerobic exercise induced peroxisome proliferatoractivated receptor gamma coactivator 1-alpha (PGC1-α) expression and improved insulin resistance [27]. High-intensity aerobic exercise increases the ratio of type 1 muscle fibers. Gielen et al. investigated how the expression of cathepsin-L, E3 ligases MuRF-1, and MaFbx changed after exercise training among HF patients, and compared them with healthy subjects [28]. As a result, the expression of MuRF-1 in HF patients was significantly higher than that in healthy subjects. In addition, after four weeks of exercise training, the expression of MuRF-1 mRNA in HF patients was reduced by 32.8% and 37.0% in people aged ≤55 years and ≥ 65 years, respectively. In another study, they investigated the expression of inflammatory cytokines (TNF-α, IL-6, and IL-1-β) before and after exercise training in HF patients [29].

**6**

#### **Figure 1.**

*It is thepossibility of treatment for HF using combinations of exercise training and other therapies. This figure presents the effects of exercise training added by other factors. The square box shows the other factors and the dotted box shows treatment effect. EAA: essential amino acids, HMB: β-Hydroxy-β-methylbutyrate, SPPB: short physical performance battery, RAA inhibitors: Renin-angiotensin-aldosterone inhibitors, SARM: selective androgen receptor modulators.*

described that, in the group receiving pulmonary rehabilitation plus an oral nutritional supplement enriched with HMB, the mean and maximum handgrip and fat free mass significantly increased at 12 weeks [40]. The review of HMB supplementation in humans suggested that this agent has positive effects in patients with chronic pulmonary disease, hip fracture, and AIDS- related and cancer-related cachexia [41], but HF was not mentioned. However, a recent study suggested that a high-protein oral nutritional supplement containing HMB increased the body weight at day 30 in HF patients [42]. As described above, protein supplementation for HF patients increases the body mass and improves muscle function.

In recent years, the role of microelements has gained attention. Magnesium insufficiency increase the risk of HF. Sasiwarang et al. examined the relationship between the onset of HF and magnesium concentration in healthy subjects for 15 years [43]. Serum magnesium was inversely related to the risk of incident of HF. Moreover, HF patients with hypomagnesemia had high levels of IL-6 and von Willebrand factor (VWF). VWF is a marker for endothelial dysfunction and the serum level of VWF in HF patients is high. The author suggested that the magnesium concentration influences the inflammatory reaction. Microelements also play an important role in the treatment of sarcopenia [44]. Magnesium supplementation was reported to possibly improve the functional indices such as quadriceps torque [45]. In addition, the walking speed of healthy elderly women in the magnesium supplementation group became significantly faster than that of those in the control group (the supplementation group:∆ 0.21 ± 0.27 m/s, the control group ∆ 0.14 ± 0.003). However, there are no studies on the effects of combination therapy of exercise training and magnesium supplementation in HF patients. As magnesium is commonly used for the treatment of arrhythmia and HF, it may be useful for the treatment of muscle dysfunction in HF*.*

#### *3.1.2 Ghrelin*

Ghrelin can improve the physical function of patients with HF. Ghrelin is produced in the fundic gland of the stomach, and stimulates *gastric* acid secretion and *motility.* Ghrelin has anabolic, orexigenic, and anti-inflammatory effects [46]. Ghrelin levels are lower in older people, especially in those with sarcopenia [47].

**9**

*Exercise Therapy for Patients with Heart Failure: Focusing on the Pathophysiology of Skeletal…*

A study on anamorelin, a selective ghrelin receptor agonist, demonstrated a significant effect on body weight and food intake, but not on muscle strength in patients with cancer cachexia [48]. Nagaya et al. reported that the injection of synthesized ghrelin (2 μg/kg twice a day for 3 weeks) to HF patients increased the LVEF without adverse events, and increased the peak workload and oxygen consumption during exercise [49]. On the other hand, rikkunshito, a Japanese herbal medicine, is a ghrelin potentiator. Fujitsuka et al. reviewed the promotion of ghrelin activity by rikkunshito [50]. Several clinical trials demonstrated that the administration of rikkunshito increased the plasma ghrelin levels. These studies support the potential use of rikkunshito for improving skeletal muscle function and exercise capacity. However, rikkunshito and dipotassium glycyrrhizinate are structural components of licorice extract. The accumulation of dipotassium glycyrrhizinate in the body may cause pseudo aldosteronism and exacerbate the condition of HF. Thus, rikkunshito should be administered carefully. The satisfactory amount of rikkunshito

Vitamin D administration improves the exercise capacity in HF patients. The role of vitamin D is to maintain homeostatic function of the calcium-phosphorus balance and regulate bone metabolism. In recent years, vitamin D was confirmed to play an essential role in skeletal muscle function. Vitamin D deficiency or mutated vitamin D receptor causes skeletal muscle atrophy [51]. Vitamin D receptors are involved in gene expression in skeletal muscle, and regulate muscle anabolism and metabolism. The receptors act on calcium channels and directly regulate muscle contraction. Therefore, vitamin D deficiency results in lipid accumulation in skeletal muscle and atrophy of type 2 myofibers. Hayakawa et al. reported that the administration of 1α25 (OH)2D3 to human muscle cells inhibited the gene expression of MaFbx and MuRF-1 [52]. Antoniak et al. examined the effects of combination therapy of vitamin D administration and exercise training in comparison with exercise training and vitamin D alone [53]. They found that *lower extremity muscle power* increased more in the combination therapy group than in the exercise training alone group. In addition, the score of the short physical performance battery, skeletal muscle power, and femur density increased more in the combination therapy group than in the vitamin D

On the other hand, a meta-analysis demonstrated that vitamin D administration reduced the levels of TNF-α, CRP, and thyroid hormone, but did not improve exercise performance [54]. Bauer et al. reported the effects of combination therapy using vitamin D and leucine-enriched whey protein on physical function in older people with sarcopenia [55]. The active group (n = 184) received vitamin D at 800 IU, 20 g of whey protein, and 9 g of leucine twice a day for 13 weeks. In the active group, the score for the chair-stand test (1.0 second on average) and muscle mass (0.19 kg on average) significantly improved when compared with the control group. Other several studies using healthy elderly subjects reported the improvement of physical functions using a combination of vitamin D and amino acids, but

Standard therapeutics for HF can improve the skeletal function in HF patients. This section explains the three types of medicines for HF that may be useful for

*DOI: http://dx.doi.org/10.5772/intechopen.97291*

*3.1.3 Vitamin D*

alone group.

not in HF patients.

skeletal muscle.

**3.2 Combination with standard therapeutics for HF**

should be investigated to manage HF effectively and safely.

*Exercise Therapy for Patients with Heart Failure: Focusing on the Pathophysiology of Skeletal… DOI: http://dx.doi.org/10.5772/intechopen.97291*

A study on anamorelin, a selective ghrelin receptor agonist, demonstrated a significant effect on body weight and food intake, but not on muscle strength in patients with cancer cachexia [48]. Nagaya et al. reported that the injection of synthesized ghrelin (2 μg/kg twice a day for 3 weeks) to HF patients increased the LVEF without adverse events, and increased the peak workload and oxygen consumption during exercise [49]. On the other hand, rikkunshito, a Japanese herbal medicine, is a ghrelin potentiator. Fujitsuka et al. reviewed the promotion of ghrelin activity by rikkunshito [50]. Several clinical trials demonstrated that the administration of rikkunshito increased the plasma ghrelin levels. These studies support the potential use of rikkunshito for improving skeletal muscle function and exercise capacity. However, rikkunshito and dipotassium glycyrrhizinate are structural components of licorice extract. The accumulation of dipotassium glycyrrhizinate in the body may cause pseudo aldosteronism and exacerbate the condition of HF. Thus, rikkunshito should be administered carefully. The satisfactory amount of rikkunshito should be investigated to manage HF effectively and safely.

#### *3.1.3 Vitamin D*

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

described that, in the group receiving pulmonary rehabilitation plus an oral nutritional supplement enriched with HMB, the mean and maximum handgrip and fat free mass significantly increased at 12 weeks [40]. The review of HMB supplementation in humans suggested that this agent has positive effects in patients with chronic pulmonary disease, hip fracture, and AIDS- related and cancer-related cachexia [41], but HF was not mentioned. However, a recent study suggested that a high-protein oral nutritional supplement containing HMB increased the body weight at day 30 in HF patients [42]. As described above, protein supplementation

*It is thepossibility of treatment for HF using combinations of exercise training and other therapies. This figure presents the effects of exercise training added by other factors. The square box shows the other factors and the dotted box shows treatment effect. EAA: essential amino acids, HMB: β-Hydroxy-β-methylbutyrate, SPPB: short physical performance battery, RAA inhibitors: Renin-angiotensin-aldosterone inhibitors, SARM: selective* 

In recent years, the role of microelements has gained attention. Magnesium insufficiency increase the risk of HF. Sasiwarang et al. examined the relationship between the onset of HF and magnesium concentration in healthy subjects for 15 years [43]. Serum magnesium was inversely related to the risk of incident of HF. Moreover, HF patients with hypomagnesemia had high levels of IL-6 and von Willebrand factor (VWF). VWF is a marker for endothelial dysfunction and the serum level of VWF in HF patients is high. The author suggested that the magnesium concentration influences the inflammatory reaction. Microelements also play an important role in the treatment of sarcopenia [44]. Magnesium supplementation was reported to possibly improve the functional indices such as quadriceps torque [45]. In addition, the walking speed of healthy elderly women in the magnesium supplementation group became significantly faster than that of those in the control group (the supplementation group:∆ 0.21 ± 0.27 m/s, the control group ∆ 0.14 ± 0.003). However, there are no studies on the effects of combination therapy of exercise training and magnesium supplementation in HF patients. As magnesium is commonly used for the treatment of arrhythmia and HF, it may be useful for the

Ghrelin can improve the physical function of patients with HF. Ghrelin is produced in the fundic gland of the stomach, and stimulates *gastric* acid secretion and *motility.* Ghrelin has anabolic, orexigenic, and anti-inflammatory effects [46]. Ghrelin levels are lower in older people, especially in those with sarcopenia [47].

for HF patients increases the body mass and improves muscle function.

**8**

*3.1.2 Ghrelin*

**Figure 1.**

*androgen receptor modulators.*

treatment of muscle dysfunction in HF*.*

Vitamin D administration improves the exercise capacity in HF patients. The role of vitamin D is to maintain homeostatic function of the calcium-phosphorus balance and regulate bone metabolism. In recent years, vitamin D was confirmed to play an essential role in skeletal muscle function. Vitamin D deficiency or mutated vitamin D receptor causes skeletal muscle atrophy [51]. Vitamin D receptors are involved in gene expression in skeletal muscle, and regulate muscle anabolism and metabolism. The receptors act on calcium channels and directly regulate muscle contraction. Therefore, vitamin D deficiency results in lipid accumulation in skeletal muscle and atrophy of type 2 myofibers. Hayakawa et al. reported that the administration of 1α25 (OH)2D3 to human muscle cells inhibited the gene expression of MaFbx and MuRF-1 [52]. Antoniak et al. examined the effects of combination therapy of vitamin D administration and exercise training in comparison with exercise training and vitamin D alone [53]. They found that *lower extremity muscle power* increased more in the combination therapy group than in the exercise training alone group. In addition, the score of the short physical performance battery, skeletal muscle power, and femur density increased more in the combination therapy group than in the vitamin D alone group.

On the other hand, a meta-analysis demonstrated that vitamin D administration reduced the levels of TNF-α, CRP, and thyroid hormone, but did not improve exercise performance [54]. Bauer et al. reported the effects of combination therapy using vitamin D and leucine-enriched whey protein on physical function in older people with sarcopenia [55]. The active group (n = 184) received vitamin D at 800 IU, 20 g of whey protein, and 9 g of leucine twice a day for 13 weeks. In the active group, the score for the chair-stand test (1.0 second on average) and muscle mass (0.19 kg on average) significantly improved when compared with the control group. Other several studies using healthy elderly subjects reported the improvement of physical functions using a combination of vitamin D and amino acids, but not in HF patients.

#### **3.2 Combination with standard therapeutics for HF**

Standard therapeutics for HF can improve the skeletal function in HF patients. This section explains the three types of medicines for HF that may be useful for skeletal muscle.

#### *3.2.1 Beta-blockers*

Beta-blockers can prevent weight loss in HF patients. Beta-blockers, which have inhibitory action against left ventricular remodeling, are used for the treatment of HF and hypertension. Bisoprolol is a beta one-selective blocker and carvedilol is an alpha-beta blocker, and they are commonly used in the treatment of HF.

Beta-blockers were recently reported to inhibit muscle atrophy. A study using cancer cachexia model mice revealed that the administration of bisoprolol inhibited the loss of skeletal muscle mass [56]. This study also reported that bisoprolol improved physical activity and oral intake. In patients with rectal cancer or small cell carcinoma, the administration of espindolol twice a day improved their life prognosis and weight loss [57].

Clark et al. investigated the effects of the administration of carvedilol on body weight loss in patients with HF [58]. Carvedilol was initially administered at 3.1 mg (twice a day) and later increased to a maximum of 25 mg per dose (twice a day). As a result, the administration of carvedilol resulted in body weight gain (1 kg on average) after one year.

Based on these studies, beta-blockers may maintain skeletal muscle quality and improve skeletal muscle mass in HF patients.

#### *3.2.2 Renin-angiotensin-aldosterone (RAA) inhibitors: angiotensin converting enzyme inhibitors (ACEI)/angiotensin-receptor blockers (ARB)*

RAA inhibitors can improve muscle function in HF patients. Angiotensin converting enzyme inhibitors (ACEI) and angiotensin receptor blockers (ARB) are commonly used for the treatment of hypertension. Saitoh et al. reported the protective action of ACEI for muscle function [59]. Several studies reported that ACEI prevent body weight loss due to HF, and improved muscle power and physical functions [60, 61]. Sumukadas et al. investigated the effects of a combination of ACEI therapy and exercise training on physical functions in older people [62]. However, the 6-minute walk distance, score of the Short Physical Performance Battery, handgrip and quadriceps strength, and QOL at 10-week and 20-week follow-up did not improve in the intervention group. In this study, some bias was considered to have caused the insufficient effects of combination therapy.

RAA inhibitors have direct positive effects on muscle function in HF patients. Thus, high-quality studies examining both RAA inhibitors and exercise training in HF patients are warranted.

#### *3.2.3 Selective androgen receptor modulators (SARM)*

Testosterone may have a positive effect on skeletal muscle mass. On the other hand, serious side effects, such as increased risks of developing prostate cancer and myocardial infarction, have been reported [63]. Therefore, selective androgen receptor repair agents known as selective androgen receptor modulators (SARM) were developed. They exert testosterone-induced muscle mass gain effects with less stimulation of the prostate. SARM may increase skeletal muscle mass in HF patients. The clinical trial SARMsMK-0773 examined their effects on skeletal muscle in female patients with sarcopenia [64]. Muscle mass in the intervention group increased significantly compared with that in the placebo group, but no effects on physical functions or muscle power were observed in this study.

No studies have investigated the effects of combination therapy of SARM and exercise training on skeletal muscle mass and physical function in HF patients.

**11**

Japan

**Author details**

Nobuo Morotomi1

Kawasaki, Kanagawa, Japan

Technology, Tokyo, Japan

\*, Kunihiro Sakuma2

\*Address all correspondence to: uoratubon@gmail.com

provided the original work is properly cited.

1 Department of Rehabilitation medicine, Shin-Yurigaoka General Hospital,

3 Setagaya Memorial Hospital, Department of Rehabilitation Medicine, Tokyo,

© 2021 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,

2 Institute for Liberal Arts, Environment and Society, Tokyo Institute of

and Kotomi Sakai3

*Exercise Therapy for Patients with Heart Failure: Focusing on the Pathophysiology of Skeletal…*

The pathophysiology of skeletal muscle in patients with HF is complex and remains unclear. However, recent studies clarified several points. The mechanisms do not function independently and instead interact with each other. In the management of HF, it is important to assess skeletal muscle and physical functions, and to consider treatment combinations including exercise training, electrical stimulation, medicine, and supplements. Large-scale, high-quality studies are warranted to elucidate the pathophysiology and establish effective treatments for HF patients.

*DOI: http://dx.doi.org/10.5772/intechopen.97291*

I do not have any conflict of interest.

**4. Conclusions**

**Conflict of interest**

*Exercise Therapy for Patients with Heart Failure: Focusing on the Pathophysiology of Skeletal… DOI: http://dx.doi.org/10.5772/intechopen.97291*

### **4. Conclusions**

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

Beta-blockers can prevent weight loss in HF patients. Beta-blockers, which have inhibitory action against left ventricular remodeling, are used for the treatment of HF and hypertension. Bisoprolol is a beta one-selective blocker and carvedilol is an

Beta-blockers were recently reported to inhibit muscle atrophy. A study using cancer cachexia model mice revealed that the administration of bisoprolol inhibited the loss of skeletal muscle mass [56]. This study also reported that bisoprolol improved physical activity and oral intake. In patients with rectal cancer or small cell carcinoma, the administration of espindolol twice a day improved their life

Clark et al. investigated the effects of the administration of carvedilol on body weight loss in patients with HF [58]. Carvedilol was initially administered at 3.1 mg (twice a day) and later increased to a maximum of 25 mg per dose (twice a day). As a result, the administration of carvedilol resulted in body weight gain (1 kg on

Based on these studies, beta-blockers may maintain skeletal muscle quality and

*3.2.2 Renin-angiotensin-aldosterone (RAA) inhibitors: angiotensin converting enzyme inhibitors (ACEI)/angiotensin-receptor blockers (ARB)*

have caused the insufficient effects of combination therapy.

*3.2.3 Selective androgen receptor modulators (SARM)*

RAA inhibitors can improve muscle function in HF patients. Angiotensin converting enzyme inhibitors (ACEI) and angiotensin receptor blockers (ARB) are commonly used for the treatment of hypertension. Saitoh et al. reported the protective action of ACEI for muscle function [59]. Several studies reported that ACEI prevent body weight loss due to HF, and improved muscle power and physical functions [60, 61]. Sumukadas et al. investigated the effects of a combination of ACEI therapy and exercise training on physical functions in older people [62]. However, the 6-minute walk distance, score of the Short Physical Performance Battery, handgrip and quadriceps strength, and QOL at 10-week and 20-week follow-up did not improve in the intervention group. In this study, some bias was considered to

RAA inhibitors have direct positive effects on muscle function in HF patients. Thus, high-quality studies examining both RAA inhibitors and exercise training in

Testosterone may have a positive effect on skeletal muscle mass. On the other hand, serious side effects, such as increased risks of developing prostate cancer and myocardial infarction, have been reported [63]. Therefore, selective androgen receptor repair agents known as selective androgen receptor modulators (SARM) were developed. They exert testosterone-induced muscle mass gain effects with less stimulation of the prostate. SARM may increase skeletal muscle mass in HF patients. The clinical trial SARMsMK-0773 examined their effects on skeletal muscle in female patients with sarcopenia [64]. Muscle mass in the intervention group increased significantly compared with that in the placebo group, but no effects on physical functions or muscle power were observed in

No studies have investigated the effects of combination therapy of SARM and exercise training on skeletal muscle mass and physical function in HF patients.

alpha-beta blocker, and they are commonly used in the treatment of HF.

*3.2.1 Beta-blockers*

prognosis and weight loss [57].

improve skeletal muscle mass in HF patients.

average) after one year.

HF patients are warranted.

**10**

this study.

The pathophysiology of skeletal muscle in patients with HF is complex and remains unclear. However, recent studies clarified several points. The mechanisms do not function independently and instead interact with each other. In the management of HF, it is important to assess skeletal muscle and physical functions, and to consider treatment combinations including exercise training, electrical stimulation, medicine, and supplements. Large-scale, high-quality studies are warranted to elucidate the pathophysiology and establish effective treatments for HF patients.

### **Conflict of interest**

I do not have any conflict of interest.

### **Author details**

Nobuo Morotomi1 \*, Kunihiro Sakuma2 and Kotomi Sakai3

1 Department of Rehabilitation medicine, Shin-Yurigaoka General Hospital, Kawasaki, Kanagawa, Japan

2 Institute for Liberal Arts, Environment and Society, Tokyo Institute of Technology, Tokyo, Japan

3 Setagaya Memorial Hospital, Department of Rehabilitation Medicine, Tokyo, Japan

\*Address all correspondence to: uoratubon@gmail.com

© 2021 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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[8] Levine B, Kalman J, Mayer L, Fillit HM, Packer M (1990) Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N Engl J Med. 323: 236-241.

[9] Reid MB, Li YP (2001) Tumor necrosis factor-alpha and muscle wasting: a cellular perspective. Respir Res. 2: 269-272.

[10] Chojkier M (2005) Inhibition of albumin synthesis in chronic diseases: molecular mechanisms. J Clin Gastroenterol. 39: S143-S146.

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Angiotensin ІІ type 1 receptor blockade attenuates TGF-β induced failure of muscle regeneration in multiple myopathic states. Nat Med. 13: 204-210.

[20] Kadoguchi T, Kinugawa S, Takada S, Fukushima A, Furihata T, Homma T, Masaki Y, Mizushima W, Nishikawa M, Takahashi M, Yokota T, Matsushima S, Okita K, Tsutsui H (2015) Angiotensin ІІ can directly induce mitochondrial dysfunction, decrease oxidative fibre number and induce atrophy in mouse hindlimb skeletal muscle. Exp Physiol.

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[21] Valentim L, Laurence KM, Townsend PA, Carroll CJ, Soond S,

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Scarabelli TM, Knight RA,

[19] Fukushima A, Kinugawa S, Takada S, Matsushima S, Sobrin MA, Ono T, Takahashi M, Suga T, Homma T, Masaki Y, Furihata T, Kadogushi T, Yokota T, Okita K, Tsutsui H (2014) (Pro) renin receptor in skeletal muscle is involved in the development of insulin resistance associated with postinfarct heart failure in mice. Am J Physiol Endocrinol Metab. 307: E503-E514.

*DOI: http://dx.doi.org/10.5772/intechopen.97291*

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Aggravating Heart Failure. Wien Klin

[14] Höllriegel R, Beck EB, Linke A, Adams V, Möbius-Winkler S, Mangner N, Sandri M, Gielen S, Gutberlet M, Hambrecht R, Schuler G, Erbs S (2013) Anabolic effects of exercise training in patients with advanced chronic heart failure (NYHA ІІІ b): impact on ubiquitin-ligases expression and skeletal muscle size. Int J

[15] Sakuma K, Yamaguchi A. Molecular mechanisms controlling skeletal muscle mass. In: Sakuma K, editor. Muscle cell and Tissue. IntechOpen; 2015. p.143-

[16] Kakutani N, Takada S, Nambu H, Matsumoto J, Furihata T, Yokota T, Fukushima A, Kinugawa S (2020) Angiotensin-converting-enzyme inhibitor prevents skeletal muscle fibrosis in myocardial infarction mice.

[17] Rouleau JL, Bichent D, Dagenais GR, Arnold JM, Parker JO, Bernstein V, Lamas G, Nadeau C (1993) Activation

Wochenschr. 128: 497-504.

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of neurohumoral systems in

1183-1189.

*Exercise Therapy for Patients with Heart Failure: Focusing on the Pathophysiology of Skeletal… DOI: http://dx.doi.org/10.5772/intechopen.97291*

Health ABC study (2009) Higher inflammatory marker levels in older persons: associations with 5-year change in muscle mass and muscle strength. J Gerontol A Biol Sci Med Sci. 64: 1183-1189.

[12] Langhaus W, Hrupka B (1999) Interleukins and tumor factor as inhibitors of food intake. Neuropeptides. 33: 415-424.

[13] Saitoh M, Santos MPD, Ebner N, Emami A, Konishi M, Ishida J, Valentova M, Sandek A, Doehner W, Anker SD, Haehling SV(2016) Nutritional status and its effects on muscle wasting in patients with chronic heart failure: insights from Studies Investigating Co-morbidities Aggravating Heart Failure. Wien Klin Wochenschr. 128: 497-504.

[14] Höllriegel R, Beck EB, Linke A, Adams V, Möbius-Winkler S, Mangner N, Sandri M, Gielen S, Gutberlet M, Hambrecht R, Schuler G, Erbs S (2013) Anabolic effects of exercise training in patients with advanced chronic heart failure (NYHA ІІІ b): impact on ubiquitin-ligases expression and skeletal muscle size. Int J Cardiol. 167: 975-980.

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[16] Kakutani N, Takada S, Nambu H, Matsumoto J, Furihata T, Yokota T, Fukushima A, Kinugawa S (2020) Angiotensin-converting-enzyme inhibitor prevents skeletal muscle fibrosis in myocardial infarction mice. Skelet Muscle. 10:11.

[17] Rouleau JL, Bichent D, Dagenais GR, Arnold JM, Parker JO, Bernstein V, Lamas G, Nadeau C (1993) Activation of neurohumoral systems in

postinfarction left ventricular dysfunction. J Am Coll Cardiol. 22: 390-398.

[18] Cohn RD, Erp CV, Habashi JP, Soleimani AA, Klein EC, Lisi MT, Gamradt M, Rhys CM, Holm TM, Loeys BL, Ramirez F, Judge DP, Ward CW, Dietz HC (2007) Angiotensin ІІ type 1 receptor blockade attenuates TGF-β induced failure of muscle regeneration in multiple myopathic states. Nat Med. 13: 204-210.

[19] Fukushima A, Kinugawa S, Takada S, Matsushima S, Sobrin MA, Ono T, Takahashi M, Suga T, Homma T, Masaki Y, Furihata T, Kadogushi T, Yokota T, Okita K, Tsutsui H (2014) (Pro) renin receptor in skeletal muscle is involved in the development of insulin resistance associated with postinfarct heart failure in mice. Am J Physiol Endocrinol Metab. 307: E503-E514.

[20] Kadoguchi T, Kinugawa S, Takada S, Fukushima A, Furihata T, Homma T, Masaki Y, Mizushima W, Nishikawa M, Takahashi M, Yokota T, Matsushima S, Okita K, Tsutsui H (2015) Angiotensin ІІ can directly induce mitochondrial dysfunction, decrease oxidative fibre number and induce atrophy in mouse hindlimb skeletal muscle. Exp Physiol. 100: 312-322.

[21] Valentim L, Laurence KM, Townsend PA, Carroll CJ, Soond S, Scarabelli TM, Knight RA, Latchman DS, Stephanou A (2006) Urocortin inhibits Beclin1-mediated autophagic cell death in cardiac myocytes exposed to ischemia/ reperfusion injury. J Mol Cell Cardiol. 40: 846-885.

[22] Zhu H, Tannous P, Johnstone JL, Kong Y, Shelton JM, Richardson JA, Le V, Levine B, Rothermel BA, Hill JA (2007) Cardiac autophagy is a maladaptive response to hemodynamic stress. J Clin Invest. 117: 1782-1793.

**12**

15: 95-101.

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

heart failure (SICA-HF). Eur Heart J. 34:

[5] Kinugawa S, Takada S,

Matsushima S, Okita K, Tsutsui H (2015) Skeletal muscle abnormalities in Heart Failure. Int Heart J. 56: 475-484.

[6] Brown DA, Perry JB, Allen ME, Sabbah HN, Stauffer BL, Shaikh SR, Cleland JGF, Colucci WS, Butler J, Voors AA, Anker SD, Pitt B, Pieske B, Filippatos G, Greene SJ, Gheorgehiade M (2017) Expert consensus document: Mitochondrial function as a therapeutic target in heart failure. Nat Rev Cardiol.

[7] Takada S, Masaki Y, Kinugawa S, Matsumoto J, Furihata T, Mizushima W, Kadoguchi T, Fukushima A, Homma T,

Takahashi M, Harashima S, Matsushita M, Yokota T, Tanaka S, Okita K, Tsutsui H (2016) Dipeptidyl peptidase-4 inhibitor improved exercise capacity and mitochondrial biogenesis in mice with heart failure via activation of glucagon-like peptide-1 receptor signalling. Cardiovasc Res. 111: 338-347.

[8] Levine B, Kalman J, Mayer L, Fillit HM, Packer M (1990) Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N

[9] Reid MB, Li YP (2001) Tumor necrosis factor-alpha and muscle wasting: a cellular perspective. Respir

[10] Chojkier M (2005) Inhibition of albumin synthesis in chronic diseases:

[11] Schaap LA, Pluijm SMF, Deeg DJH,

Newman AB, Colbert LH, Pahor M, Rubin SM, Tylavsky FA, Visser M,

molecular mechanisms. J Clin Gastroenterol. 39: S143-S146.

Harris TB, Kritchevsky SB,

Engl J Med. 323: 236-241.

Res. 2: 269-272.

512-519.

14: 238-250.

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Doehner W, Hilfiker-Kleiner D, Force T, Anker SD (2014) Prevention of liver cancer cachexia-induced cardiac wasting and heart failure. Eur Heart J.

[57] Coats AJ, Fuang HG, Prabhash K, Haehling S von, Tilson J, Brown R, Beadle J, Anker SD & for and on behalf of the ACT-ONE study group (2016) Espindlol for the treatment and

training and vitamin D3

BMJ Open. 7: e014619.

Cardiol. 39: 56-61.

Assoc. 16: 740-747.

35: 932-941.

36: 71-80.

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Mol Metab. 4: 437-460.

Health Aging. 19: 669-672.

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[52] Hayakawa N, Fukumura J,

Yasuno H, Fujimoto-Ouchi K, Kitamura

and cachexia (ROMANA1 and ROMANA2): results from two randomized, double blind, phase 3 trials. Lancet Oncol. 17: 519-531.

110: 3674-3679.

Muscle. 8: 686-701.

Perez-Tilve D, Pfluger PT, Schwartz TW, Seeley RJ, Sleeman M, Sun Y, Sussel L, Tong J, Thorner MO, van der Lely AJ, van der Ploeg LHT, Zigman JM, Kojima M, Kangawa K, Smith RG, Horvath T, Tschop MH (2015) Ghrelin.

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**16**

[58] Clark AL, Krum H, Katus HA, Mohacsi P, Salekin D, Schultz MK, Packer M, Anker SD (2017) Effect of beta-adrenergic blockade with carvedilol on cachexia in severe chronic heart failure: results from the COPERNICUS trial. J Cachexia Sarcopenia Muscle. 8: 549-556.

[59] Saitoh M, Ebner N, Haehling SV, Anker SD, Springer J (2018) Therapeutic considerations of sarcopenia in heart failure patients. Expert Rev Cardiovasc Ther. 16: 133-142.

[60] Anker SD, Negassa A, Coats AJ, Poole-Wilson PA, Cohn JN, Yusuf S (2003) Prognostic importance of weight loss in chronic heart failure and the effect of treatment with angiotensinconverting-enzyme inhibitors: an observational study. Lancet. 361: 1077-1083.

[61] Zhou LS, Xu LJ, Wang XQ, Huhang YH, Xiao Q (2015) Effect of angiotensin-converting-enzyme inhibitors on physical function in elderly subjects: A systematic review and meta-analysis. Drugs Aging. 32: 727-735.

[62] Sumukadas D, Band M, Miller S, Cvoro V, Witham M, Struthers A, McConnachie A, Lloyd SM, McMurdo M (2014) Do ACE inhibitors improve the response to exercise training in functionally impaired older adults? A randomized controlled trial. J Gerontol A Biol Sci Med Sci. 69: 736-743.

[63] Borst SE, Shuster JJ, Zou B, Ye F, Jia H, Wokhlu A, Yarrow JF (2014) Cardiovascular risks and elevation of serum DHT vary by route of

testosterone administration: a systematic review and meta-analysis. BMC Med. 12: 211.

[64] Papanicolaou DA, Ather SN, Zhu H, Zhou Y, Lutkiewicz J, Scott BB, Chandler J (2013) A phase ІІA randomized, placebo-controlled clinical trial to study the efficacy and safety of the selective androgen receptor modulator (SARM), MK-0773 in female participants with sarcopenia. J Nutr Health Aging. 17: 533-543.

**19**

**Chapter 2**

**Abstract**

Evidence for the Role of Cell

Reprogramming in Naturally

Pulmonary arterial hypertension (PAH) is a fatal disease without a cure. If untreated, increased pulmonary vascular resistance kills patients within several years due to right heart failure. Even with the currently available therapies, survival durations remain short. By the time patients are diagnosed with this disease, the damage to the right ventricle (RV) has already developed. Therefore, agents that repair the damaged RV have therapeutic potential. We previously reported that cardiac fibrosis that occurs in the RV of adult Sprague–Dawley rats with PAH could naturally be reversed. We herein investigated the mechanism of this remarkable cardiac repair process. Counting of cardiomyocytes showed that the elimination of cardiac fibrosis is associated with the increased RV myocyte number, suggesting that new cardiomyocytes were generated. Immunohistochemistry showed the expression of α-smooth muscle actin and Sox-2 in RV myocytes of rats with PAH. Transmission electron microscopy detected the structure that resembles maturing cardiomyocytes in both the RV of PAH rats and cultured cardiomyocytes derived from induced pluripotent stem cells. We propose that the damaged RV in PAH can be repaired by activating the cell reprogramming mechanism that converts resident

**Keywords:** cardiac repair, cardiomyocyte regeneration, cell reprogramming,

time patients are diagnosed, RV damage has often already occurred.

Pulmonary arterial hypertension (PAH) affects males and females of any age, including children. Despite the availability of approved drugs, PAH remains a fatal disease without a cure [1, 2]. The major pathogenic features that increase the pulmonary vascular resistance in PAH include the vasoconstriction and the development of vascular remodeling, in which pulmonary artery (PA) walls are thickened and the lumens are narrowed or occluded. Increased resistance puts strain on the right ventricle (RV), and right heart failure is the major cause of death among PAH patients [3, 4]. The median overall survival for patients diagnosed with PAH is 2.8 years from the time of diagnosis (3-year survival: 48%) if untreated [5, 6]. Even with currently available therapies, the prognosis remains poor with only 58–75% of PAH patients surviving for 3 years [7–10]. PAH is a progressive disease; and by the

Occurring Cardiac Repair

*Nataliia V. Shults and Yuichiro J. Suzuki*

cardiac fibroblasts into induced cardiomyocytes.

pulmonary hypertension, right ventricle

**1. Introduction**

#### **Chapter 2**

## Evidence for the Role of Cell Reprogramming in Naturally Occurring Cardiac Repair

*Nataliia V. Shults and Yuichiro J. Suzuki*

#### **Abstract**

Pulmonary arterial hypertension (PAH) is a fatal disease without a cure. If untreated, increased pulmonary vascular resistance kills patients within several years due to right heart failure. Even with the currently available therapies, survival durations remain short. By the time patients are diagnosed with this disease, the damage to the right ventricle (RV) has already developed. Therefore, agents that repair the damaged RV have therapeutic potential. We previously reported that cardiac fibrosis that occurs in the RV of adult Sprague–Dawley rats with PAH could naturally be reversed. We herein investigated the mechanism of this remarkable cardiac repair process. Counting of cardiomyocytes showed that the elimination of cardiac fibrosis is associated with the increased RV myocyte number, suggesting that new cardiomyocytes were generated. Immunohistochemistry showed the expression of α-smooth muscle actin and Sox-2 in RV myocytes of rats with PAH. Transmission electron microscopy detected the structure that resembles maturing cardiomyocytes in both the RV of PAH rats and cultured cardiomyocytes derived from induced pluripotent stem cells. We propose that the damaged RV in PAH can be repaired by activating the cell reprogramming mechanism that converts resident cardiac fibroblasts into induced cardiomyocytes.

**Keywords:** cardiac repair, cardiomyocyte regeneration, cell reprogramming, pulmonary hypertension, right ventricle

#### **1. Introduction**

Pulmonary arterial hypertension (PAH) affects males and females of any age, including children. Despite the availability of approved drugs, PAH remains a fatal disease without a cure [1, 2]. The major pathogenic features that increase the pulmonary vascular resistance in PAH include the vasoconstriction and the development of vascular remodeling, in which pulmonary artery (PA) walls are thickened and the lumens are narrowed or occluded. Increased resistance puts strain on the right ventricle (RV), and right heart failure is the major cause of death among PAH patients [3, 4]. The median overall survival for patients diagnosed with PAH is 2.8 years from the time of diagnosis (3-year survival: 48%) if untreated [5, 6]. Even with currently available therapies, the prognosis remains poor with only 58–75% of PAH patients surviving for 3 years [7–10]. PAH is a progressive disease; and by the time patients are diagnosed, RV damage has often already occurred.

RV failure is the major cause of death among patients with PAH. However, no treatment strategies are available to manage the RV dysfunctions. Physiologically, the RV needs to cope with fold changes in PA pressure. Thus, the RV is capable of adapting to increased pressure. Similarly to human patients with PAH, we found that the RV of Sprague–Dawley (SD) rats treated with SU5416 and hypoxia to produce PAH suffer from severe cardiac fibrosis at 8 to 17 weeks after the initiation of the SU5416/hypoxia treatment [11]. Remarkably, at 35 weeks after the initiation of the SU5416/hypoxia treatment, RV fibrosis was found to be resolved in these rats, despite the RV pressure remained high [12]. Thus, the RV remodeling can naturally be reversed in these animals, providing an interesting model of RV repair. Understanding the mechanism of such naturally occurring events should shed a light on developing therapeutic strategies to repair the cardiac damage in human patients. The present study examined the mechanism of this cardiac repair process.

#### **2. Materials and methods**

#### **2.1 Experimental animals**

Male adult SD rats (Charles River Laboratories International, Inc., Wilmington, MA, USA) were subcutaneously injected with SU5416 (20 mg/kg body weight; MedChem Express, Monmouth Junction, NJ, USA), maintained in hypoxia for 3 weeks [11, 12] and then in normoxia for up to 32 weeks (35-week time points). Animals were subjected to hypoxia in a chamber (30"w x 20"d x 20"h) regulated by an OxyCycler Oxygen Profile Controller (Model A84XOV; BioSpherix, Redfield, NY, USA) set to maintain 10% O2 with an influx of N2 gas, located in the animal care facility at the Georgetown University Medical Center [11, 12]. Ventilation to the outside of the chamber was adjusted to remove CO2, such that its level did not exceed 5000 ppm. Control animals were subjected to ambient 21% O2 (normoxia) in another chamber. Animals were fed normal rat chow. Animals were anesthetized and euthanized by excising the heart and the lungs.

The Georgetown University Animal Care and Use Committee approved all animal experiments, and the investigation conformed to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.

#### **2.2 Immunohistochemistry (IHC)**

RV tissues were immersed in buffered 10% formalin at room temperature, and were embedded in paraffin. These paraffin-embedded tissues were cut and mounted on glass slides. IHC was performed using horseradish peroxidase (HRP) labeled polymer and 3,3′-diaminobenzidine (DAB) chromagen (Agilent Technologies, Santa Clara, CA, USA) with α-smooth muscle actin (αSMA; Catalog # ab32575) and Sox2 (Catalog # ab97959) antibodies (Abcam, Cambridge, UK).

#### **2.3 Transmission electron microscopy (TEM)**

The RV free wall tissues of rats subjected to SU5416/hypoxia as well as cultured cardiomyocytes derived from human induced pluripotent stem cells (iPSCs) purchased from Cell Applications, Inc. (San Diego, CA) were fixed in the 2.5% glutaraldehyde/0.05 M cacodylate solution, post-fixed with 1% osmium tetroxide and embedded in EmBed812. Ultrathin sections (70 nm) were post-stained with uranyl acetate and lead citrate and examined in the Talos F200X FEG transmission electron

**21**

*Evidence for the Role of Cell Reprogramming in Naturally Occurring Cardiac Repair*

La Jolla, CA, USA). P < 0.05 was considered to be significant.

microscope (FEI, Hillsboro, OR, USA) at 80 KV located at the George Washington University Nanofabrication and Imaging Center. Digital electron micrographs were

Means and standard errors were calculated. Comparisons between three groups were analyzed by using one-way analysis of variance (ANOVA) with a Student– Newman–Keuls post-hoc test using the GraphPad Prism (GraphPad Software, Inc.,

Since the initial discovery that treating SD rats with the SU5416 injection plus chronic hypoxia promoted severe PAH with pulmonary vascular lesions that resemble those of human patients [13], this experimental model has become a gold standard in the research field of PAH. Experimental design usually involves the single subcutaneous injection of SU5416, followed by subjecting to chronic hypoxia for 3 weeks. In many studies, rats are then maintained in normoxia for 2 to 5 weeks, and PAH and pulmonary vascular remodeling are observed [13–17]. At this stage, some laboratories including ours have reported that the RV is severely damaged with fibrosis [11, 14, 18, 19]. At 8 to 17 weeks after the SU5416 injection, however, we found that, despite the occurrence of severe fibrosis, the RV contractility is maintained or even improved, perhaps due to the formation of 'super' RV myocytes [11]. Moreover, our more recent results demonstrated that, at 35 weeks (3 weeks hypoxia followed by 32 weeks of normoxia), RV fibrosis was largely resolved, despite the RV pressure remained high [12]. The number of myofibroblasts that contribute to the formation of fibrosis, as detected using a well utilized marker αSMA, is increased in non-vessel regions of the RV of SD rats with severe PAH as well as RV fibrosis, and this expression declined in rats with repaired RV at 35 week after the SU5416 injection [12]. Thus, the SU5416/hypoxia treatment does not cause the death of SD rats and the damaged RV can be repaired naturally. Since RV myocytes were not hypertrophied at 35-weeks, the number of myocytes must have been increased to fill the post-fibrotic areas. Indeed counting cardiomyocytes indicated that, compared to the 17-week time point when fibrosis was present, the number of cardiomyocytes increased at 35-weeks when the RV

Based on these results, we hypothesize that these SD rats possess a mechanism that repairs the damaged heart in response to PAH. This raises a question on where regenerated cardiomyocytes come from when the RV repair occurs. It is now known that some adult cardiomyocytes are capable of proliferating [20]. However, the cardiac renewal through the cardiomyocyte proliferation would be too slow to replace large fibrotic areas that were seen in our experimental model. Another possibility is that new cardiomyocytes were regenerated from cardiac progenitor cells. We have identified c-kit and isl1-positive cardiac progenitor cells in the RV of SD rats,

Our previous experiments described in Zungu-Edmondson et al. [12] using the αSMA antibody were originally performed for the purpose of detecting myofibroblasts, which express αSMA. By examining αSMA IHC slides from RVs of SD rats with PAH (at 17-weeks) with a larger magnification (x1,000) as shown in **Figure 2A** of Zungu-Edmondson et al. [12], we noticed that some brown

however, their levels were not altered in PAH rats (data not shown).

*DOI: http://dx.doi.org/10.5772/intechopen.94740*

recorded with the TIA software (FEI).

**2.4 Statistical analysis**

was repaired (**Figure 1**).

**3. Results**

microscope (FEI, Hillsboro, OR, USA) at 80 KV located at the George Washington University Nanofabrication and Imaging Center. Digital electron micrographs were recorded with the TIA software (FEI).

#### **2.4 Statistical analysis**

Means and standard errors were calculated. Comparisons between three groups were analyzed by using one-way analysis of variance (ANOVA) with a Student– Newman–Keuls post-hoc test using the GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA). P < 0.05 was considered to be significant.

#### **3. Results**

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

**2. Materials and methods**

and euthanized by excising the heart and the lungs.

**2.3 Transmission electron microscopy (TEM)**

**2.2 Immunohistochemistry (IHC)**

Health (NIH) Guide for the Care and Use of Laboratory Animals.

**2.1 Experimental animals**

RV failure is the major cause of death among patients with PAH. However, no treatment strategies are available to manage the RV dysfunctions. Physiologically, the RV needs to cope with fold changes in PA pressure. Thus, the RV is capable of adapting to increased pressure. Similarly to human patients with PAH, we found that the RV of Sprague–Dawley (SD) rats treated with SU5416 and hypoxia to produce PAH suffer from severe cardiac fibrosis at 8 to 17 weeks after the initiation of the SU5416/hypoxia treatment [11]. Remarkably, at 35 weeks after the initiation of the SU5416/hypoxia treatment, RV fibrosis was found to be resolved in these rats, despite the RV pressure remained high [12]. Thus, the RV remodeling can naturally be reversed in these animals, providing an interesting model of RV repair. Understanding the mechanism of such naturally occurring events should shed a light on developing therapeutic strategies to repair the cardiac damage in human patients. The present study examined the mechanism of this cardiac repair process.

Male adult SD rats (Charles River Laboratories International, Inc., Wilmington, MA, USA) were subcutaneously injected with SU5416 (20 mg/kg body weight; MedChem Express, Monmouth Junction, NJ, USA), maintained in hypoxia for 3 weeks [11, 12] and then in normoxia for up to 32 weeks (35-week time points). Animals were subjected to hypoxia in a chamber (30"w x 20"d x 20"h) regulated by an OxyCycler Oxygen Profile Controller (Model A84XOV; BioSpherix, Redfield, NY, USA) set to maintain 10% O2 with an influx of N2 gas, located in the animal care facility at the Georgetown University Medical Center [11, 12]. Ventilation to the outside of the chamber was adjusted to remove CO2, such that its level did not exceed 5000 ppm. Control animals were subjected to ambient 21% O2 (normoxia) in another chamber. Animals were fed normal rat chow. Animals were anesthetized

The Georgetown University Animal Care and Use Committee approved all animal experiments, and the investigation conformed to the National Institutes of

RV tissues were immersed in buffered 10% formalin at room temperature, and were embedded in paraffin. These paraffin-embedded tissues were cut and mounted on glass slides. IHC was performed using horseradish peroxidase (HRP) labeled polymer and 3,3′-diaminobenzidine (DAB) chromagen (Agilent Technologies, Santa Clara, CA, USA) with α-smooth muscle actin (αSMA; Catalog # ab32575) and Sox2 (Catalog # ab97959) antibodies (Abcam, Cambridge, UK).

The RV free wall tissues of rats subjected to SU5416/hypoxia as well as cultured

cardiomyocytes derived from human induced pluripotent stem cells (iPSCs) purchased from Cell Applications, Inc. (San Diego, CA) were fixed in the 2.5% glutaraldehyde/0.05 M cacodylate solution, post-fixed with 1% osmium tetroxide and embedded in EmBed812. Ultrathin sections (70 nm) were post-stained with uranyl acetate and lead citrate and examined in the Talos F200X FEG transmission electron

**20**

Since the initial discovery that treating SD rats with the SU5416 injection plus chronic hypoxia promoted severe PAH with pulmonary vascular lesions that resemble those of human patients [13], this experimental model has become a gold standard in the research field of PAH. Experimental design usually involves the single subcutaneous injection of SU5416, followed by subjecting to chronic hypoxia for 3 weeks. In many studies, rats are then maintained in normoxia for 2 to 5 weeks, and PAH and pulmonary vascular remodeling are observed [13–17]. At this stage, some laboratories including ours have reported that the RV is severely damaged with fibrosis [11, 14, 18, 19]. At 8 to 17 weeks after the SU5416 injection, however, we found that, despite the occurrence of severe fibrosis, the RV contractility is maintained or even improved, perhaps due to the formation of 'super' RV myocytes [11]. Moreover, our more recent results demonstrated that, at 35 weeks (3 weeks hypoxia followed by 32 weeks of normoxia), RV fibrosis was largely resolved, despite the RV pressure remained high [12]. The number of myofibroblasts that contribute to the formation of fibrosis, as detected using a well utilized marker αSMA, is increased in non-vessel regions of the RV of SD rats with severe PAH as well as RV fibrosis, and this expression declined in rats with repaired RV at 35 week after the SU5416 injection [12]. Thus, the SU5416/hypoxia treatment does not cause the death of SD rats and the damaged RV can be repaired naturally. Since RV myocytes were not hypertrophied at 35-weeks, the number of myocytes must have been increased to fill the post-fibrotic areas. Indeed counting cardiomyocytes indicated that, compared to the 17-week time point when fibrosis was present, the number of cardiomyocytes increased at 35-weeks when the RV was repaired (**Figure 1**).

Based on these results, we hypothesize that these SD rats possess a mechanism that repairs the damaged heart in response to PAH. This raises a question on where regenerated cardiomyocytes come from when the RV repair occurs. It is now known that some adult cardiomyocytes are capable of proliferating [20]. However, the cardiac renewal through the cardiomyocyte proliferation would be too slow to replace large fibrotic areas that were seen in our experimental model. Another possibility is that new cardiomyocytes were regenerated from cardiac progenitor cells. We have identified c-kit and isl1-positive cardiac progenitor cells in the RV of SD rats, however, their levels were not altered in PAH rats (data not shown).

Our previous experiments described in Zungu-Edmondson et al. [12] using the αSMA antibody were originally performed for the purpose of detecting myofibroblasts, which express αSMA. By examining αSMA IHC slides from RVs of SD rats with PAH (at 17-weeks) with a larger magnification (x1,000) as shown in **Figure 2A** of Zungu-Edmondson et al. [12], we noticed that some brown

#### **Figure 1.**

*Restoration of RV cardiomyocytes. SU5416-injected SD rats were subjected to 3 weeks hypoxia and then maintained in normoxia to promote PAH. 17 and 35 weeks after the SU5416 injection, RV myocardium tissues were fixed in formalin, embedded in paraffin, and subjected to H&E staining. The number of cardiomyocytes were counted and expressed as % of the control rats (Cont). SD rats with PAH at 17 weeks after the initiation of SU5416/hypoxia had significantly reduced RV myocyte number compared to healthy controls. This decrease in RV myocyte number was restored at 35 weeks. The symbol \* denotes that the values are significantly different from each other at p < 0.05.*

#### **Figure 2.**

*Discovery of naturally-occurring induced cardiomyocytes (iCMs)? SU5416-injected SD rats were subjected to 3 weeks hypoxia and then maintained in normoxia to promote PAH. 17 weeks after the injection, myocardium tissues were fixed in formalin, embedded in paraffin, and subjected to IHC using the antibody against* α*SMA (brown stains). (A and B)* α*SMA IHC results shown at x200 and x1,000 magnifications, which clearly show the brown stains in RV cardiomyocytes of SD rats with PAH. (C) This brown* α*SMA stain was not observed in control rat RVs without PAH. Scale bars indicate 200* μ*m for x200 and 50* μ*m for x1,000.*

αSMA stains, in addition to myofibroblasts indicated by arrows, also occurred on cardiomyocytes. We initially discarded these observations by thinking that they are non-specific artifacts. However, further examinations of a number of IHC slides made us convinced that cardiomyocytes are indeed stained with the αSMA antibody. This seems to occur regionally as a group in the myocardial walls (**Figure 2A**). **Figure 2B** shows the amplified view of **Figure 2A** clearly demonstrating that these cardiomyocytes with clear striations express αSMA. By

**23**

**Figure 3.**

*Evidence for the Role of Cell Reprogramming in Naturally Occurring Cardiac Repair*

of cardiomyocytes with the αSMA antibody (**Figure 2C**).

contrast, control RVs from healthy rats without PAH did not exhibit the staining

Cell reprogramming is defined as the conversion of one specific cell type to another. This technology has gained considerable attention when Prof. Shinya Yamanaka discovered the means to convert fibroblasts into iPSCs and received the Nobel Prize [21]. In their study, stem cell-related transcription factors including Oct4 and Sox2 were used to convert somatic cells to pluripotent cells that can be differentiated into various cell types including cardiomyocytes [22]. More recently, a combination of cardiac-specific transcription factors was found to directly convert fibroblasts into cardiomyocytes [23, 24]. **Figure 3A** shows our TEM study of cardiomyocytes derived from iPSCs. These induced cardiomyocytes (iCMs) are capable of beating, express contractile proteins, and exhibit an organized sarcomere structure with clear striations and Z-lines (**Figure 3A**). In addition, some regions with the not well-defined sarcomere organization, which could be in the process of maturing into iCMs were also identified in these TEM images (**Figure 3B**). iCMs have been shown to express αSMA [25], while normal adult cardiomyocytes do not. Thus, αSMA-positive cardiomyocytes we observed in adult SD rats with PAH could be iCM-like cells. Consistently with this hypothesis, our examination of TEM images revealed that the structure resembling cardiomyocytes maturing from iPSCs as observed in cultured cells (**Figure 3B**) also occurs in the

These results would support the concept that RVs of SD rats with PAH have iCMs-like cells that are produced via cell reprogramming. However, the αSMA expression can be induced by the fetal gene program mechanism that is associated with cardiac hypertrophy, independent of cell reprogramming. Thus, we examined if factors more directly related to cell reprogramming are expressed in these cardiomyocytes. We found that cardiomyocytes of RV tissues from SD rats with PAH also express Sox2 (**Figure 5A** and **B**), a stem cell-related transcription factor that has been used to generate iPSCs [22]. By contrast, no Sox2 stains were observed

*TEM image of cardiomyocytes derived from iPSCs. The reprogramming technology was used to convert cultured human fibroblasts into iPSCs, then to cardiomyocytes (cell applications, Inc.). Fixed iPSC-derived cardiomyocytes were observed under a transmission electron microscope. (A) the representative image shows cardiomyocytes with clear striations and sarcomere structures (arrow), indicating that iPSCs indeed can become matured cardiomyocytes. Magnification, x5,500. (B) we also identified some regions with the not well defined sarcomere organization (arrows), which may be in the process of maturing into cardiomyocytes. Magnification, x14,000.*

*DOI: http://dx.doi.org/10.5772/intechopen.94740*

RV of SD rats with PAH (**Figure 4B**).

in healthy control SD rats without PAH (**Figure 5C**).

#### *Evidence for the Role of Cell Reprogramming in Naturally Occurring Cardiac Repair DOI: http://dx.doi.org/10.5772/intechopen.94740*

contrast, control RVs from healthy rats without PAH did not exhibit the staining of cardiomyocytes with the αSMA antibody (**Figure 2C**).

Cell reprogramming is defined as the conversion of one specific cell type to another. This technology has gained considerable attention when Prof. Shinya Yamanaka discovered the means to convert fibroblasts into iPSCs and received the Nobel Prize [21]. In their study, stem cell-related transcription factors including Oct4 and Sox2 were used to convert somatic cells to pluripotent cells that can be differentiated into various cell types including cardiomyocytes [22]. More recently, a combination of cardiac-specific transcription factors was found to directly convert fibroblasts into cardiomyocytes [23, 24]. **Figure 3A** shows our TEM study of cardiomyocytes derived from iPSCs. These induced cardiomyocytes (iCMs) are capable of beating, express contractile proteins, and exhibit an organized sarcomere structure with clear striations and Z-lines (**Figure 3A**). In addition, some regions with the not well-defined sarcomere organization, which could be in the process of maturing into iCMs were also identified in these TEM images (**Figure 3B**). iCMs have been shown to express αSMA [25], while normal adult cardiomyocytes do not. Thus, αSMA-positive cardiomyocytes we observed in adult SD rats with PAH could be iCM-like cells. Consistently with this hypothesis, our examination of TEM images revealed that the structure resembling cardiomyocytes maturing from iPSCs as observed in cultured cells (**Figure 3B**) also occurs in the RV of SD rats with PAH (**Figure 4B**).

These results would support the concept that RVs of SD rats with PAH have iCMs-like cells that are produced via cell reprogramming. However, the αSMA expression can be induced by the fetal gene program mechanism that is associated with cardiac hypertrophy, independent of cell reprogramming. Thus, we examined if factors more directly related to cell reprogramming are expressed in these cardiomyocytes. We found that cardiomyocytes of RV tissues from SD rats with PAH also express Sox2 (**Figure 5A** and **B**), a stem cell-related transcription factor that has been used to generate iPSCs [22]. By contrast, no Sox2 stains were observed in healthy control SD rats without PAH (**Figure 5C**).

#### **Figure 3.**

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

αSMA stains, in addition to myofibroblasts indicated by arrows, also occurred on cardiomyocytes. We initially discarded these observations by thinking that they are non-specific artifacts. However, further examinations of a number of IHC slides made us convinced that cardiomyocytes are indeed stained with the αSMA antibody. This seems to occur regionally as a group in the myocardial walls (**Figure 2A**). **Figure 2B** shows the amplified view of **Figure 2A** clearly demonstrating that these cardiomyocytes with clear striations express αSMA. By

*control rat RVs without PAH. Scale bars indicate 200* μ*m for x200 and 50* μ*m for x1,000.*

*Discovery of naturally-occurring induced cardiomyocytes (iCMs)? SU5416-injected SD rats were subjected to 3 weeks hypoxia and then maintained in normoxia to promote PAH. 17 weeks after the injection, myocardium tissues were fixed in formalin, embedded in paraffin, and subjected to IHC using the antibody against* α*SMA (brown stains). (A and B)* α*SMA IHC results shown at x200 and x1,000 magnifications, which clearly show the brown stains in RV cardiomyocytes of SD rats with PAH. (C) This brown* α*SMA stain was not observed in* 

*Restoration of RV cardiomyocytes. SU5416-injected SD rats were subjected to 3 weeks hypoxia and then maintained in normoxia to promote PAH. 17 and 35 weeks after the SU5416 injection, RV myocardium tissues were fixed in formalin, embedded in paraffin, and subjected to H&E staining. The number of cardiomyocytes were counted and expressed as % of the control rats (Cont). SD rats with PAH at 17 weeks after the initiation of SU5416/hypoxia had significantly reduced RV myocyte number compared to healthy controls. This decrease in RV myocyte number was restored at 35 weeks. The symbol \* denotes that the values are significantly different* 

**22**

**Figure 2.**

**Figure 1.**

*from each other at p < 0.05.*

*TEM image of cardiomyocytes derived from iPSCs. The reprogramming technology was used to convert cultured human fibroblasts into iPSCs, then to cardiomyocytes (cell applications, Inc.). Fixed iPSC-derived cardiomyocytes were observed under a transmission electron microscope. (A) the representative image shows cardiomyocytes with clear striations and sarcomere structures (arrow), indicating that iPSCs indeed can become matured cardiomyocytes. Magnification, x5,500. (B) we also identified some regions with the not well defined sarcomere organization (arrows), which may be in the process of maturing into cardiomyocytes. Magnification, x14,000.*

#### **Figure 4.**

*TEM identification of maturing ICM-like cells in the RV of PAH rats. SD rats were injected with SU5416, subjected to 3 weeks hypoxia, and maintained in normoxia. 20 weeks after the injection, RV tissues were fixed and analyzed by TEM. (A) the image shows normal cardiomyocytes with clear striations and sarcomere structures (arrow). Magnification, x5,500. (B) we also identified some regions with the not well defined sarcomere organization (arrows), which may be in the process of maturing into iCMs, similar to the structure shown in Figure 3B. Magnification, x14,000.*

#### **Figure 5.**

*Expression of Sox2. SU5416-injected SD rats were subjected to 3 weeks hypoxia and then maintained in normoxia to promote PAH. 17 weeks after the injection, hearts were fixed and subjected to IHC with the antibody against Sox2 (brown stains). (A and B) Sox2 IHC results shown at x200 and at x1,000 magnifications, which clearly show the brown stains in RV cardiomyocytes of SD rats with PAH. (C) This brown Sox2 stain was not observed in control rat RVs without PAH. Scale bars indicate 200* μ*m for x200 and 50* μ*m for x1,000.*

#### **4. Discussion**

In response to pressure overload, the heart ventricles undergo a series of adaptive events. In response to systemic and pulmonary hypertension, the left ventricle (LV) and the RV, respectively hypertrophy in order to increase the force of muscle contraction. Concentric hypertrophy is the first change that occurs in response to chronic pressure overload, and this compensatory mechanism allows for improved cardiac output. Exercise-induced cardiac hypertrophy, for example, increases the force of contraction in accordance the needs associated with strenuous exercise and training. This adaptive feature is reversible. However, in chronic disease conditions, this compensatory mechanism thickens the ventricular wall too much in a manner that decreases the stroke volume and thus the cardiac output. This results in the second adaptation to decrease the ventricular wall thickness. In case of the LV, the transition from concentric to eccentric hypertrophy predominates,

**25**

**Figure 6.**

myocytes to save lives.

*elicit strong muscle contraction.*

*Evidence for the Role of Cell Reprogramming in Naturally Occurring Cardiac Repair*

the RV where cardiomyocytes die get filled with fibrosis.

however, further work is needed to prove this concept.

resulting in the dilated LV with the thin ventricular wall. This event, however, does not seem to occur in the RV in response to chronic pulmonary hypertension and the hallmark of cor pulmonale is that the RV myocytes remain concentrically hypertrophied at the time of heart failure. In the RV, the attempt to decrease the RV wall thickness is expected to be related to the promotion of cell death, although it is unclear whether apoptosis really occurs in the RV of human patients with pulmonary hypertension [26]. However, apoptotic cardiomyocytes have been detected in the RV of a rat model of pulmonary hypertension [11]. In these rats, regions of

Using well-studied model of PAH, in which SD rats are treated with the SU5416

Further, remarkably at 35 weeks after the SU5416/hypoxia initiation, these fibrosis regions disappear and are filled with newly formed cardiomyocytes [12]. The present study indeed showed that the number of cardiomyocytes were increased at the 35-week time point, compared to the 17-week time point. This increased number of cardiomyocytes was found to be associated with the production of RV cardiomyocytes that express αSMA and Sox2 as well as the occurrence of the structure visible in TEM that resembles maturing iCMs similar to those observed in cultured cardiomyocytes derived from iPSCs. From these results, we hypothesize that the damaged RV due to PAH can naturally be repaired in SD rats through a mechanism that involves the conversion of resident cardiac fibroblasts into iCMs via cell reprogramming (**Figure 6**). Our finding also suggests that the nature possesses the ability for maturing iCMs into functional cardiomyocytes that are capable of eliciting strong muscle contraction through a "functionalization" process (**Figure 6**). Our results so far provided evidence to support this novel mechanism of cardiac regeneration,

Understanding of the endogenous means to repair the heart is important, not only to provide basic knowledge of cardiac physiology, but also to develop therapeutic strategies to treat conditions in which cardiomyocytes are damaged. Along with the cardiomyocyte proliferation and the involvement of cardiac progenitor cells, our results suggest a novel mechanism of cardiac regeneration through cell reprogramming. Defining this naturally occurring mechanism should contribute to the development of technologies to convert resident cardiac fibroblasts into cardio-

*Scheme depicting our hypothesis. SD rats possess an RV repair mechanism, in which naturally occurring cell reprogramming process converts resident fibroblasts into* α*SMA-and Sox2-positive induced cardiomyocytes (iCMs). These rats also possess the functionalization mechanism that makes functional cardiomyocytes that can* 

injection and chronic hypoxia, we previously found that RVs were capable of maintaining sufficient force of muscle contraction even severe fibrosis occurred at 8 to 17 weeks after the initiation of the SU5416/hypoxia treatment [11]. We postulated that this is due to the formation of "super RV myocytes" that are capable of eliciting stronger force of contraction via a mechanism involving the downregulation of calsequestrin 2, the major Ca2+-binding protein of the sarcoplasmic reticulum.

*DOI: http://dx.doi.org/10.5772/intechopen.94740*

#### *Evidence for the Role of Cell Reprogramming in Naturally Occurring Cardiac Repair DOI: http://dx.doi.org/10.5772/intechopen.94740*

resulting in the dilated LV with the thin ventricular wall. This event, however, does not seem to occur in the RV in response to chronic pulmonary hypertension and the hallmark of cor pulmonale is that the RV myocytes remain concentrically hypertrophied at the time of heart failure. In the RV, the attempt to decrease the RV wall thickness is expected to be related to the promotion of cell death, although it is unclear whether apoptosis really occurs in the RV of human patients with pulmonary hypertension [26]. However, apoptotic cardiomyocytes have been detected in the RV of a rat model of pulmonary hypertension [11]. In these rats, regions of the RV where cardiomyocytes die get filled with fibrosis.

Using well-studied model of PAH, in which SD rats are treated with the SU5416 injection and chronic hypoxia, we previously found that RVs were capable of maintaining sufficient force of muscle contraction even severe fibrosis occurred at 8 to 17 weeks after the initiation of the SU5416/hypoxia treatment [11]. We postulated that this is due to the formation of "super RV myocytes" that are capable of eliciting stronger force of contraction via a mechanism involving the downregulation of calsequestrin 2, the major Ca2+-binding protein of the sarcoplasmic reticulum.

Further, remarkably at 35 weeks after the SU5416/hypoxia initiation, these fibrosis regions disappear and are filled with newly formed cardiomyocytes [12]. The present study indeed showed that the number of cardiomyocytes were increased at the 35-week time point, compared to the 17-week time point. This increased number of cardiomyocytes was found to be associated with the production of RV cardiomyocytes that express αSMA and Sox2 as well as the occurrence of the structure visible in TEM that resembles maturing iCMs similar to those observed in cultured cardiomyocytes derived from iPSCs. From these results, we hypothesize that the damaged RV due to PAH can naturally be repaired in SD rats through a mechanism that involves the conversion of resident cardiac fibroblasts into iCMs via cell reprogramming (**Figure 6**). Our finding also suggests that the nature possesses the ability for maturing iCMs into functional cardiomyocytes that are capable of eliciting strong muscle contraction through a "functionalization" process (**Figure 6**). Our results so far provided evidence to support this novel mechanism of cardiac regeneration, however, further work is needed to prove this concept.

Understanding of the endogenous means to repair the heart is important, not only to provide basic knowledge of cardiac physiology, but also to develop therapeutic strategies to treat conditions in which cardiomyocytes are damaged. Along with the cardiomyocyte proliferation and the involvement of cardiac progenitor cells, our results suggest a novel mechanism of cardiac regeneration through cell reprogramming. Defining this naturally occurring mechanism should contribute to the development of technologies to convert resident cardiac fibroblasts into cardiomyocytes to save lives.

#### **Figure 6.**

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

**24**

**4. Discussion**

*50* μ*m for x1,000.*

**Figure 5.**

**Figure 4.**

*shown in Figure 3B. Magnification, x14,000.*

In response to pressure overload, the heart ventricles undergo a series of adaptive events. In response to systemic and pulmonary hypertension, the left ventricle (LV) and the RV, respectively hypertrophy in order to increase the force of muscle contraction. Concentric hypertrophy is the first change that occurs in response to chronic pressure overload, and this compensatory mechanism allows for improved cardiac output. Exercise-induced cardiac hypertrophy, for example, increases the force of contraction in accordance the needs associated with strenuous exercise and training. This adaptive feature is reversible. However, in chronic disease conditions, this compensatory mechanism thickens the ventricular wall too much in a manner that decreases the stroke volume and thus the cardiac output. This results in the second adaptation to decrease the ventricular wall thickness. In case of the LV, the transition from concentric to eccentric hypertrophy predominates,

*Expression of Sox2. SU5416-injected SD rats were subjected to 3 weeks hypoxia and then maintained in normoxia to promote PAH. 17 weeks after the injection, hearts were fixed and subjected to IHC with the antibody against Sox2 (brown stains). (A and B) Sox2 IHC results shown at x200 and at x1,000 magnifications, which clearly show the brown stains in RV cardiomyocytes of SD rats with PAH. (C) This brown Sox2 stain was not observed in control rat RVs without PAH. Scale bars indicate 200* μ*m for x200 and* 

*TEM identification of maturing ICM-like cells in the RV of PAH rats. SD rats were injected with SU5416, subjected to 3 weeks hypoxia, and maintained in normoxia. 20 weeks after the injection, RV tissues were fixed and analyzed by TEM. (A) the image shows normal cardiomyocytes with clear striations and sarcomere structures (arrow). Magnification, x5,500. (B) we also identified some regions with the not well defined sarcomere organization (arrows), which may be in the process of maturing into iCMs, similar to the structure* 

*Scheme depicting our hypothesis. SD rats possess an RV repair mechanism, in which naturally occurring cell reprogramming process converts resident fibroblasts into* α*SMA-and Sox2-positive induced cardiomyocytes (iCMs). These rats also possess the functionalization mechanism that makes functional cardiomyocytes that can elicit strong muscle contraction.*

### **5. Conclusion**

The present study generated evidence to support a fascinating and novel mechanism of naturally occurring cardiac repair that should be important for the development of effective therapeutic strategies to treat cardiac failure. This novel mechanism involves the conversion of resident cardiac fibroblasts into iCMs through the cell reprogramming process that appears to share events occurring in iPSC biology.

#### **Acknowledgements**

This work was supported in part by the NIH (grant numbers R01HL072844, R21AI142649, R03AG059554, and R03AA026516) to Y.J.S. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

#### **Author details**

Nataliia V. Shults and Yuichiro J. Suzuki\* Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington DC, USA

\*Address all correspondence to: ys82@georgetown.edu

© 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.

**27**

*Evidence for the Role of Cell Reprogramming in Naturally Occurring Cardiac Repair*

in incident and prevalent cohorts of patients with pulmonary arterial hypertension. Eur Respir J.

[9] Thenappan T, Shah SJ, Rich S, Tian L, Archer SL, Gomberg-Maitland M. Survival in pulmonary arterial

hypertension: a reappraisal of the NIH risk stratification equation. Eur Respir J.

Pulmonary Hypertension (COMPERA).

[11] Zungu-Edmondson M, Shults NV, Wong CM, Suzuki YJ. Modulators of right ventricular apoptosis and contractility in a rat model of pulmonary hypertension.

[12] Zungu-Edmondson M, Shults NV, Melnyk O, Suzuki YJ. Natural reversal of pulmonary vascular remodeling and right ventricular remodeling in SU5416/ hypoxia-treated Sprague-Dawley rats.

Circulation. 2014;129:57-65.

Cardiovasc Res. 2016;110:30-39.

PLoS One. 2017;12:e0182551.

[13] Taraseviciene-Stewart L, Kasahara Y, Alger L, Hirth P, Mc Mahon G, Waltenberger J, Voelkel NF, Tuder RM. Inhibition of the VEGF receptor 2 combined with chronic hypoxia causes cell death-dependent pulmonary endothelial cell proliferation and severe pulmonary hypertension.

FASEB J. 2001;15:427-438.

2010;36:549-555.

2010;35:1079-1087.

[10] Olsson KM, Delcroix M, Ghofrani HA, Tiede H, Huscher D, Speich R, Grünig E, Staehler G, Rosenkranz S, Halank M, Held M, Lange TJ, Behr J, Klose H, Claussen M, Ewert R, Opitz CF, Vizza CD, Scelsi L, Vonk-Noordegraaf A, Kaemmerer H, Gibbs JS, Coghlan G, Pepke-Zaba J, Schulz U, Gorenflo M, Pittrow D, Hoeper MM. Anticoagulation and survival in pulmonary arterial hypertension: results from the Comparative, Prospective Registry of Newly Initiated Therapies for

*DOI: http://dx.doi.org/10.5772/intechopen.94740*

[1] Fallah F. Recent strategies in treatment of pulmonary arterial hypertension, a review. Glob J Health

[2] Rosenkranz S. Pulmonary hypertension 2015: current definitions, terminology, and novel treatment options. Clin Res Cardiol. 2015;104:

[3] Delcroix M, Naeije R. Optimising the management of pulmonary arterial hypertension patients:

emergency treatments. Eur Respir Rev.

[4] McLaughlin VV, Shah SJ, Souza R, Humbert M. Management of pulmonary arterial hypertension. J Am Coll Cardiol.

[5] D'Alonzo GE, Barst RJ, Ayres SM,

Detre KM, Fishman AP, Goldring RM, Groves BM, Kernis JT, et al. Survival in patients with primary pulmonary hypertension. Results from a national prospective registry. Ann Intern Med.

Bergofsky EH, Brundage BH,

[6] Runo JR, Loyd JE. Primary pulmonary hypertension. Lancet.

[7] Benza RL, Miller DP, Frost A, Barst RJ, Krichman AM, and McGoon MD. Analysis of the lung allocation score estimation of risk of death in patients with pulmonary arterial hypertension using data from the REVEAL Registry. Transplantation.

[8] Humbert M, Sitbon O, Yaïci A, Montani D, O'Callaghan DS, Jaïs X, Parent F, Savale L, Natali D, Günther S, Chaouat A, Chabot F, Cordier JF, Habib G, Gressin V, Jing ZC, Souza R, Simonneau

G; French Pulmonary Arterial Hypertension Network. Survival

Sci. 2015;7:307-322.

**References**

197-207.

2010;19:204-211.

2015;65:1976-97.

1991;115:343-349.

2003;361:1533-1544.

2010;90:298-305.

*Evidence for the Role of Cell Reprogramming in Naturally Occurring Cardiac Repair DOI: http://dx.doi.org/10.5772/intechopen.94740*

#### **References**

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

The present study generated evidence to support a fascinating and novel mechanism of naturally occurring cardiac repair that should be important for the development of effective therapeutic strategies to treat cardiac failure. This novel mechanism involves the conversion of resident cardiac fibroblasts into iCMs through the cell reprogramming process that appears to share events occurring in iPSC biology.

This work was supported in part by the NIH (grant numbers R01HL072844, R21AI142649, R03AG059554, and R03AA026516) to Y.J.S. The content is solely the responsibility of the authors and does not necessarily represent the official views of

**26**

**Author details**

**5. Conclusion**

**Acknowledgements**

the NIH.

Nataliia V. Shults and Yuichiro J. Suzuki\*

provided the original work is properly cited.

\*Address all correspondence to: ys82@georgetown.edu

Center, Washington DC, USA

Department of Pharmacology and Physiology, Georgetown University Medical

© 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,

[1] Fallah F. Recent strategies in treatment of pulmonary arterial hypertension, a review. Glob J Health Sci. 2015;7:307-322.

[2] Rosenkranz S. Pulmonary hypertension 2015: current definitions, terminology, and novel treatment options. Clin Res Cardiol. 2015;104: 197-207.

[3] Delcroix M, Naeije R. Optimising the management of pulmonary arterial hypertension patients: emergency treatments. Eur Respir Rev. 2010;19:204-211.

[4] McLaughlin VV, Shah SJ, Souza R, Humbert M. Management of pulmonary arterial hypertension. J Am Coll Cardiol. 2015;65:1976-97.

[5] D'Alonzo GE, Barst RJ, Ayres SM, Bergofsky EH, Brundage BH, Detre KM, Fishman AP, Goldring RM, Groves BM, Kernis JT, et al. Survival in patients with primary pulmonary hypertension. Results from a national prospective registry. Ann Intern Med. 1991;115:343-349.

[6] Runo JR, Loyd JE. Primary pulmonary hypertension. Lancet. 2003;361:1533-1544.

[7] Benza RL, Miller DP, Frost A, Barst RJ, Krichman AM, and McGoon MD. Analysis of the lung allocation score estimation of risk of death in patients with pulmonary arterial hypertension using data from the REVEAL Registry. Transplantation. 2010;90:298-305.

[8] Humbert M, Sitbon O, Yaïci A, Montani D, O'Callaghan DS, Jaïs X, Parent F, Savale L, Natali D, Günther S, Chaouat A, Chabot F, Cordier JF, Habib G, Gressin V, Jing ZC, Souza R, Simonneau G; French Pulmonary Arterial Hypertension Network. Survival

in incident and prevalent cohorts of patients with pulmonary arterial hypertension. Eur Respir J. 2010;36:549-555.

[9] Thenappan T, Shah SJ, Rich S, Tian L, Archer SL, Gomberg-Maitland M. Survival in pulmonary arterial hypertension: a reappraisal of the NIH risk stratification equation. Eur Respir J. 2010;35:1079-1087.

[10] Olsson KM, Delcroix M, Ghofrani HA, Tiede H, Huscher D, Speich R, Grünig E, Staehler G, Rosenkranz S, Halank M, Held M, Lange TJ, Behr J, Klose H, Claussen M, Ewert R, Opitz CF, Vizza CD, Scelsi L, Vonk-Noordegraaf A, Kaemmerer H, Gibbs JS, Coghlan G, Pepke-Zaba J, Schulz U, Gorenflo M, Pittrow D, Hoeper MM. Anticoagulation and survival in pulmonary arterial hypertension: results from the Comparative, Prospective Registry of Newly Initiated Therapies for Pulmonary Hypertension (COMPERA). Circulation. 2014;129:57-65.

[11] Zungu-Edmondson M, Shults NV, Wong CM, Suzuki YJ. Modulators of right ventricular apoptosis and contractility in a rat model of pulmonary hypertension. Cardiovasc Res. 2016;110:30-39.

[12] Zungu-Edmondson M, Shults NV, Melnyk O, Suzuki YJ. Natural reversal of pulmonary vascular remodeling and right ventricular remodeling in SU5416/ hypoxia-treated Sprague-Dawley rats. PLoS One. 2017;12:e0182551.

[13] Taraseviciene-Stewart L, Kasahara Y, Alger L, Hirth P, Mc Mahon G, Waltenberger J, Voelkel NF, Tuder RM. Inhibition of the VEGF receptor 2 combined with chronic hypoxia causes cell death-dependent pulmonary endothelial cell proliferation and severe pulmonary hypertension. FASEB J. 2001;15:427-438.

[14] Oka M, Homma N, Taraseviciene-Stewart L, Morris KG, Kraskauskas D, Burns N, Voelkel NF, McMurtry IF. Rho kinase-mediated vasoconstriction is important in severe occlusive pulmonary arterial hypertension in rats. Circ Res. 2007;100:923-929.

[15] Ibrahim YF, Wong CM, Pavlickova L, Liu L, Trasar L, Bansal G, Suzuki YJ. Mechanism of the susceptibility of remodeled pulmonary vessels to drug-induced cell killing. J Am Heart Assoc. 2014;3:e000520.

[16] Alzoubi A, Toba M, Abe K, O'Neill KD, Rocic P, Fagan KA McMurtry IF, Oka M. Dehydroepiandrosterone restores right ventricular structure and function in rats with severe pulmonary arterial hypertension. Am J Physiol Heart Circ Physiol. 2013;304:H1708-H1718.

[17] Wang X, Ibrahim YF, Das D, Zungu-Edmondson M, Shults NV, Suzuki YJ. Carfilzomib reverses pulmonary arterial hypertension. Cardiovasc Res. 2016;110:188-199.

[18] Bogaard HJ, Natarajan R, Henderson SC, Long CS, Kraskauskas D, Smithson L, Ockaili R, McCord JM, Voelkel NF. Chronic pulmonary artery pressure elevation is insufficient to explain right heart failure. Circulation. 2009;120:1951-1960.

[19] Bogaard HJ, Natarajan R, Mizuno S, Abbate A, Chang PJ, Chau VQ, Hoke NN, Kraskauskas D, Kasper M, Salloum FN, Voelkel NF. Adrenergic receptor blockade reverses right heart remodeling and dysfunction in pulmonary hypertensive rats. Am J Respir Crit Care Med. 2010;182:652-660.

[20] Yuan X, Braun T. Multimodal regulation of cardiac myocyte proliferation. Circ Res. 2017;121:293-309. [21] Yamanaka S. Induced pluripotent stem cells: Past, present, and future. Cell Stem Cell. 2012;10:678-684.

[22] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663-676.

[23] Ieda M, Fu JD, Delgado-Olguin P, Vedantham V, Hayashi Y, Bruneau BG, Srivastava D. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell. 2010;142:375-386.

[24] Song K, Nam YJ, Luo X, Qi X, Tan W, Huang GN, Acharya A, Smith CL, Tallquist MD, Neilson EG, Hill JA, Bassel-Duby R, Olson EN. Heart repair by reprogramming non-myocytes with cardiac transcription factors. Nature. 2012;485:599-604.

[25] Cao N, Huang Y, Zheng J, Spencer CI, Zhang Y, Fu JD, Nie B, Xie M, Zhang M, Wang H, Ma T, Xu T, Shi G, Srivastava D, Ding S. Conversion of human fibroblasts into functional cardiomyocytes by small molecules. Science. 2016;352:1216-1220.

[26] Voelkel NF, Quaife RA, Leinwand LA, Barst RJ, McGoon MD, Meldrum DR, Dupuis J, Long CS, Rubin LJ, Smart FW, Suzuki YJ, Gladwin M, Denholm EM, Gail DB; Right ventricular function and failure: report of a National Heart, Lung, and Blood Institute working group on cellular and molecular mechanisms of right heart failure. Circulation. 2006;114:1883-1891.

**29**

**Chapter 3**

**Abstract**

cell (MSC)-based therapies.

**1. Introduction**

life in the elderly.

Interventional Strategies to Delay

Aging affects bones, cartilage, muscles, and other connective tissue in the musculoskeletal system, leading to numerous age-related pathologies including osteoporosis, osteoarthritis, and sarcopenia. Understanding healthy aging may therefore open new therapeutic targets, thereby leading to the development of novel approaches to prevent several age-related orthopaedic diseases. It is well recognized that aging-related stem cell depletion and dysfunction leads to reduced regenerative capacity in various musculoskeletal tissues. However, more recent evidence suggests that dysregulated autophagy and cellular senescence might be fundamental mechanisms associated with aging-related musculoskeletal decline. The mammalian/mechanical target of Rapamycin (mTOR) is known to be an essential negative regulator of autophagy, and its inhibition has been demonstrated to promote longevity in numerous species. Besides, several reports demonstrate that selective elimination of senescent cells and their cognate Senescence-Associated Secretory Phenotype (SASP) can mitigate musculoskeletal tissue decline. Therefore, senolytic drugs/agents that can specifically target senescent cells, may offer a novel therapeutic strategy to treat a litany of age-related orthopaedic conditions. This chapter focuses on osteoarthritis and osteoporosis, very common debilitating orthopaedic conditions, and reviews current concepts highlighting new therapeutic strategies, including the mTOR inhibitors, senolytic agents, and mesenchymal stem

**Keywords:** Stem cells, senescence, mTOR, Osteoarthritis, Osteoporosis, aging

Aging is a process of progressive loss of physiological function and reserve, characterized by cellular senescence, stem cell exhaustion, DNA damage, telomere attrition, and deregulated nutrient sensing [1]. It is well known that agingrelated stem cell depletion and dysfunction leads to reduced tissue regenerative capacity in various stem cell populations. Osteoarthritis (OA) and osteoporosis (OP) are two common aging-related skeletal diseases that influence the quality of

Musculoskeletal System

*William S. Hambright, Sudheer Ravuri,* 

*Marc J. Philippon and Johnny Huard*

*Naomasa Fukase, Ingrid K. Stake, Yoichi Murata,* 

Aging-Related Dysfunctions of the

#### **Chapter 3**

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

[21] Yamanaka S. Induced pluripotent stem cells: Past, present, and future. Cell Stem Cell. 2012;10:678-684.

[22] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell.

[23] Ieda M, Fu JD, Delgado-Olguin P, Vedantham V, Hayashi Y, Bruneau BG, Srivastava D. Direct reprogramming

cardiomyocytes by defined factors. Cell.

of fibroblasts into functional

Nature. 2012;485:599-604.

[25] Cao N, Huang Y, Zheng J, Spencer CI, Zhang Y, Fu JD, Nie B, Xie M, Zhang M, Wang H, Ma T, Xu T, Shi G, Srivastava D, Ding S. Conversion of human fibroblasts into functional cardiomyocytes by small molecules.

Science. 2016;352:1216-1220.

[26] Voelkel NF, Quaife RA,

2006;114:1883-1891.

Leinwand LA, Barst RJ, McGoon MD, Meldrum DR, Dupuis J, Long CS, Rubin LJ, Smart FW, Suzuki YJ, Gladwin M, Denholm EM, Gail DB; Right ventricular function and failure: report of a National Heart, Lung, and Blood Institute working group on cellular and molecular mechanisms of right heart failure. Circulation.

[24] Song K, Nam YJ, Luo X, Qi X, Tan W, Huang GN, Acharya A, Smith CL, Tallquist MD, Neilson EG, Hill JA, Bassel-Duby R, Olson EN. Heart repair by reprogramming non-myocytes with cardiac transcription factors.

2006;126:663-676.

2010;142:375-386.

[14] Oka M, Homma N,

2007;100:923-929.

[15] Ibrahim YF, Wong CM, Pavlickova L, Liu L, Trasar L,

Heart Assoc. 2014;3:e000520.

[17] Wang X, Ibrahim YF, Das D, Zungu-Edmondson M, Shults NV, Suzuki YJ. Carfilzomib reverses pulmonary arterial hypertension. Cardiovasc Res. 2016;110:188-199.

[18] Bogaard HJ, Natarajan R,

[19] Bogaard HJ, Natarajan R,

[20] Yuan X, Braun T. Multimodal regulation of cardiac myocyte

proliferation. Circ Res. 2017;121:293-309.

2009;120:1951-1960.

Henderson SC, Long CS, Kraskauskas D, Smithson L, Ockaili R, McCord JM, Voelkel NF. Chronic pulmonary artery pressure elevation is insufficient to explain right heart failure. Circulation.

Mizuno S, Abbate A, Chang PJ, Chau VQ, Hoke NN, Kraskauskas D, Kasper M, Salloum FN, Voelkel NF. Adrenergic receptor blockade reverses right heart remodeling and dysfunction in pulmonary hypertensive rats. Am J Respir Crit Care Med. 2010;182:652-660.

Taraseviciene-Stewart L, Morris KG, Kraskauskas D, Burns N, Voelkel NF, McMurtry IF. Rho kinase-mediated vasoconstriction is important in severe occlusive pulmonary arterial hypertension in rats. Circ Res.

Bansal G, Suzuki YJ. Mechanism of the susceptibility of remodeled pulmonary vessels to drug-induced cell killing. J Am

[16] Alzoubi A, Toba M, Abe K, O'Neill KD, Rocic P, Fagan KA McMurtry IF, Oka M. Dehydroepiandrosterone restores right ventricular structure and function in rats with severe pulmonary arterial hypertension. Am J Physiol Heart Circ Physiol. 2013;304:H1708-H1718.

**28**

## Interventional Strategies to Delay Aging-Related Dysfunctions of the Musculoskeletal System

*Naomasa Fukase, Ingrid K. Stake, Yoichi Murata, William S. Hambright, Sudheer Ravuri, Marc J. Philippon and Johnny Huard*

### **Abstract**

Aging affects bones, cartilage, muscles, and other connective tissue in the musculoskeletal system, leading to numerous age-related pathologies including osteoporosis, osteoarthritis, and sarcopenia. Understanding healthy aging may therefore open new therapeutic targets, thereby leading to the development of novel approaches to prevent several age-related orthopaedic diseases. It is well recognized that aging-related stem cell depletion and dysfunction leads to reduced regenerative capacity in various musculoskeletal tissues. However, more recent evidence suggests that dysregulated autophagy and cellular senescence might be fundamental mechanisms associated with aging-related musculoskeletal decline. The mammalian/mechanical target of Rapamycin (mTOR) is known to be an essential negative regulator of autophagy, and its inhibition has been demonstrated to promote longevity in numerous species. Besides, several reports demonstrate that selective elimination of senescent cells and their cognate Senescence-Associated Secretory Phenotype (SASP) can mitigate musculoskeletal tissue decline. Therefore, senolytic drugs/agents that can specifically target senescent cells, may offer a novel therapeutic strategy to treat a litany of age-related orthopaedic conditions. This chapter focuses on osteoarthritis and osteoporosis, very common debilitating orthopaedic conditions, and reviews current concepts highlighting new therapeutic strategies, including the mTOR inhibitors, senolytic agents, and mesenchymal stem cell (MSC)-based therapies.

**Keywords:** Stem cells, senescence, mTOR, Osteoarthritis, Osteoporosis, aging

#### **1. Introduction**

Aging is a process of progressive loss of physiological function and reserve, characterized by cellular senescence, stem cell exhaustion, DNA damage, telomere attrition, and deregulated nutrient sensing [1]. It is well known that agingrelated stem cell depletion and dysfunction leads to reduced tissue regenerative capacity in various stem cell populations. Osteoarthritis (OA) and osteoporosis (OP) are two common aging-related skeletal diseases that influence the quality of life in the elderly.

Osteoarthritis is one of the most common chronic degenerative joint diseases that cause joint pain and dysfunction. Multiple factors, including mechanical, genetic, and aging-related factors, are involved in the development of OA [2]. At the cellular level, it is characterized by loss of tissue cellularity, and phenotypic changes to chondrocytes, and subsequent damage to the extracellular matrix (ECM) [3]. Chondrocytes are resident cells in cartilage tissue and are involved in both the synthesis and turnover of the ECM [4]; therefore, maintaining chondrocyte health is an essential factor in preventing articular cartilage degeneration. With age, articular cartilage ECM degrades and remodels with the fragmentation of the principal proteoglycan protein aggrecan, chondroitin sulfate, and relative increases in keratan sulfate and hyaluronan deposition [5, 6]. Chondrocytes also exhibit an autolytic phenotype causing the proteolytic cleavage of various collagen molecules [7]. Collectively, such ECM perturbations during aging lead to a concomitant decrease in water content, affecting tensile strength of the cartilage and transmission of load.

Osteoporosis is a systemic bone degenerative disease characterized by a progressive loss of bone mass accompanied by a significant reduction in the mechanical strength of the bones [8]. Bone loss during aging is also characterized by changes in bone shape, mineral content, adiposity, mineral turnover, and reduction in bone forming osteoblasts [9, 10]. Another bi-product of aging is a reduction of bone healing capacity exacerbated by chronic inflammation inherent with advancing age [11] that also disrupts homeostatic interactions between signaling factors and bone cells, resulting in a state of dysregulated remodeling and bone turnover. Thus, aging is one of the factors most closely associated with the development of OP [12]. Brittle bones, as a result of OP in the elderly have been known to cause fractures triggered by minor trauma, significantly worsening the quality of life and even reduce life expectancy [13].

Recent research is beginning to unravel the mechanisms of how aging makes bones and joints more susceptible to the development of these diseases. Understanding the underlying mechanisms may provide new treatments to delay or prevent the development of these aging-related orthopedic disorders. This chapter focuses on OA and OP among aging-related dysfunctions of the skeletal system and reviews current concepts on new therapeutic strategies, especially the mammalian/ mechanistic target of Rapamycin (mTOR) inhibitors, senolytic agents, and mesenchymal stem cells (MSCs).

#### **2. Autophagy as a therapeutic target in aging-related orthopedic diseases**

#### **2.1 Autophagy and mTOR in OA**

Autophagy is an essential homeostatic process for the clearance of damaged intracellular components [14]. It is conserved evolutionarily across species and is commonly known to be activated under stress conditions, including nutrientdeficiency, to generate energy [15]. When cells face nutritional stress, the autophagy pathway is activated to degrade unnecessary intracellular components while maintaining only the minimum essential components to prevent energy loss. Recent evidence has linked autophagy in the pathophysiology of OA. Inhibition of autophagy has been reported to promote the expression of OA-like genes induced by interleukin-1β (IL-1β), whereas activation of autophagy reduces the intracellular reactive oxygen species (ROS) by removing damaged mitochondria, thereby protecting chondrocytes from OA-like changes [16]. During aging and especially in OA, autophagic flux is diminished, likely due to decreases in autophagy regulator genes ULK1, beclin-1, and LC [17], and is associated with increased chondrocyte

**31**

the modulation of autophagy.

*Interventional Strategies to Delay Aging-Related Dysfunctions of the Musculoskeletal System*

apoptosis and mitochondrial dysfunction [18]. These results suggested that increased autophagy is an adaptive response to protect chondrocytes from stress and that autophagy regulates OA-like gene expression changes via the modulation of apoptosis and ROS [19]. Thus, the modulators of autophagy, such as mTOR, may

shown to reduce the severity of OA in pre-clinical animal models [24–27].

Rapamycin is the oldest known natural mTOR inhibitor that has been traditionally used clinically as an immunosuppressive agent [28]. Rapamycin acts through binding of FK506-binding proteins and primarily destabilizes mTORC1, but to some extent can prevent the phosphorylation of downstream targets of mTOR1 [29–31]. Since its FDA approval in 1999, Rapamycin has been used by millions of patients. There is a litany of clinical evidence suggesting that Rapamycin is a safe and effective drug with few side effects for which all are reversible [31, 32]. Interestingly, recent studies have shown that Rapamycin acts to activate human chondrocyte autophagy *in vitro* primarily by inhibiting mTOR complex 1 (mTORC1) and suppressing the development of OA-like changes [19]. Furthermore, systemic administration of Rapamycin has been shown to reduce the

Our group has found that intra-articular injection of Rapamycin was a safe and effective therapeutic delivery method to protect articular cartilage from osteoarthritic changes in a mouse model of OA [27]. On the other hand, deletion of mTOR has been shown to up-regulate autophagy and protect mice from OA in inducible cartilage-specific mTOR knockout mice [24]. Torin 1, a selective ATP-competitive inhibitor of mTOR, which can cause induction of autophagy, is also regarded as a potent inhibitor of both mTORC1 and mTORC2. In a rabbit model of OA, intraarticular injection of Torin 1 was shown to reduce articular cartilage degeneration [33, 34]. Another agent with demonstrated ability to reduce mTOR activity is Metformin, which is FDA-approved for the treatment of type 2 diabetes [35]. Metformin has been shown to activate 5' AMP-activated protein kinase (AMPK), a negative regulator of mTOR. Recently, Metformin has also been shown to inhibit cartilage degeneration in OA mouse models by downregulating mTOR [36]. In another murine study, Metformin was shown to reduce OA structural worsening and reduce pain scores [37]. Therefore, multiple lines of evidence support the theory of mTOR signaling as a promising therapeutic target for OA, mainly through

**2.2 Pre-clinical studies targeting mTOR for the treatment of OA**

severity of OA through activation of autophagy in a mouse model [26].

mTOR is a serine/threonine protein kinase that is a negative regulator of autophagy, integrates inputs from nutrients and growth factors, and regulates many basic cellular processes through two distinct protein complexes, mTORC1 and mTORC2. mTOR is an established longevity axis, and its inhibition either pharmacologically or genetically, has been demonstrated to extend lifespan in numerous species [20–22]. Recent studies suggest that mTOR plays an important role in cartilage growth and development, alters articular cartilage homeostasis, and contributes significantly to the cartilage degenerative process associated with OA [23] mTOR expression has been shown to be elevated in OA models and has been associated with increased chondrocyte apoptosis [24]. Given that mTOR is a negative regulator of autophagy, the deleterious effects of age-associated increases in mTOR activity can be linked to decreased autophagy in chondrocytes during OA. Pre-clinical experiments in rat OA models have also shown that suppressing the PI3K/AKT/mTOR signaling pathway promotes articular chondrocyte autophagy and alleviates inflammation [25]. Both pharmacological and genetic approaches for inhibiting mTOR signaling have been

*DOI: http://dx.doi.org/10.5772/intechopen.97311*

represent key targets to for the treatment of OA.

#### *Interventional Strategies to Delay Aging-Related Dysfunctions of the Musculoskeletal System DOI: http://dx.doi.org/10.5772/intechopen.97311*

apoptosis and mitochondrial dysfunction [18]. These results suggested that increased autophagy is an adaptive response to protect chondrocytes from stress and that autophagy regulates OA-like gene expression changes via the modulation of apoptosis and ROS [19]. Thus, the modulators of autophagy, such as mTOR, may represent key targets to for the treatment of OA.

mTOR is a serine/threonine protein kinase that is a negative regulator of autophagy, integrates inputs from nutrients and growth factors, and regulates many basic cellular processes through two distinct protein complexes, mTORC1 and mTORC2. mTOR is an established longevity axis, and its inhibition either pharmacologically or genetically, has been demonstrated to extend lifespan in numerous species [20–22]. Recent studies suggest that mTOR plays an important role in cartilage growth and development, alters articular cartilage homeostasis, and contributes significantly to the cartilage degenerative process associated with OA [23] mTOR expression has been shown to be elevated in OA models and has been associated with increased chondrocyte apoptosis [24]. Given that mTOR is a negative regulator of autophagy, the deleterious effects of age-associated increases in mTOR activity can be linked to decreased autophagy in chondrocytes during OA. Pre-clinical experiments in rat OA models have also shown that suppressing the PI3K/AKT/mTOR signaling pathway promotes articular chondrocyte autophagy and alleviates inflammation [25]. Both pharmacological and genetic approaches for inhibiting mTOR signaling have been shown to reduce the severity of OA in pre-clinical animal models [24–27].

#### **2.2 Pre-clinical studies targeting mTOR for the treatment of OA**

Rapamycin is the oldest known natural mTOR inhibitor that has been traditionally used clinically as an immunosuppressive agent [28]. Rapamycin acts through binding of FK506-binding proteins and primarily destabilizes mTORC1, but to some extent can prevent the phosphorylation of downstream targets of mTOR1 [29–31]. Since its FDA approval in 1999, Rapamycin has been used by millions of patients. There is a litany of clinical evidence suggesting that Rapamycin is a safe and effective drug with few side effects for which all are reversible [31, 32].

Interestingly, recent studies have shown that Rapamycin acts to activate human chondrocyte autophagy *in vitro* primarily by inhibiting mTOR complex 1 (mTORC1) and suppressing the development of OA-like changes [19]. Furthermore, systemic administration of Rapamycin has been shown to reduce the severity of OA through activation of autophagy in a mouse model [26].

Our group has found that intra-articular injection of Rapamycin was a safe and effective therapeutic delivery method to protect articular cartilage from osteoarthritic changes in a mouse model of OA [27]. On the other hand, deletion of mTOR has been shown to up-regulate autophagy and protect mice from OA in inducible cartilage-specific mTOR knockout mice [24]. Torin 1, a selective ATP-competitive inhibitor of mTOR, which can cause induction of autophagy, is also regarded as a potent inhibitor of both mTORC1 and mTORC2. In a rabbit model of OA, intraarticular injection of Torin 1 was shown to reduce articular cartilage degeneration [33, 34]. Another agent with demonstrated ability to reduce mTOR activity is Metformin, which is FDA-approved for the treatment of type 2 diabetes [35]. Metformin has been shown to activate 5' AMP-activated protein kinase (AMPK), a negative regulator of mTOR. Recently, Metformin has also been shown to inhibit cartilage degeneration in OA mouse models by downregulating mTOR [36]. In another murine study, Metformin was shown to reduce OA structural worsening and reduce pain scores [37]. Therefore, multiple lines of evidence support the theory of mTOR signaling as a promising therapeutic target for OA, mainly through the modulation of autophagy.

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

Osteoarthritis is one of the most common chronic degenerative joint diseases that cause joint pain and dysfunction. Multiple factors, including mechanical, genetic, and aging-related factors, are involved in the development of OA [2]. At the cellular level, it is characterized by loss of tissue cellularity, and phenotypic changes to chondrocytes, and subsequent damage to the extracellular matrix (ECM) [3]. Chondrocytes are resident cells in cartilage tissue and are involved in both the synthesis and turnover of the ECM [4]; therefore, maintaining chondrocyte health is an essential factor in preventing articular cartilage degeneration. With age, articular cartilage ECM degrades and remodels with the fragmentation of the principal proteoglycan protein aggrecan, chondroitin sulfate, and relative increases in keratan sulfate and hyaluronan deposition [5, 6]. Chondrocytes also exhibit an autolytic phenotype causing the proteolytic cleavage of various collagen molecules [7]. Collectively, such ECM perturbations during aging lead to a concomitant decrease in water content, affecting tensile strength of the cartilage and transmission of load. Osteoporosis is a systemic bone degenerative disease characterized by a progressive loss of bone mass accompanied by a significant reduction in the mechanical strength of the bones [8]. Bone loss during aging is also characterized by changes in bone shape, mineral content, adiposity, mineral turnover, and reduction in bone forming osteoblasts [9, 10]. Another bi-product of aging is a reduction of bone healing capacity exacerbated by chronic inflammation inherent with advancing age [11] that also disrupts homeostatic interactions between signaling factors and bone cells, resulting in a state of dysregulated remodeling and bone turnover. Thus, aging is one of the factors most closely associated with the development of OP [12]. Brittle bones, as a result of OP in the elderly have been known to cause fractures triggered by minor trauma, significantly worsening the quality of life and even reduce life

Recent research is beginning to unravel the mechanisms of how aging makes bones and joints more susceptible to the development of these diseases. Understanding the underlying mechanisms may provide new treatments to delay or prevent the development of these aging-related orthopedic disorders. This chapter focuses on OA and OP among aging-related dysfunctions of the skeletal system and reviews current concepts on new therapeutic strategies, especially the mammalian/

mechanistic target of Rapamycin (mTOR) inhibitors, senolytic agents, and

**2. Autophagy as a therapeutic target in aging-related orthopedic diseases**

Autophagy is an essential homeostatic process for the clearance of damaged intracellular components [14]. It is conserved evolutionarily across species and is commonly known to be activated under stress conditions, including nutrientdeficiency, to generate energy [15]. When cells face nutritional stress, the

autophagy pathway is activated to degrade unnecessary intracellular components while maintaining only the minimum essential components to prevent energy loss. Recent evidence has linked autophagy in the pathophysiology of OA. Inhibition of autophagy has been reported to promote the expression of OA-like genes induced by interleukin-1β (IL-1β), whereas activation of autophagy reduces the intracellular reactive oxygen species (ROS) by removing damaged mitochondria, thereby protecting chondrocytes from OA-like changes [16]. During aging and especially in OA, autophagic flux is diminished, likely due to decreases in autophagy regulator genes ULK1, beclin-1, and LC [17], and is associated with increased chondrocyte

**30**

expectancy [13].

mesenchymal stem cells (MSCs).

**2.1 Autophagy and mTOR in OA**

#### **2.3 Autophagy and mTOR in OP**

Multiple proteins involved in the autophagic activity are essential for the survival, differentiation, and function of bone cells, including osteocytes, osteoblasts, and osteoclasts [38]. Autophagy is critical for the necessary crosstalk between bone resident cells and thus plays a critical role in the signaling dynamics for bone synthesis (osteoblasts) and degradation (osteoclasts). Dysregulation in the level of autophagic activity has been found to disrupt the balance between bone formation and bone resorption linked to the onset and progression of OP [38–42]. In addition, autophagy plays an important role in MSC function and lineage determination from adipogenesis to osteoblastogenesis, [43] and it has been linked to the increased adipogenic differentiation in bone MSCs [44]. Autophagic activity is known to decrease with age, especially in bone cells, [45] hence the regulation of autophagic activity is considered a promising strategy for the prevention and treatment of OP [14, 46].

Osteoblast dysfunction is a significant cause of aging-related bone loss, but the mechanisms underlying osteoblast dysfunction with aging are not fully elucidated. mTOR has been shown, through in-vitro studies, to regulate osteogenic genes (Runx2, Osterix), stemness genes (Oct3/4, Nanog), and mineralization through alkaline phosphatase production [47].

#### **2.4 Pre-clinical studies targeting mTOR for the treatment of OP**

Several studies have investigated the efficacy of targeting mTOR for the treatment of OP. Systemic delivery of autophagy modulators such as Rapamycin and its analogs have been tested in a number of animal models. In a study using 24-month-old rats, micro-CT showed that Rapamycin effectively inhibited agingrelated bone loss in trabecular bone. In this study, Rapamycin treatment resulted in a significant decrease in the number of osteoclasts, as well as the induction of osteoclast autophagy and a decrease in osteocyte apoptosis compared to the control group [48]. Besides, mTORC2 signaling stimulates osteoblast differentiation and is involved in aging-related OP [49]. The expression of Rictor, a specific component of mTORC2, is decreased in osteoblasts during aging, which may contribute to aging-related bone loss, and deletion of Rictor in osteoblasts has been shown to accelerate aging-related bone loss in a mouse model [49]. The use of Everolimus, a Rapamycin analog and predominant mTORC1 inhibitor, has been shown to protect against OP onset in ovariectomized rats through the reduction of osteoclast formation and cathepsin K mediated matrix degradation [50]. Rapamycin has also been shown to reduce the severity of age-related bone conditions in trabecular bones of aged male rats by activating osteocyte autophagy [48]. Taken together, mTOR may play a critical role in aging-related OP and represents a promising therapeutic target.

#### **3. Cellular senescence: a new therapeutic strategy for the treatment of aging-related musculoskeletal decline**

#### **3.1 Senescent cells**

Senescent cells play a vital role in the aging process and promote degenerative diseases, geriatric syndromes, and potentially malignancy through the production of a Senescence Associated Secretory Phenotype (SASP) characterized by proinflammatory and catabolic anti-regenerative factors [51]. Senescence is a state

**33**

*matrix protein; HA, Hyaluronic acid.*

**Figure 1.**

*Interventional Strategies to Delay Aging-Related Dysfunctions of the Musculoskeletal System*

defined by replicative arrest and resistance to apoptosis with altered metabolic activity, and several factors, often related to cell or tissue damage, can induce senescence, including DNA lesions, mechanical/shear stress, reactive metabolites, proteotoxic stress, and inflammation [51]. When present, these factors can activate one or more pathways through the p16Ink4a/retinoblastoma protein, p53/p21Cip1, or other transcription factor cascades, resulting in cell cycle arrest, metabolic shifts, altered gene expression, and the production of deleterious SASP

Senescent cells accumulate in tissues throughout the lifespan and are normally removed by the immune system [53]. However, inefficient removal due to chronic stress or pathology may exceed the capacity of the immune system, especially during aging, where chronic inflammation disrupts the homeostatic immunologic clearance mechanisms [51, 53–55]. SASP factors from senescent cells are especially deleterious and include various cytokines, chemokines proteins, growth factors, and tissue degrading matrix metalloproteinases (MMPs) [51, 56, 57]. These factors stimulate inflammation, ECM degradation, fibrosis, and secondary senescence in surrounding cells [51, 54, 58]. SASP components can be cell-type specific, and senescence triggers are influenced by several factors, including hormones, stress, drugs, and pathogens [51]. A schema showing the association between senescence and OA is shown in **Figure 1**. With an increased number of senescent cells, a higher amount of secreted SASP components may cause an inflammatory, apoptotic, and cell- and tissuedestructing effect, eventually resulting in aging and chronic diseases. For these reasons, targeting senescent cells has garnered significant attention for the treatment

of age-associated pathologies, especially musculoskeletal conditions [59].

According to the "geroscience hypothesis" [60], targeting senescent cells is appealing as they are a fundamental property of aging and may thus delay or reverse physiological consequences of the aging process or the development of aging-related diseases [51, 58]. However, there are no established markers

*Schema of the association between senescence and OA. Aging is accompanied by the secretion of the senescenceassociated secretory phenotype (SASP), including various chemokines, cytokines, proteases, and growth factors, which act alone or together to cause degenerative changes in the subchondral bone, synovial fold, and articular cartilage, ultimately leading to OA. IL, interleukin; RANKL, Receptor activator of NF-*κβ *ligand; TNF, tumor necrosis factor; MMPs, Matrix metalloproteinases; TGFβ, Transforming Growth Factor-β; IGF, insulin-like growth factor; OPN, Osteopontin; SOST, Sclerostin; OC, Osteocalcin; PGE, Prostaglandin E; BMPs, Bone morphogenetic proteins; GM-CSF, Granulocyte Macrophage colony-stimulating Factor; CCL, C-C motif chemokine ligand; VEGF, vascular endothelial growth factor; GMCSF, Granulocyte-macrophagecolonystimulating factor; ADAMTS, A disintegrin and metalloproteinase with thrombospondin motifs; GROα, Growth-related oncogene-α; CS-846, The 846 epitope of chondroitin sulfate; COMP, Cartilage oligomeric* 

*DOI: http://dx.doi.org/10.5772/intechopen.97311*

factors [51, 52].

*Interventional Strategies to Delay Aging-Related Dysfunctions of the Musculoskeletal System DOI: http://dx.doi.org/10.5772/intechopen.97311*

defined by replicative arrest and resistance to apoptosis with altered metabolic activity, and several factors, often related to cell or tissue damage, can induce senescence, including DNA lesions, mechanical/shear stress, reactive metabolites, proteotoxic stress, and inflammation [51]. When present, these factors can activate one or more pathways through the p16Ink4a/retinoblastoma protein, p53/p21Cip1, or other transcription factor cascades, resulting in cell cycle arrest, metabolic shifts, altered gene expression, and the production of deleterious SASP factors [51, 52].

Senescent cells accumulate in tissues throughout the lifespan and are normally removed by the immune system [53]. However, inefficient removal due to chronic stress or pathology may exceed the capacity of the immune system, especially during aging, where chronic inflammation disrupts the homeostatic immunologic clearance mechanisms [51, 53–55]. SASP factors from senescent cells are especially deleterious and include various cytokines, chemokines proteins, growth factors, and tissue degrading matrix metalloproteinases (MMPs) [51, 56, 57]. These factors stimulate inflammation, ECM degradation, fibrosis, and secondary senescence in surrounding cells [51, 54, 58]. SASP components can be cell-type specific, and senescence triggers are influenced by several factors, including hormones, stress, drugs, and pathogens [51]. A schema showing the association between senescence and OA is shown in **Figure 1**. With an increased number of senescent cells, a higher amount of secreted SASP components may cause an inflammatory, apoptotic, and cell- and tissuedestructing effect, eventually resulting in aging and chronic diseases. For these reasons, targeting senescent cells has garnered significant attention for the treatment of age-associated pathologies, especially musculoskeletal conditions [59].

According to the "geroscience hypothesis" [60], targeting senescent cells is appealing as they are a fundamental property of aging and may thus delay or reverse physiological consequences of the aging process or the development of aging-related diseases [51, 58]. However, there are no established markers

#### **Figure 1.**

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

Multiple proteins involved in the autophagic activity are essential for the survival, differentiation, and function of bone cells, including osteocytes, osteoblasts, and osteoclasts [38]. Autophagy is critical for the necessary crosstalk between bone resident cells and thus plays a critical role in the signaling dynamics for bone synthesis (osteoblasts) and degradation (osteoclasts). Dysregulation in the level of autophagic activity has been found to disrupt the balance between bone formation and bone resorption linked to the onset and progression of OP [38–42]. In addition, autophagy plays an important role in MSC function and lineage determination from adipogenesis to osteoblastogenesis, [43] and it has been linked to the increased adipogenic differentiation in bone MSCs [44]. Autophagic activity is known to decrease with age, especially in bone cells, [45] hence the regulation of autophagic activity is considered a promising strategy for the prevention and treatment of OP

Osteoblast dysfunction is a significant cause of aging-related bone loss, but the mechanisms underlying osteoblast dysfunction with aging are not fully elucidated. mTOR has been shown, through in-vitro studies, to regulate osteogenic genes (Runx2, Osterix), stemness genes (Oct3/4, Nanog), and mineralization through

Several studies have investigated the efficacy of targeting mTOR for the treatment of OP. Systemic delivery of autophagy modulators such as Rapamycin and its analogs have been tested in a number of animal models. In a study using 24-month-old rats, micro-CT showed that Rapamycin effectively inhibited agingrelated bone loss in trabecular bone. In this study, Rapamycin treatment resulted in a significant decrease in the number of osteoclasts, as well as the induction of osteoclast autophagy and a decrease in osteocyte apoptosis compared to the control group [48]. Besides, mTORC2 signaling stimulates osteoblast differentiation and is involved in aging-related OP [49]. The expression of Rictor, a specific component of mTORC2, is decreased in osteoblasts during aging, which may contribute to aging-related bone loss, and deletion of Rictor in osteoblasts has been shown to accelerate aging-related bone loss in a mouse model [49]. The use of Everolimus, a Rapamycin analog and predominant mTORC1 inhibitor, has been shown to protect against OP onset in ovariectomized rats through the reduction of osteoclast formation and cathepsin K mediated matrix degradation [50]. Rapamycin has also been shown to reduce the severity of age-related bone conditions in trabecular bones of aged male rats by activating osteocyte autophagy [48]. Taken together, mTOR may play a critical role in aging-related OP and represents a

**3. Cellular senescence: a new therapeutic strategy for the treatment of** 

Senescent cells play a vital role in the aging process and promote degenerative diseases, geriatric syndromes, and potentially malignancy through the production of a Senescence Associated Secretory Phenotype (SASP) characterized by proinflammatory and catabolic anti-regenerative factors [51]. Senescence is a state

**2.4 Pre-clinical studies targeting mTOR for the treatment of OP**

**2.3 Autophagy and mTOR in OP**

alkaline phosphatase production [47].

promising therapeutic target.

**3.1 Senescent cells**

**aging-related musculoskeletal decline**

[14, 46].

**32**

*Schema of the association between senescence and OA. Aging is accompanied by the secretion of the senescenceassociated secretory phenotype (SASP), including various chemokines, cytokines, proteases, and growth factors, which act alone or together to cause degenerative changes in the subchondral bone, synovial fold, and articular cartilage, ultimately leading to OA. IL, interleukin; RANKL, Receptor activator of NF-*κβ *ligand; TNF, tumor necrosis factor; MMPs, Matrix metalloproteinases; TGFβ, Transforming Growth Factor-β; IGF, insulin-like growth factor; OPN, Osteopontin; SOST, Sclerostin; OC, Osteocalcin; PGE, Prostaglandin E; BMPs, Bone morphogenetic proteins; GM-CSF, Granulocyte Macrophage colony-stimulating Factor; CCL, C-C motif chemokine ligand; VEGF, vascular endothelial growth factor; GMCSF, Granulocyte-macrophagecolonystimulating factor; ADAMTS, A disintegrin and metalloproteinase with thrombospondin motifs; GROα, Growth-related oncogene-α; CS-846, The 846 epitope of chondroitin sulfate; COMP, Cartilage oligomeric matrix protein; HA, Hyaluronic acid.*

universally specific to senescent cells [51, 54]. Higher expression of p16Ink4a and p21Cip1 usually occur in senescent cells [61, 62]. One of the prominent hallmarks, however, is resistance to apoptosis [63]. Zhu et al. reported that senescent cells anti apoptotic pathway (SCAP) networks are expressed at higher levels compared to non-senescent cells, playing an essential role in the resistance to apoptosis [64]. Of 39 transcripts targeted by small interfering RNAs (siRNAs), six transcripts (ephrin ligand (EFN) B1, EFNB3, p21Cip1, plasminogen-activated inhibitor-2 (PAI-2), phosphatidylinositol-4,5-bisphosphate 3-kinase delta catalytic subunit (PI3KCD), and BCL-xL) were found to downregulate SCAPs and elicit apoptosis in senescent cells. Targeting several SCAP pathways may also be necessary for senescent cell removal and may further increase specificity for senescent cells while not affecting healthy non-senescent cells [51, 64].

#### **3.2 Treatment of aging-related skeletal diseases with senolytic agents**

The use of senolytic agents is a promising approach to delay aging and reduce the severity of chronic diseases through senescent cell depletion [58]. Various drugs have now been characterized that demonstrate the ability to eliminate senescent cells, namely through reduction in anti-apoptotic signaling [65]. For example, Fisetin is a recently characterized senolytic flavonoid phytonutrient found in fruits and vegetables that can extend health and lifespan in naturally aged and progeroid mice [66]. Yousefzadeh et al. tested ten flavonoids and found that Fisetin demonstrated the highest senolytic activity [66]. Fisetin has been found to target senescence associated pathways such as SIRT1, [67] BCL-2/BCL-XL, [68, 69] HIF-1α, [70] p53/MDM2, [69, 71] and AKT, [69, 72] leading to elimination of senescent cells. However, it also has the ability to reduce oxidative stress via SIRT1/Nrf2, [73] decrease mitochondria-derived ROS via inhibition of GSK3β, [73] and exhibit anti-inflammatory effects. Another example, Metformin, is a widely prescribed anti-diabetic drug with lifespan extending effects in mice, [74] and exhibits senolytic activity via targeting some similar, but also several different senescence associated pathways from Fisetin. Metformin is known to activate AMPK, which indirectly inhibits mTOR, the most established nutrient sensing longevity regulating pathway to date [75]. In addition, Metformin reduces oxidative stress and SASP related inflammation through inhibition of NF-kB activity, [75, 76] and inhibits insulin and IGF-1 signaling, which are all hyperactivated during aging [75, 77]. Thus, many senolytic drugs have pleiotropic effects that mitigate age-related cellular and physiological dysfunction. Furthermore, senolytic agents can be administered intermittently since they do not interfere with a receptor or an enzyme, thereby reducing potential side effects [54]. Accordingly, intermittent treatment may maintain the positive effect of senescent cells on wound healing, cancer prevention, and homeostasis [78–81].

The list of agents with senolytic potential is growing. Zhu et al. tested 46 potential senolytic agents and found that Dasatinib and Quercetin demonstrated particularly good results in terms of reducing senescent cells [64]. Dasatinib is a tyrosine kinase inhibitor used in cancer treatment and is known to interfere with ephrin-dependent suppression of apoptosis [82, 83]. Quercetin is a naturally occurring flavonoid found in several fruits and vegetables known to inhibit PI3K, kinases, and serpines [64]. Noteworthy, Dasatinib, in combination with Quercetin was able to induce apoptosis more effectively in a broader range of senescent cell types than either one alone [84]. Navitoclax, a lymphoma drug, inhibits the anti-apoptotic proteins in the Bcl-2 family and has been used in cancer treatment [85]. It has demonstrated senolytic effects in several cell types *in vitro*. However, in aged mice,

**35**

*Interventional Strategies to Delay Aging-Related Dysfunctions of the Musculoskeletal System*

it demonstrated reduced trabecular bone volume and impaired osteoblast function [86]. Additionally, Navitoclax is directed against a small number of SCAP network molecules and has shown poor specificity for senescent cells with increased risk of side effects, including thrombocytopenia due to its effect on platelets [87]. Thus, not all drugs with senolytic ability are clinically suitable due to a range of potential side effects or drug-to-drug interactions with existing medications. However, the ability to transiently dose and the existence of natural compounds with favorable safety profiles (ex. Fisetin, Quercetin, Metformin, Rapamycin, Geldanamycin etc.)

Some senolytic agents have been tested for their effects on the musculoskeletal system, demonstrating a potential to reduce aging-related diseases and conditions. Here we will focus on the use of senolytic agents in the setting of OA and OP.

Senescent cells have long been associated with OA [88, 89]. Articular cartilage from patients with advanced OA have a significant number of senescent chondrocytes, [90, 91] with canonical hallmarks of senescence, including metabolic dysfunction, telomere attrition, and decreased autophagy [51, 52]. Aging-related oxidative stress, in addition to mechanical stress from cartilage loading, may contribute to chondrocyte senescence [88]. The cell's ability to compensate for stress reduces with aging, and an accumulation of senescent cells results in a loss of homeostasis with increased secretion of inflammatory mediators, reduced matrix synthesis, and impaired response to growth factor stimulation. This may result in the development of OA. The link between senescence and OA was demonstrated by Xu et al. [92]. Three months after injection of senescent cells into the knee joint of mice, they found changes suggestive of OA, including damage to cartilage and menisci, osteophytes, and changes of the subchondral bone. The mice also demonstrated pain and reduced function. These changes were not found in the control group, suggesting that high concentrations of senescent cells may act detrimentally on the articular cartilage homeostasis [92]. In addition, other groups have shown that local clearance of senescent cells genetically within the intra-articular space significantly reduced the development of injury-induced OA and promoted a pro-

The role of the senescence marker p16Ink4a was studied by Diekman et al. [94]. The mRNA expression of p16Ink4a was significantly higher in aged mice than in young mice, which inhibited chondrocyte proliferation. Interestingly, SASP factors correlated to the expression of p16Ink4a regardless of age; however, inhibition of p16Ink4a did not affect the SASP production or prevent the development of age-induced or post-traumatic OA of the knee joint. In another study, Zheng et al. studied the effect of Fisetin *in vitro* on IL-1β stimulated human chondrocytes and *in vivo* in murine OA models [95]. In the IL-1β stimulated human chondrocytes, Fisetin increased the expression of the enzyme silent information regulator (SIRT) 1 and thereby inhibited the IL-1β induced increased levels of NO, PGE2, IL-6, TNFα. Additionally, the mRNA expression and the protein levels of the IL-1β induced iNOs, COX-2, MMP-3, MMP-13, ADAMTS-5 were inhibited, and the induced downregulation of SOX-9, aggrecan, and collagen-II degradation was increased. Sirtinol, an inhibitor of SIRT1, reversed the effects of Fisetin on chondrocytes. In the murine OA models, Fisetin demonstrated findings of attenuated progression of

OA, thus highlighting its potential as a senolytic treatment for OA [67].

*DOI: http://dx.doi.org/10.5772/intechopen.97311*

offer the potential for continuous administration.

*3.2.1.1 Pre-clinical studies of senolytic agents for the treatment of OA*

*3.2.1 Agents for the treatment of OA*

regenerative environment [93].

#### *Interventional Strategies to Delay Aging-Related Dysfunctions of the Musculoskeletal System DOI: http://dx.doi.org/10.5772/intechopen.97311*

it demonstrated reduced trabecular bone volume and impaired osteoblast function [86]. Additionally, Navitoclax is directed against a small number of SCAP network molecules and has shown poor specificity for senescent cells with increased risk of side effects, including thrombocytopenia due to its effect on platelets [87]. Thus, not all drugs with senolytic ability are clinically suitable due to a range of potential side effects or drug-to-drug interactions with existing medications. However, the ability to transiently dose and the existence of natural compounds with favorable safety profiles (ex. Fisetin, Quercetin, Metformin, Rapamycin, Geldanamycin etc.) offer the potential for continuous administration.

Some senolytic agents have been tested for their effects on the musculoskeletal system, demonstrating a potential to reduce aging-related diseases and conditions. Here we will focus on the use of senolytic agents in the setting of OA and OP.

#### *3.2.1 Agents for the treatment of OA*

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

healthy non-senescent cells [51, 64].

prevention, and homeostasis [78–81].

universally specific to senescent cells [51, 54]. Higher expression of p16Ink4a and p21Cip1 usually occur in senescent cells [61, 62]. One of the prominent hallmarks, however, is resistance to apoptosis [63]. Zhu et al. reported that senescent cells anti apoptotic pathway (SCAP) networks are expressed at higher levels compared to non-senescent cells, playing an essential role in the resistance to apoptosis [64]. Of 39 transcripts targeted by small interfering RNAs (siRNAs), six transcripts (ephrin ligand (EFN) B1, EFNB3, p21Cip1, plasminogen-activated inhibitor-2 (PAI-2), phosphatidylinositol-4,5-bisphosphate 3-kinase delta catalytic subunit (PI3KCD), and BCL-xL) were found to downregulate SCAPs and elicit apoptosis in senescent cells. Targeting several SCAP pathways may also be necessary for senescent cell removal and may further increase specificity for senescent cells while not affecting

**3.2 Treatment of aging-related skeletal diseases with senolytic agents**

The use of senolytic agents is a promising approach to delay aging and reduce the severity of chronic diseases through senescent cell depletion [58]. Various drugs have now been characterized that demonstrate the ability to eliminate senescent cells, namely through reduction in anti-apoptotic signaling [65]. For example, Fisetin is a recently characterized senolytic flavonoid phytonutrient found in fruits and vegetables that can extend health and lifespan in naturally aged and progeroid mice [66]. Yousefzadeh et al. tested ten flavonoids and found that Fisetin demonstrated the highest senolytic activity [66]. Fisetin has been found to target senescence associated pathways such as SIRT1, [67] BCL-2/BCL-XL, [68, 69] HIF-1α, [70] p53/MDM2, [69, 71] and AKT, [69, 72] leading to elimination of senescent cells. However, it also has the ability to reduce oxidative stress via SIRT1/Nrf2, [73] decrease mitochondria-derived ROS via inhibition of GSK3β, [73] and exhibit anti-inflammatory effects. Another example, Metformin, is a widely prescribed anti-diabetic drug with lifespan extending effects in mice, [74] and exhibits senolytic activity via targeting some similar, but also several different senescence associated pathways from Fisetin. Metformin is known to activate AMPK, which indirectly inhibits mTOR, the most established nutrient sensing longevity regulating pathway to date [75]. In addition, Metformin reduces oxidative stress and SASP related inflammation through inhibition of NF-kB activity, [75, 76] and inhibits insulin and IGF-1 signaling, which are all hyperactivated during aging [75, 77]. Thus, many senolytic drugs have pleiotropic effects that mitigate age-related cellular and physiological dysfunction. Furthermore, senolytic agents can be administered intermittently since they do not interfere with a receptor or an enzyme, thereby reducing potential side effects [54]. Accordingly, intermittent treatment may maintain the positive effect of senescent cells on wound healing, cancer

The list of agents with senolytic potential is growing. Zhu et al. tested 46 potential senolytic agents and found that Dasatinib and Quercetin demonstrated particularly good results in terms of reducing senescent cells [64]. Dasatinib is a tyrosine kinase inhibitor used in cancer treatment and is known to interfere with ephrin-dependent suppression of apoptosis [82, 83]. Quercetin is a naturally occurring flavonoid found in several fruits and vegetables known to inhibit PI3K, kinases, and serpines [64]. Noteworthy, Dasatinib, in combination with Quercetin was able to induce apoptosis more effectively in a broader range of senescent cell types than either one alone [84]. Navitoclax, a lymphoma drug, inhibits the anti-apoptotic proteins in the Bcl-2 family and has been used in cancer treatment [85]. It has demonstrated senolytic effects in several cell types *in vitro*. However, in aged mice,

**34**

#### *3.2.1.1 Pre-clinical studies of senolytic agents for the treatment of OA*

Senescent cells have long been associated with OA [88, 89]. Articular cartilage from patients with advanced OA have a significant number of senescent chondrocytes, [90, 91] with canonical hallmarks of senescence, including metabolic dysfunction, telomere attrition, and decreased autophagy [51, 52]. Aging-related oxidative stress, in addition to mechanical stress from cartilage loading, may contribute to chondrocyte senescence [88]. The cell's ability to compensate for stress reduces with aging, and an accumulation of senescent cells results in a loss of homeostasis with increased secretion of inflammatory mediators, reduced matrix synthesis, and impaired response to growth factor stimulation. This may result in the development of OA. The link between senescence and OA was demonstrated by Xu et al. [92]. Three months after injection of senescent cells into the knee joint of mice, they found changes suggestive of OA, including damage to cartilage and menisci, osteophytes, and changes of the subchondral bone. The mice also demonstrated pain and reduced function. These changes were not found in the control group, suggesting that high concentrations of senescent cells may act detrimentally on the articular cartilage homeostasis [92]. In addition, other groups have shown that local clearance of senescent cells genetically within the intra-articular space significantly reduced the development of injury-induced OA and promoted a proregenerative environment [93].

The role of the senescence marker p16Ink4a was studied by Diekman et al. [94]. The mRNA expression of p16Ink4a was significantly higher in aged mice than in young mice, which inhibited chondrocyte proliferation. Interestingly, SASP factors correlated to the expression of p16Ink4a regardless of age; however, inhibition of p16Ink4a did not affect the SASP production or prevent the development of age-induced or post-traumatic OA of the knee joint. In another study, Zheng et al. studied the effect of Fisetin *in vitro* on IL-1β stimulated human chondrocytes and *in vivo* in murine OA models [95]. In the IL-1β stimulated human chondrocytes, Fisetin increased the expression of the enzyme silent information regulator (SIRT) 1 and thereby inhibited the IL-1β induced increased levels of NO, PGE2, IL-6, TNFα. Additionally, the mRNA expression and the protein levels of the IL-1β induced iNOs, COX-2, MMP-3, MMP-13, ADAMTS-5 were inhibited, and the induced downregulation of SOX-9, aggrecan, and collagen-II degradation was increased. Sirtinol, an inhibitor of SIRT1, reversed the effects of Fisetin on chondrocytes. In the murine OA models, Fisetin demonstrated findings of attenuated progression of OA, thus highlighting its potential as a senolytic treatment for OA [67].

SIRT1 has been reported to be important for cartilage homeostasis by promoting chondrocyte survival and ECM homeostasis [96]. However, data suggest that SIRT1 is proteolytically inactivated during OA [97]. Batshon et al. demonstrated that the NT/CT SIRT1 fragments were found in serum, and an elevated serum NT/CT SIRT1 ratio was associated with both post-traumatic and aging-related OA in mice [98]. A similar increase was found in humans with OA. Further analysis confirmed that the elevated NT/CT SIRT1 fragments are derived from chondrocytes. Senolytic treatment decreased the serum NT/CT SIRT1 ratio and enhanced the intracellular level of SIRT1 in chondrocytes, which correlated with the reduced severity of OA. Dai et al. showed that the combination of Dasatinib and Quercetin to remove senescent chondroprogenitor cells can inhibit SASP formation and thus effectively improve the results of distraction arthroplasty in vitro and in vivo [84]. Recently, it has also been found that intra-articular injection of Navitoclax in post-traumatic OA rats can reduce inflammation, remove senescent chondrocytes in OA, and promote chondrogenic phenotype [99]. Jeon et al. recently tested UBX0101, a senolytic that was found to selectively eliminate senescent cells, found that intra-articular administration of UBX0101 reduced the incidence of post-traumatic OA and associated pain resulting in the development of a prochondrogenic environment [93]. Faust et al. reported that IL-17 expression was increased in mice with post-traumatic OA and in aged mice, and that there was a correlation between senescent cells and IL-17. In their study, senescent fibroblasts increased the level of Th17 cells, when stimulated by IL-6, IL-1β, and TGF-β, and Th17 cells induced senescence in fibroblasts. Inhibition of senescent cells in mice reduced Th17 cells and IL-17; however, both local (UBX0101) and systemic (Navitoclax) senolytic treatment was necessary to reduce cartilage degeneration in aged mice [100]. These findings would provide markers for diagnostic screening and targets for senolytic agents in the treatment of OA.

#### *3.2.1.2 Clinical studies of senolytic agents for the treatment of OA*

Several Phase 1 and 2 clinical trials on senolytic agents for the treatment of OA are underway (ClinicalTrials.gov ID: NCT03513016, NCT04229225, NCT04129944, NCT04210986). At our clinic, we are carrying out multiple Phase I/II randomized controlled trials examining the efficacy of Fisetin in the setting of knee OA with (NCT04210986) or without co-treatment with bone marrow concentrate (BMC). In these studies, patients diagnosed with Kellgren-Lawrence grade II-IV knee OA and a numerical rating scale (NRS) pain score of 4–10 are included. Outcome measures include safety and tolerability of Fisetin administration for two days on and 28 days off (20 mg/kg), as well as patient report pain and function indices, OA and SASP related biomarkers, and magnetic resonance imaging (MRI) of cartilage. There have also been multiple other trials using the senolytic agent UBX0101 carried out by Unity Biotechnology (NCT04229225, NCT04129944, NCT03513016, NCT03100799). Many of these studies demonstrated the safety and tolerability of UBX0101 injected intra-articularly with different dosing regimens (single- vs. multidose, ascending doses) (https://doi.org/10.1016/j.joca.2020.02.752). However, none of the studies demonstrated a significant reduction in pain up to 12 weeks as assessed by the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scoring system. A long-term outcome trial measuring safety and tolerability of UBX0101 at one year was also terminated, failing to meet primary or secondary objectives. (https://clinicaltrials.gov/ct2/show/NCT04349956) While many of these trials are underway, senolytic treatment for OA may nonetheless be a potentially groundbreaking novel treatment strategy to ameliorate the onset and/or progression of OA. More studies are needed to better understand therapeutic delivery (oral vs.

**37**

*Interventional Strategies to Delay Aging-Related Dysfunctions of the Musculoskeletal System*

senescent cells of the joint may provide a more effective intervention.

*3.2.2.1 Pre-clinical studies of senolytic agents for the treatment of OP*

intra-articular), dosing, and senolytic drug of choice. It stands to reason that a combination of senolytic agents or alternative senolytics with higher potency to eliminate

Several studies have located senescent cells in bone with aging [101, 102]. Farr et al. reported a higher expression of the senolytic markers p16Ink4a, p21Cip1, and p53, especially in osteocytes and myeloid cells, in aged mice compared to young mice [101]. Corresponding with the senescent osteocytes, the aged mice also demonstrated higher levels of SASP genes. Similar results were reported in bone biopsies from humans [103], suggesting that senescent osteocytes and SASPs may play an important role in aging-related OP. The role of senescent cells was further linked using a transgenic mouse line carrying a suicide transgene (INK-ATTAC) whereby p16Ink4a cells could be eliminated with the treatment of a drug (AP20187). The results showed that the AP20187 treated aged mice demonstrated clearance of senescent cells, lower osteoclast numbers, and improved trabecular bone of the spine and femur compared to the vehicle treated mice. In contrast, treatment with AP20187 in young mice did not change the bone quality. Further, this study showed similar results with oral senolytic treatment using Dasatinib and Quercetin, which led to significantly lower p16Ink4a mRNA expression and percentage of senescent osteocytes in bone compared to vehicle. They also found that the use of the JAK inhibitor Ruxolitinib reduced the SASP factors IL-6, IL-8, and PAI-1 with concomitant improved spine and femur bone microarchitecture [104]. These results suggest that targeting senescent cells or SASP from senescent cells may reduce bone resorption and maintain or enhance bone formation. Therefore, senolytic drugs may be a

*DOI: http://dx.doi.org/10.5772/intechopen.97311*

*3.2.2 Senolytic agents for the treatment of OP*

promising alternative for treating aging-related OP.

*3.2.2.2 Clinical studies of senolytic agents for the treatment of OP*

Osteoporosis is a debilitating disease that significantly increases the risk of fracture, costing an estimated 13.8 billion USD annually, and directly increases the mortality rate by more than 30% in elderly Americans [105]. Pharmacological treatment for OP consists of two main categories; antiresorptive (bisphosphonates, estrogen agonists, etc.) and anabolic drugs, all with the intent to reduce fracture incidence [106]. However, current therapies are limited given the widely known side effects of chronic use, including the functional decline of the gastrointestinal tract and kidneys, osteonecrosis, esophageal cancer, osteogenic sarcoma, atrial fibrillation, and venous thromboembolism [107]. Further, anti-resorptive therapies, the general first-line approach, are uniformly associated with a concomitant reduction in bone formation, which prevents optimal fracture healing [108]. Thus, disease modifying alternatives with a better safety profile (or that require less dosing) is certainly needed for the treatment of OP. Like OA, cellular senescence is thought to be a fundamental driver of age-associated decline in a bone [104]. Several clinical trials are investigating the use of senotherapeutic drugs in the setting of OP.

In one Phase 2 randomized controlled trial, the senolytic drugs Dasatinib plus Quercetin and Fisetin alone are being tested in healthy elderly women aged 70+, when bone density is known to be reduced (ClinicalTrials.gov ID: NCT04313634). Bone turnover serum markers CTX-I and P2NP are measured with and without senolytic treatment. There are also two other active trials examining the effects of Fisetin for the treatment of frailty syndrome (NCT03675724, NCT03430037), an

intra-articular), dosing, and senolytic drug of choice. It stands to reason that a combination of senolytic agents or alternative senolytics with higher potency to eliminate senescent cells of the joint may provide a more effective intervention.

#### *3.2.2 Senolytic agents for the treatment of OP*

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

*3.2.1.2 Clinical studies of senolytic agents for the treatment of OA*

Several Phase 1 and 2 clinical trials on senolytic agents for the treatment of OA are underway (ClinicalTrials.gov ID: NCT03513016, NCT04229225, NCT04129944, NCT04210986). At our clinic, we are carrying out multiple Phase I/II randomized controlled trials examining the efficacy of Fisetin in the setting of knee OA with (NCT04210986) or without co-treatment with bone marrow concentrate (BMC). In these studies, patients diagnosed with Kellgren-Lawrence grade II-IV knee OA and a numerical rating scale (NRS) pain score of 4–10 are included. Outcome measures include safety and tolerability of Fisetin administration for two days on and 28 days off (20 mg/kg), as well as patient report pain and function indices, OA and SASP related biomarkers, and magnetic resonance imaging (MRI) of cartilage. There have also been multiple other trials using the senolytic agent UBX0101 carried out by Unity Biotechnology (NCT04229225, NCT04129944, NCT03513016, NCT03100799). Many of these studies demonstrated the safety and tolerability of UBX0101 injected intra-articularly with different dosing regimens (single- vs. multidose, ascending doses) (https://doi.org/10.1016/j.joca.2020.02.752). However, none of the studies demonstrated a significant reduction in pain up to 12 weeks as assessed by the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scoring system. A long-term outcome trial measuring safety and tolerability of UBX0101 at one year was also terminated, failing to meet primary or secondary objectives. (https://clinicaltrials.gov/ct2/show/NCT04349956) While many of these trials are underway, senolytic treatment for OA may nonetheless be a potentially groundbreaking novel treatment strategy to ameliorate the onset and/or progression of OA. More studies are needed to better understand therapeutic delivery (oral vs.

SIRT1 has been reported to be important for cartilage homeostasis by promoting chondrocyte survival and ECM homeostasis [96]. However, data suggest that SIRT1 is proteolytically inactivated during OA [97]. Batshon et al. demonstrated that the NT/CT SIRT1 fragments were found in serum, and an elevated serum NT/CT SIRT1 ratio was associated with both post-traumatic and aging-related OA in mice [98]. A similar increase was found in humans with OA. Further analysis confirmed that the elevated NT/CT SIRT1 fragments are derived from chondrocytes. Senolytic treatment decreased the serum NT/CT SIRT1 ratio and enhanced the intracellular level of SIRT1 in chondrocytes, which correlated with the reduced severity of OA. Dai et al. showed that the combination of Dasatinib and Quercetin to remove senescent chondroprogenitor cells can inhibit SASP formation and thus effectively improve the results of distraction arthroplasty in vitro and in vivo [84]. Recently, it has also been found that intra-articular injection of Navitoclax in post-traumatic OA rats can reduce inflammation, remove senescent chondrocytes in OA, and promote chondrogenic phenotype [99]. Jeon et al. recently tested UBX0101, a senolytic that was found to selectively eliminate senescent cells, found that intra-articular administration of UBX0101 reduced the incidence of post-traumatic OA and associated pain resulting in the development of a prochondrogenic environment [93]. Faust et al. reported that IL-17 expression was increased in mice with post-traumatic OA and in aged mice, and that there was a correlation between senescent cells and IL-17. In their study, senescent fibroblasts increased the level of Th17 cells, when stimulated by IL-6, IL-1β, and TGF-β, and Th17 cells induced senescence in fibroblasts. Inhibition of senescent cells in mice reduced Th17 cells and IL-17; however, both local (UBX0101) and systemic (Navitoclax) senolytic treatment was necessary to reduce cartilage degeneration in aged mice [100]. These findings would provide markers for diagnostic screening and targets for senolytic agents in the treat-

**36**

ment of OA.

#### *3.2.2.1 Pre-clinical studies of senolytic agents for the treatment of OP*

Several studies have located senescent cells in bone with aging [101, 102]. Farr et al. reported a higher expression of the senolytic markers p16Ink4a, p21Cip1, and p53, especially in osteocytes and myeloid cells, in aged mice compared to young mice [101]. Corresponding with the senescent osteocytes, the aged mice also demonstrated higher levels of SASP genes. Similar results were reported in bone biopsies from humans [103], suggesting that senescent osteocytes and SASPs may play an important role in aging-related OP. The role of senescent cells was further linked using a transgenic mouse line carrying a suicide transgene (INK-ATTAC) whereby p16Ink4a cells could be eliminated with the treatment of a drug (AP20187). The results showed that the AP20187 treated aged mice demonstrated clearance of senescent cells, lower osteoclast numbers, and improved trabecular bone of the spine and femur compared to the vehicle treated mice. In contrast, treatment with AP20187 in young mice did not change the bone quality. Further, this study showed similar results with oral senolytic treatment using Dasatinib and Quercetin, which led to significantly lower p16Ink4a mRNA expression and percentage of senescent osteocytes in bone compared to vehicle. They also found that the use of the JAK inhibitor Ruxolitinib reduced the SASP factors IL-6, IL-8, and PAI-1 with concomitant improved spine and femur bone microarchitecture [104]. These results suggest that targeting senescent cells or SASP from senescent cells may reduce bone resorption and maintain or enhance bone formation. Therefore, senolytic drugs may be a promising alternative for treating aging-related OP.

#### *3.2.2.2 Clinical studies of senolytic agents for the treatment of OP*

Osteoporosis is a debilitating disease that significantly increases the risk of fracture, costing an estimated 13.8 billion USD annually, and directly increases the mortality rate by more than 30% in elderly Americans [105]. Pharmacological treatment for OP consists of two main categories; antiresorptive (bisphosphonates, estrogen agonists, etc.) and anabolic drugs, all with the intent to reduce fracture incidence [106]. However, current therapies are limited given the widely known side effects of chronic use, including the functional decline of the gastrointestinal tract and kidneys, osteonecrosis, esophageal cancer, osteogenic sarcoma, atrial fibrillation, and venous thromboembolism [107]. Further, anti-resorptive therapies, the general first-line approach, are uniformly associated with a concomitant reduction in bone formation, which prevents optimal fracture healing [108]. Thus, disease modifying alternatives with a better safety profile (or that require less dosing) is certainly needed for the treatment of OP. Like OA, cellular senescence is thought to be a fundamental driver of age-associated decline in a bone [104]. Several clinical trials are investigating the use of senotherapeutic drugs in the setting of OP.

In one Phase 2 randomized controlled trial, the senolytic drugs Dasatinib plus Quercetin and Fisetin alone are being tested in healthy elderly women aged 70+, when bone density is known to be reduced (ClinicalTrials.gov ID: NCT04313634). Bone turnover serum markers CTX-I and P2NP are measured with and without senolytic treatment. There are also two other active trials examining the effects of Fisetin for the treatment of frailty syndrome (NCT03675724, NCT03430037), an


**39**

**Senolytic Agent** Navitoclax (N) (ABT-263)

BCL-2/BCL-XL family

rat

IA

OA

N/A

N/A

N/A

N/A

N/A

[59, 99, 109]

N: 0 25–5 <sup>μ</sup>M, 4 injections within 4 to 6 weeks post-op.

UBX0101 (U)

MDM2/p53

mice

IA

OA

NCT03513016 Phase I

U: 0.1–0 4 mg, single dose

IA

Knee OA

Safety and tolerability of a single dose of U. (Time Frame: 12 weeks)

U: 1 mM, every other day starting from 2 weeks post-op. (5–6 shots)

NCT04229225

U: Single injection of

IA

Knee OA

Safety and

tolerability of a

single and repeat

dose of U. (Time

Frame: 24 weeks)

8.0 mg at week 0, or

two doses of 4.0 mg

(weeks 0 and 4).

NCT04129944

U: Single dose of

IA

Knee OA

Change in

(WOMAC-A)

Score from

baseline to week

12. (Time Frame:

12 weeks)

0.5 mg, 2 mg, or 4 mg

at week 0.

Phase II

*IA, intra-articular injection.*

**Table 1.**

*Senolytics as Potential Therapeutic Agents for OA and OP.*

Phase I

**Mechanism of Action**

**Species Dose**

**Preclinical Animal Model**

**Route**

**Condition**

**ID (Phase)**

**Dose**

**Route**

**Target Population**

**Primary Endpoint**

**References**

**Clinical Trial**

*Interventional Strategies to Delay Aging-Related Dysfunctions of the Musculoskeletal System*

*DOI: http://dx.doi.org/10.5772/intechopen.97311*

[59, 93, 109]


*Interventional Strategies to Delay Aging-Related Dysfunctions of the Musculoskeletal System DOI: http://dx.doi.org/10.5772/intechopen.97311*

*IA, intra-articular injection.*

**Table 1.**

*Senolytics as Potential Therapeutic Agents for OA and OP.*

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

**38**

**Senolytic** 

**Mechanism** 

**Species Dose**

**Agent**

Dasatinib

BCR-ABL,

rat

Mixed solution

IA

OA

NCT04313634

1. D + Q treatment

oral

elderly

Percent changes

[59, 84, 109]

in serum bone

turnover markers

C-terminal

telopeptide

of type I

collagen and

amino-terminal

propeptide of

type I collagen.

(Time Frame:

20 weeks)

2. F treatment group:

oral

elderly

women

Intermittent dosing

of F (20 mg/kg/day

for 3 consecutive

days) every 28 days,

repeated 5 times in

total.

women

group: Intermittent

dosing of D

(100 mg × 2 days) + Q

(1000 mg × 3

consecutive days)

every 28 days,

repeated 5 times in

total.

Phase II

(1 ml) of D

(500 nM) + Q

(100 μM,

weekly

injection

until 4 weeks

post-op.

Quercetin

BCL-2/

(Q )

BCL-XL

family, PI3K/

AKT, ROS,

MDM2/p53/

p21/serpine

(PAI-1&2),

HIF-1α

Fisetin (F)

BCL-2/

mice

F: 20 mg/kg

oral

OA

NCT04210986

F; 20 mg/kg for two

oral

Knee OA

Evaluation of

[59, 95, 109]

liver and kidney

toxicity and

Tumor Lysis

Syndrome by

measuring

peripheral blood

chemistry.

(Time Frame:

12 months)

consecutive days,

followed by 28 days

off, then 2 more

consecutive days,

Phase I/II

daily

BCL-XL

family, PI3K/

AKT, ROS,

MDM2/p53/

p21/serpine

(PAI-1&2),

HIF-1α,

SIRT1, IL-1β

SRC, c-KIT,

ephrin A

receptor, P53

and PAI-2

(D)

**of Action**

**Preclinical Animal Model**

**Route**

**Condition**

**ID (Phase)**

**Dose**

**Route**

**Target** 

**Primary** 

**References**

**Endpoint**

**Population**

**Clinical Trial**

age-related condition characterized by sarcopenia and decreased bone density. In these studies, primary endpoints include serum inflammatory markers and mobility based on a 6-min walk test.

Another promising senolytic drug is Metformin. There are at least four active trials measuring the effects of Metformin on pre-frail to frail patients (NCT03451006, NCT02570672, NCT04221750, NCT02325245). Primary endpoints in these studies are mobility and motor skills functions, including gait speed, balance ability, and grip strength test, geriatric depression score, and weight loss. Similar to OA trials, many of these trials are not complete, but there is compelling evidence that senolytic agents might benefit a litany of age-related skeletal decline. Details of preclinical animal studies and clinical trials of major senolytics in OA and OP and their mechanisms are summarized in **Table 1** [59, 109].

#### **4. Mesenchymal stem cell (MSC)-based therapy for the treatment of aging-related musculoskeletal decline**

#### **4.1 Biological mechanisms of MSCs**

Mesenchymal stem cells (MSCs) are present in a variety of human tissues, including bone marrow, adipose tissue, synovial tissue, and cord blood [110–112]. However, there is currently a lack of conclusive evidence regarding the potential biological mechanisms of MSCs for the treatment of aging-related musculoskeletal diseases. Understanding the function of MSCs opens up the possibility of developing robust MSC-based therapies for musculoskeletal regenerative medicine. Until now, there are two theories on the mechanism of function: (1) Direct adherence and incorporation into the host tissue and (2) trophic effects resulting from the MSCderived secretomes.

#### *4.1.1 Direct adherence and incorporation of MSCs*

One of the primary advantages of MSCs is their ability to interact with various chemokine receptors (such as CXCR4, which is involved in MSC migration), integrins, selectins, and vascular cell adhesion molecule-1, to home damaged tissues [113–119]. The original hypothesis regarding the tissue regeneration mechanism of MSCs was that implanted cells would migrate directly to injury sites, where they would differentiate into functional cells and eventually promote repair of damaged connective tissue [120]. In support of this hypothesis, it has been reported that injected MSCs have the potential to adhere to the injury site and repair the host cartilage through regeneration, and interestingly, MSCs may also migrate to the injury site for tissue regeneration [121, 122]. However, whether the introduced MSCs are actually taken up into the host tissue and act directly on the damaged tissue has yet to be verified.

#### *4.1.2 Trophic effects of MSCs*

With decades of research on the underlying functionality of MSCs, it has been found that there exists a discrepancy between the frequency and duration of transplants and the remarkable healing power of MSCs [120]. Numerous studies have been conducted to resolve this conundrum, presenting the concept that MSCs possess the ability to maintain the proliferation and survival of certain cell types by secreting trophic factors, and regulating certain aspects of the immune system, thereby ushering MSC-based therapies into a new phase [123].

**41**

vitro [131, 132].

*Interventional Strategies to Delay Aging-Related Dysfunctions of the Musculoskeletal System*

**4.2 Properties and benefits of each type of MSCs on aging-related** 

Bone marrow is generally considered to be the home of hematopoietic stem cells and is known to contain MSCs as part of the stromal fraction [125]. BM-MSCs possess a high potential for cartilage repair due to their ready availability [126]. BM-MSCs have also been widely studied as a treatment for OP due to their high

The effect of BM-MSCs on OA has been verified in numerous animal studies. Chiang et al*.* investigated the effects of intra-articular injection of allogeneic BM-MSCs in an *in vivo* rabbit OA model. They observed that the BM-MSCs transplantation group had significantly better histological scores than the hyaluronic acid injection group [127]. Furthermore, Song et al*.* compared the efficacy of bone marrow mononuclear cells (BMMCs) and BM-MSCs in a sheep OA model and demonstrated that the BM-MSCs group had smaller lesions and a relatively smoother femoral condyle. They also reported that ICRS scores showed a greater improvement in the BM-MSCs group than the BMMCs and PBS (control) groups. They further stated that the results of histology showed fewer changes to cartilage

Ichioka et al*.* demonstrated that direct injection of allogenic BM-MSCs into the bone marrow cavity of irradiated P6 sub-strain of senescence-accelerated mice (SAMP6), an osteoporotic mouse, resulted in inhibition of osteoblast and osteoclast formation in an age-dependent manner and promoted adipogenesis, increased trabecular bone and decreased bone mineral density [129]. Autologous BM-MSC transplantation has been reported to improve bone formation and strengthen osteoporotic bones in ovariectomy (OVX) -treated rabbits [130] and in estrogen-deficient goats, mimicking the postmenopausal OP that occurs in elderly women [110]. However, there is limited support for autologous BM-MSCs to treat OP in elderly patients because BM-MSCs isolated from the bone marrow of elderly patients have shown reduced proliferation and osteogenic capacity in

Analysis of the MSC secretion and proteome has revealed various paracrine factors that can reduce apoptosis and inflammation and stimulate angiogenesis and self-renewal of progenitor cells [120]. Notably, MSCs are known to act as immunosuppressive cells that can alleviate inflammation and reduce monocyte activation by releasing anti-inflammatory factors, including interleukin-1 receptor antagonist (IL-1ra) [124]. Pro-inflammatory cytokines, such as interleukin-1β (IL-1β), are widely known to play an essential role in OA progression [95] and IL-1ra confers an overall inhibitory effect on IL-1ß mediated inflammation and matrix degradation. Taken together, MSCs should confer a therapeutic potential in

*DOI: http://dx.doi.org/10.5772/intechopen.97311*

OA patients.

**musculoskeletal decline**

ability of osteogenesis.

*4.2.1 Bone marrow-derived MSCs (BM-MSCs)*

*4.2.1.2 Pre-clinical studies of BM-MSCs for OA*

and bone in the BM-MSCs group [128].

*4.2.1.3 Pre-clinical studies of bm-MSCs for OP*

*4.2.1.1 Characteristics and advantages of BM-MSCs*

*Interventional Strategies to Delay Aging-Related Dysfunctions of the Musculoskeletal System DOI: http://dx.doi.org/10.5772/intechopen.97311*

Analysis of the MSC secretion and proteome has revealed various paracrine factors that can reduce apoptosis and inflammation and stimulate angiogenesis and self-renewal of progenitor cells [120]. Notably, MSCs are known to act as immunosuppressive cells that can alleviate inflammation and reduce monocyte activation by releasing anti-inflammatory factors, including interleukin-1 receptor antagonist (IL-1ra) [124]. Pro-inflammatory cytokines, such as interleukin-1β (IL-1β), are widely known to play an essential role in OA progression [95] and IL-1ra confers an overall inhibitory effect on IL-1ß mediated inflammation and matrix degradation. Taken together, MSCs should confer a therapeutic potential in OA patients.

#### **4.2 Properties and benefits of each type of MSCs on aging-related musculoskeletal decline**

#### *4.2.1 Bone marrow-derived MSCs (BM-MSCs)*

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

OP and their mechanisms are summarized in **Table 1** [59, 109].

**aging-related musculoskeletal decline**

*4.1.1 Direct adherence and incorporation of MSCs*

**4.1 Biological mechanisms of MSCs**

derived secretomes.

to be verified.

*4.1.2 Trophic effects of MSCs*

based on a 6-min walk test.

age-related condition characterized by sarcopenia and decreased bone density. In these studies, primary endpoints include serum inflammatory markers and mobility

(NCT03451006, NCT02570672, NCT04221750, NCT02325245). Primary endpoints in these studies are mobility and motor skills functions, including gait speed, balance ability, and grip strength test, geriatric depression score, and weight loss. Similar to OA trials, many of these trials are not complete, but there is compelling evidence that senolytic agents might benefit a litany of age-related skeletal decline. Details of preclinical animal studies and clinical trials of major senolytics in OA and

Another promising senolytic drug is Metformin. There are at least four active trials measuring the effects of Metformin on pre-frail to frail patients

**4. Mesenchymal stem cell (MSC)-based therapy for the treatment of** 

Mesenchymal stem cells (MSCs) are present in a variety of human tissues, including bone marrow, adipose tissue, synovial tissue, and cord blood [110–112]. However, there is currently a lack of conclusive evidence regarding the potential biological mechanisms of MSCs for the treatment of aging-related musculoskeletal diseases. Understanding the function of MSCs opens up the possibility of developing robust MSC-based therapies for musculoskeletal regenerative medicine. Until now, there are two theories on the mechanism of function: (1) Direct adherence and incorporation into the host tissue and (2) trophic effects resulting from the MSC-

One of the primary advantages of MSCs is their ability to interact with various chemokine receptors (such as CXCR4, which is involved in MSC migration), integrins, selectins, and vascular cell adhesion molecule-1, to home damaged tissues [113–119]. The original hypothesis regarding the tissue regeneration mechanism of MSCs was that implanted cells would migrate directly to injury sites, where they would differentiate into functional cells and eventually promote repair of damaged connective tissue [120]. In support of this hypothesis, it has been reported that injected MSCs have the potential to adhere to the injury site and repair the host cartilage through regeneration, and interestingly, MSCs may also migrate to the injury site for tissue regeneration [121, 122]. However, whether the introduced MSCs are actually taken up into the host tissue and act directly on the damaged tissue has yet

With decades of research on the underlying functionality of MSCs, it has been

found that there exists a discrepancy between the frequency and duration of transplants and the remarkable healing power of MSCs [120]. Numerous studies have been conducted to resolve this conundrum, presenting the concept that MSCs possess the ability to maintain the proliferation and survival of certain cell types by secreting trophic factors, and regulating certain aspects of the immune system,

thereby ushering MSC-based therapies into a new phase [123].

**40**

#### *4.2.1.1 Characteristics and advantages of BM-MSCs*

Bone marrow is generally considered to be the home of hematopoietic stem cells and is known to contain MSCs as part of the stromal fraction [125]. BM-MSCs possess a high potential for cartilage repair due to their ready availability [126]. BM-MSCs have also been widely studied as a treatment for OP due to their high ability of osteogenesis.

#### *4.2.1.2 Pre-clinical studies of BM-MSCs for OA*

The effect of BM-MSCs on OA has been verified in numerous animal studies. Chiang et al*.* investigated the effects of intra-articular injection of allogeneic BM-MSCs in an *in vivo* rabbit OA model. They observed that the BM-MSCs transplantation group had significantly better histological scores than the hyaluronic acid injection group [127]. Furthermore, Song et al*.* compared the efficacy of bone marrow mononuclear cells (BMMCs) and BM-MSCs in a sheep OA model and demonstrated that the BM-MSCs group had smaller lesions and a relatively smoother femoral condyle. They also reported that ICRS scores showed a greater improvement in the BM-MSCs group than the BMMCs and PBS (control) groups. They further stated that the results of histology showed fewer changes to cartilage and bone in the BM-MSCs group [128].

#### *4.2.1.3 Pre-clinical studies of bm-MSCs for OP*

Ichioka et al*.* demonstrated that direct injection of allogenic BM-MSCs into the bone marrow cavity of irradiated P6 sub-strain of senescence-accelerated mice (SAMP6), an osteoporotic mouse, resulted in inhibition of osteoblast and osteoclast formation in an age-dependent manner and promoted adipogenesis, increased trabecular bone and decreased bone mineral density [129]. Autologous BM-MSC transplantation has been reported to improve bone formation and strengthen osteoporotic bones in ovariectomy (OVX) -treated rabbits [130] and in estrogen-deficient goats, mimicking the postmenopausal OP that occurs in elderly women [110]. However, there is limited support for autologous BM-MSCs to treat OP in elderly patients because BM-MSCs isolated from the bone marrow of elderly patients have shown reduced proliferation and osteogenic capacity in vitro [131, 132].

*4.2.2 Adipose-derived MSCs (A-MSCs)*

#### *4.2.2.1 Characteristics and advantages of A-MSCs*

MSCs were first reported in adipose tissue in 2001 [133] and have been touted as an attractive source of MSCs. Although A-MSCs have the advantage of being easier to isolate than BM-MSCs, [134] BM-MSCs have been shown to be prone to chondrogenic differentiation, both *in vitro* and *in vivo* [135]. Interestingly, however, it has also been reported that the addition of paracrine or cytokine factors increases the cartilage capacity of A-MSCs to a level comparable to that of BM-MSCs [136]. It is worth noting that the yield of A-MSCs and their proliferation and differentiation ability is dependent on the site of tissue collection [137] and the age of the donor [138].

#### *4.2.2.2 Pre-clinical studies of A-MSCs for OA*

The effect of A-MSCs on OA has been investigated in numerous animal studies. Tang et al. compared the efficacy of three types of intra-articular injections, subcutaneous A-MSCs, visceral A-MSCs, and PBS (control), in a rat model of OA. Subcutaneous They found that A-MSCs injection decreased osteophyte and fibrous tissue formation compared to PBS or visceral A-MSCs. In addition, histologically, a smooth cartilage surface and distribution of lacunae and chondrocytes were observed in rats treated with subcutaneous A-MSCs [139]. Kuroda et al. verified the efficacy of A-MSCs for OA treatment using a rabbit model. They found that nearly normal cartilage was observed in the A-MSCs group, with less cartilage damage than in the control group. Further, the proportion of MMP-13 positive cells was significantly lower in sections of the A-MSCs group than in the control group [140].

#### *4.2.2.3 Pre-clinical studies of A-MSCs for OP*

The effects of A-MSCs have also been evaluated in OP animal models. Mirsaidi et al*.* performed the A-MSCs injection to SAMP6 mice and observed improvement of trabecular bone quality [141]. Additionally, Cho et al*.* studied the efficacy of human A-MSCs using OVX nude mice, showing that human A-MSCs could inhibit OVX-induced bone loss over eight weeks [142]. Furthermore, Ye et al*.* found that autologous A-MSCs enhanced bone regeneration in an OVX-induced rabbit model of OP, suggesting that this was due not only to autologous osteogenic differentiation but also to the promotion of osteogenesis and inhibition of adipogenesis through the activation of BMP-2 and BMPR-IB signaling pathways [143].

#### *4.2.3 Synovium-derived MSCs (S-MSCs)*

#### *4.2.3.1 Characteristics and advantages of S-MSCs*

In 2001, De Bari *et al*. isolated the first MSCs from the synovium of the human knee joint [144]. Since then, S-MSCs have attracted attention because they are more readily accessible, possess a higher growth rate, and are less immunogenic compared with MSCs from other origins [145]. Sakaguchi et al*.* compared the yield, expandability, differentiation potential, and epitope profiles of human MSCs derived from five different mesenchymal tissue sources: bone marrow, synovium, periosteum, adipose tissue, and muscle, and concluded that S-MSCs had the highest capacity for chondrogenesis [146].

**43**

female hosts [163].

*Interventional Strategies to Delay Aging-Related Dysfunctions of the Musculoskeletal System*

The beneficial effects of S-MSCs in promoting cartilage regeneration have been reported in pig, [146] leporin, [135] and canine models [147]. In a recent systematic review of *in vivo* studies on synovium-derived mesenchymal stem cell transplantation for cartilage regeneration, To et al*.* showed, in 4 human and 16 animal articles, that S-MSCs possess overall good chondrogenic potential and positive effect for treating chondral lesions and preventing OA [148]. Ozeki et al. found that intraarticular injection of S-MSCs in a rat model of OA could inhibit the OA progression and attenuate synovitis when administered once a week instead of a single dose [149]. Accumulating evidence that S-MSCs possess a strong chondrogenic potential and the fact that MSCs derived from synovial tissue is specific to target joints have led to a growing interest in the application of S-MSCs for a stem cell therapy of

As it pertains to osteogenic potential, Sakaguchi et al*.* showed that S-MSCs possessed a higher capacity than adipose tissue- and muscle-derived cells, comparable to bone marrow-, and periosteal-derived [150]. However, S-MSC-based therapy for

MDSCs/MDSPCs are pluripotent cells isolated from postnatal skeletal muscle via established preplating techniques. They are characterized by multiple critical features such as long-term proliferation/self-renewing capacity, resistance to oxidative and inflammatory stress, and multilineage differentiation potential [151–153]. Recently, it has been shown that skeletal muscle-derived MSCs from OA patients exhibit superior biological properties compared to the bone-derived MSCs counterpart, making them a promising candidate for autologous stem cell therapy [154]. MDSPCs have been shown to improve the regenerative capacity of various tissues, including bone, cartilage, skeletal muscle, and cardiac muscle, by promoting angiogenesis [155–159]. Of note, several studies have investigated differences in the proliferation and differentiation ability of MDSCs by sex and age [158, 160, 161]. It has been shown that male murine MDSPCs displayed higher chondrogenic differentiation capacity and cartilage regeneration potential than female murine MDSPCs [160]. Similarly, Corsi et al*.* showed that the osteogenesis of male murine MDSPCs was superior to that of female MDSPCs [162]. Furthermore, our group has recently found that in human MDSPCs, male MDSPCs possess a greater ability to undergo chondrogenesis

Our group, on the other hand, have reported that not only donor but also host sex affects bone regeneration; male murine hosts showed a greater amount of MDSPC-mediated ectopic bone formation and cranial defect healing than did

An interesting study by Kuroda et al. indicated that local delivery of BMP-4 by genetically engineered MDSCs promoted chondrogenesis with a significant

*4.2.4 Muscle-derived stem cells (MDSCs)/muscle-derived stem progenitor cells* 

*4.2.4.1 Characteristics and advantages of MDSCs/MDSPCs*

and osteogenesis than female MDSPCs [161].

*4.2.4.2 Pre-clinical studies of MDSCs/MDSPCs for OA*

*DOI: http://dx.doi.org/10.5772/intechopen.97311*

*4.2.3.2 Pre-clinical studies of S-MSCs for OA*

*4.2.3.3 Pre-clinical studies of S-MSCs for OP*

OP has yet to be well investigated.

*(MDSPCs)*

knee OA.

*Interventional Strategies to Delay Aging-Related Dysfunctions of the Musculoskeletal System DOI: http://dx.doi.org/10.5772/intechopen.97311*

#### *4.2.3.2 Pre-clinical studies of S-MSCs for OA*

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

MSCs were first reported in adipose tissue in 2001 [133] and have been touted as an attractive source of MSCs. Although A-MSCs have the advantage of being easier to isolate than BM-MSCs, [134] BM-MSCs have been shown to be prone to chondrogenic differentiation, both *in vitro* and *in vivo* [135]. Interestingly, however, it has also been reported that the addition of paracrine or cytokine factors increases the cartilage capacity of A-MSCs to a level comparable to that of BM-MSCs [136]. It is worth noting that the yield of A-MSCs and their proliferation and differentiation ability is dependent on the site of tissue collection [137] and the age of the

The effect of A-MSCs on OA has been investigated in numerous animal studies. Tang et al. compared the efficacy of three types of intra-articular injections, subcutaneous A-MSCs, visceral A-MSCs, and PBS (control), in a rat model of OA. Subcutaneous They found that A-MSCs injection decreased osteophyte and fibrous tissue formation compared to PBS or visceral A-MSCs. In addition, histologically, a smooth cartilage surface and distribution of lacunae and chondrocytes were observed in rats treated with subcutaneous A-MSCs [139]. Kuroda et al. verified the efficacy of A-MSCs for OA treatment using a rabbit model. They found that nearly normal cartilage was observed in the A-MSCs group, with less cartilage damage than in the control group. Further, the proportion of MMP-13 positive cells was significantly lower in sections of the A-MSCs group than in the

The effects of A-MSCs have also been evaluated in OP animal models. Mirsaidi et al*.* performed the A-MSCs injection to SAMP6 mice and observed improvement of trabecular bone quality [141]. Additionally, Cho et al*.* studied the efficacy of human A-MSCs using OVX nude mice, showing that human A-MSCs could inhibit OVX-induced bone loss over eight weeks [142]. Furthermore, Ye et al*.* found that autologous A-MSCs enhanced bone regeneration in an OVX-induced rabbit model of OP, suggesting that this was due not only to autologous osteogenic differentiation but also to the promotion of osteogenesis and inhibition of adipogenesis through

In 2001, De Bari *et al*. isolated the first MSCs from the synovium of the human knee joint [144]. Since then, S-MSCs have attracted attention because they are more readily accessible, possess a higher growth rate, and are less immunogenic compared with MSCs from other origins [145]. Sakaguchi et al*.* compared the yield, expandability, differentiation potential, and epitope profiles of human MSCs derived from five different mesenchymal tissue sources: bone marrow, synovium, periosteum, adipose tissue, and muscle, and concluded that S-MSCs had the highest

the activation of BMP-2 and BMPR-IB signaling pathways [143].

*4.2.2 Adipose-derived MSCs (A-MSCs)*

donor [138].

control group [140].

*4.2.2.1 Characteristics and advantages of A-MSCs*

*4.2.2.2 Pre-clinical studies of A-MSCs for OA*

*4.2.2.3 Pre-clinical studies of A-MSCs for OP*

*4.2.3 Synovium-derived MSCs (S-MSCs)*

capacity for chondrogenesis [146].

*4.2.3.1 Characteristics and advantages of S-MSCs*

**42**

The beneficial effects of S-MSCs in promoting cartilage regeneration have been reported in pig, [146] leporin, [135] and canine models [147]. In a recent systematic review of *in vivo* studies on synovium-derived mesenchymal stem cell transplantation for cartilage regeneration, To et al*.* showed, in 4 human and 16 animal articles, that S-MSCs possess overall good chondrogenic potential and positive effect for treating chondral lesions and preventing OA [148]. Ozeki et al. found that intraarticular injection of S-MSCs in a rat model of OA could inhibit the OA progression and attenuate synovitis when administered once a week instead of a single dose [149]. Accumulating evidence that S-MSCs possess a strong chondrogenic potential and the fact that MSCs derived from synovial tissue is specific to target joints have led to a growing interest in the application of S-MSCs for a stem cell therapy of knee OA.

#### *4.2.3.3 Pre-clinical studies of S-MSCs for OP*

As it pertains to osteogenic potential, Sakaguchi et al*.* showed that S-MSCs possessed a higher capacity than adipose tissue- and muscle-derived cells, comparable to bone marrow-, and periosteal-derived [150]. However, S-MSC-based therapy for OP has yet to be well investigated.

#### *4.2.4 Muscle-derived stem cells (MDSCs)/muscle-derived stem progenitor cells (MDSPCs)*

#### *4.2.4.1 Characteristics and advantages of MDSCs/MDSPCs*

MDSCs/MDSPCs are pluripotent cells isolated from postnatal skeletal muscle via established preplating techniques. They are characterized by multiple critical features such as long-term proliferation/self-renewing capacity, resistance to oxidative and inflammatory stress, and multilineage differentiation potential [151–153]. Recently, it has been shown that skeletal muscle-derived MSCs from OA patients exhibit superior biological properties compared to the bone-derived MSCs counterpart, making them a promising candidate for autologous stem cell therapy [154]. MDSPCs have been shown to improve the regenerative capacity of various tissues, including bone, cartilage, skeletal muscle, and cardiac muscle, by promoting angiogenesis [155–159].

Of note, several studies have investigated differences in the proliferation and differentiation ability of MDSCs by sex and age [158, 160, 161]. It has been shown that male murine MDSPCs displayed higher chondrogenic differentiation capacity and cartilage regeneration potential than female murine MDSPCs [160]. Similarly, Corsi et al*.* showed that the osteogenesis of male murine MDSPCs was superior to that of female MDSPCs [162]. Furthermore, our group has recently found that in human MDSPCs, male MDSPCs possess a greater ability to undergo chondrogenesis and osteogenesis than female MDSPCs [161].

Our group, on the other hand, have reported that not only donor but also host sex affects bone regeneration; male murine hosts showed a greater amount of MDSPC-mediated ectopic bone formation and cranial defect healing than did female hosts [163].

#### *4.2.4.2 Pre-clinical studies of MDSCs/MDSPCs for OA*

An interesting study by Kuroda et al. indicated that local delivery of BMP-4 by genetically engineered MDSCs promoted chondrogenesis with a significant

improvement of articular cartilage repair in rats [164]. This suggests that MDSCs are advantageous concerning chondrogenic differentiation potential. Furthermore, Matsumoto et al. demonstrated in a rat model that MDSCs therapy with sFlt-1 and BMP-4 promotes chondrogenesis in OA, and inhibits cartilage resorption by inhibiting angiogenesis, thus enabling sustained cartilage regeneration and repair [165].

#### *4.2.4.3 Pre-clinical studies of MDSCs/MDSPCs for OP*

Our group isolated young and old populations of gender-matched human muscle-derived stem cells (hMDSCs) to examine the effect of age on osteogenic differentiation using a critical-size calvarial bone defect mouse model. In addition, the effect of donor and host age on hMDSC-mediated bone regeneration was investigated. We showed that donor age did not impair hMDSC-mediated bone regeneration, while host age had the adverse effect. We also found that hMDSCs form functional bone regardless of the age of the donor or host, suggesting that these cells are a promising resource for bone regeneration [166].

#### **4.3 Clinical studies of MSC-based therapy for the treatment of aging-related musculoskeletal decline**

#### *4.3.1 Clinical studies of MSC-based therapy for OA*

More than 30 clinical trials on the administration of intra-articular MSCs for the treatment of OA have been completed to date, including randomized controlled trials, retrospective studies, and cohort studies (https://www.clinicaltrials.gov/), and most of the published results have shown clinical benefit [111, 112, 167–178]. Currently, B-MSCs and A-MSCs are the most commonly used cell sources in clinical trials for OA, [179] however, finding an optimal treatment with MSCs is challenging due to the great diversity of patient populations, delivery methods, cell numbers, culture expansion methodology, and follow-up periods.

Until now, no clinical trials on the benefit of intra-articular administration of S-MSCs in patients with OA have been published. Interestingly, however, Sekiya et al*.* found that arthroscopic S-MSCs transplantation improved the clinical outcome of knees with articular cartilage defects at a mean follow-up of 52 months in 10 patients based on MRI, histology, and clinical outcome score evaluation [180]. Furthermore, Shimomura et al*.* conducted the first human pilot study of implanting scaffold-free tissue-engineered constructs generated from S-MSCs to the injury site for five patients with symptomatic knee cartilage lesions, demonstrating that selfassessed clinical scores for pain, symptoms, activities of daily living, sports activities, and quality of life improved significantly at 24 months postoperatively, with no serious adverse events. In addition, second-look arthroscopy and MRI confirmed complete defect filling in all cases, and biopsy of the regenerated cartilage showed that the repair tissue consisted of hyaline cartilage [181]. These results showed that implantation of S-MSCs could repair articular cartilage damage and prevent its progression to OA, and therefore, future clinical applications in patients with OA may be promising. There is also evidence that, in a phase I/II study, repeated administration of umbilical cord-derived-MSCs improved safety and clinical outcomes for long-term pain in patients with knee OA [112].

#### *4.3.2 Clinical studies of MSC-based therapy for OP*

A recent Phase 1 clinical trial has been conducted using fucosylated BM-MSCs for patients with OP. In this study, autologous BM-MSCs were harvested 30 days

**45**

**Author details**

*Interventional Strategies to Delay Aging-Related Dysfunctions of the Musculoskeletal System*

prior to the infusion and cultured under good manufacturing practice (GMP) conditions to purify and obtain mesenchymal cell established dose range. This study is ongoing, and the recruitment of participants is currently closed. (ClinicalTrials.

Although the use of MSC products for the treatment of aging-related skeletal disorders is becoming increasingly prevalent, well-designed studies are imperative to conclusively prove their clinical benefits and identify the optimal indications, cell

Naomasa Fukase, Ingrid K. Stake, Yoichi Murata, William S. Hambright,

© 2021 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,

Sudheer Ravuri, Marc J. Philippon and Johnny Huard\* Steadman Philippon Research Institute, Vail, CO, USA

\*Address all correspondence to: jhuard@sprivail.org

provided the original work is properly cited.

*DOI: http://dx.doi.org/10.5772/intechopen.97311*

sources, delivery methods, and doses.

gov ID: NCT02566655).

*Interventional Strategies to Delay Aging-Related Dysfunctions of the Musculoskeletal System DOI: http://dx.doi.org/10.5772/intechopen.97311*

prior to the infusion and cultured under good manufacturing practice (GMP) conditions to purify and obtain mesenchymal cell established dose range. This study is ongoing, and the recruitment of participants is currently closed. (ClinicalTrials. gov ID: NCT02566655).

Although the use of MSC products for the treatment of aging-related skeletal disorders is becoming increasingly prevalent, well-designed studies are imperative to conclusively prove their clinical benefits and identify the optimal indications, cell sources, delivery methods, and doses.

#### **Author details**

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

*4.2.4.3 Pre-clinical studies of MDSCs/MDSPCs for OP*

**musculoskeletal decline**

*4.3.1 Clinical studies of MSC-based therapy for OA*

culture expansion methodology, and follow-up periods.

long-term pain in patients with knee OA [112].

*4.3.2 Clinical studies of MSC-based therapy for OP*

these cells are a promising resource for bone regeneration [166].

improvement of articular cartilage repair in rats [164]. This suggests that MDSCs are advantageous concerning chondrogenic differentiation potential. Furthermore, Matsumoto et al. demonstrated in a rat model that MDSCs therapy with sFlt-1 and BMP-4 promotes chondrogenesis in OA, and inhibits cartilage resorption by inhibiting angiogenesis, thus enabling sustained cartilage regeneration and repair [165].

Our group isolated young and old populations of gender-matched human muscle-derived stem cells (hMDSCs) to examine the effect of age on osteogenic differentiation using a critical-size calvarial bone defect mouse model. In addition, the effect of donor and host age on hMDSC-mediated bone regeneration was investigated. We showed that donor age did not impair hMDSC-mediated bone regeneration, while host age had the adverse effect. We also found that hMDSCs form functional bone regardless of the age of the donor or host, suggesting that

**4.3 Clinical studies of MSC-based therapy for the treatment of aging-related** 

More than 30 clinical trials on the administration of intra-articular MSCs for the treatment of OA have been completed to date, including randomized controlled trials, retrospective studies, and cohort studies (https://www.clinicaltrials.gov/), and most of the published results have shown clinical benefit [111, 112, 167–178]. Currently, B-MSCs and A-MSCs are the most commonly used cell sources in clinical trials for OA, [179] however, finding an optimal treatment with MSCs is challenging due to the great diversity of patient populations, delivery methods, cell numbers,

Until now, no clinical trials on the benefit of intra-articular administration of S-MSCs in patients with OA have been published. Interestingly, however, Sekiya et al*.* found that arthroscopic S-MSCs transplantation improved the clinical outcome of knees with articular cartilage defects at a mean follow-up of 52 months in 10 patients based on MRI, histology, and clinical outcome score evaluation [180]. Furthermore, Shimomura et al*.* conducted the first human pilot study of implanting scaffold-free tissue-engineered constructs generated from S-MSCs to the injury site for five patients with symptomatic knee cartilage lesions, demonstrating that selfassessed clinical scores for pain, symptoms, activities of daily living, sports activities, and quality of life improved significantly at 24 months postoperatively, with no serious adverse events. In addition, second-look arthroscopy and MRI confirmed complete defect filling in all cases, and biopsy of the regenerated cartilage showed that the repair tissue consisted of hyaline cartilage [181]. These results showed that implantation of S-MSCs could repair articular cartilage damage and prevent its progression to OA, and therefore, future clinical applications in patients with OA may be promising. There is also evidence that, in a phase I/II study, repeated administration of umbilical cord-derived-MSCs improved safety and clinical outcomes for

A recent Phase 1 clinical trial has been conducted using fucosylated BM-MSCs for patients with OP. In this study, autologous BM-MSCs were harvested 30 days

**44**

Naomasa Fukase, Ingrid K. Stake, Yoichi Murata, William S. Hambright, Sudheer Ravuri, Marc J. Philippon and Johnny Huard\* Steadman Philippon Research Institute, Vail, CO, USA

\*Address all correspondence to: jhuard@sprivail.org

© 2021 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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[175] Gupta, P.K., et al., *Efficacy and safety of adult human bone marrowderived, cultured, pooled, allogeneic mesenchymal stromal cells (Stempeucel®):* 

*osteoarthritis of the knee joint.* Arthritis

[176] Kuah, D., et al., *Safety, tolerability and efficacy of intra-articular Progenza in knee osteoarthritis: a randomized doubleblind placebo-controlled single ascending dose study.* J Transl Med, 2018. **16**(1): p. 49.

[177] Song, Y., et al., *Human adiposederived mesenchymal stem cells for* 

Med, 2018. **13**(3): p. 295-307.

2018. **20**(1).

p. 117-24.

*osteoarthritis: a pilot study with long-term follow-up and repeated injections.* Regen

[178] Spasovski, D., et al., *Intra-articular injection of autologous adipose-derived mesenchymal stem cells in the treatment of knee osteoarthritis.* J Gene Med,

[179] Wyles, C.C., et al., *Mesenchymal stem cell therapy for osteoarthritis: current perspectives.* Stem Cells Cloning, 2015. **8**:

[180] Sekiya, I., et al., *Arthroscopic Transplantation of Synovial Stem Cells Improves Clinical Outcomes in Knees With Cartilage Defects.* Clin Orthop Relat Res,

[181] Shimomura, K., et al., *First-in-Human Pilot Study of Implantation of a Scaffold-Free Tissue-Engineered Construct Generated From Autologous Synovial Mesenchymal Stem Cells for Repair of Knee Chondral Lesions.* Am J Sports Med,

2015. **473**(7): p. 2316-26.

2018. **46**(10): p. 2384-2393.

*preclinical and clinical trial in* 

Res Ther, 2016. **18**(1): p. 301.

*Interventional Strategies to Delay Aging-Related Dysfunctions of the Musculoskeletal System DOI: http://dx.doi.org/10.5772/intechopen.97311*

*intra-articular, autologous adipose tissue injections for the treatment of mild-tomoderate knee osteoarthritis compared to hyaluronic acid: a study protocol.* BMC Musculoskelet Disord, 2018. **19**(1): p. 383.

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

Arthritis Rheum, 2009. **60**(5): p.

[166] Gao, X., et al., *Influences of donor and host age on human muscle-derived stem cell-mediated bone regeneration.* Stem Cell Res Ther, 2018. **9**(1): p. 316.

[167] Koh, Y.G. and Y.J. Choi, *Infrapatellar fat pad-derived mesenchymal stem cell therapy for knee osteoarthritis.* Knee, 2012.

[168] Vega, A., et al., *Treatment of Knee Osteoarthritis With Allogeneic Bone Marrow Mesenchymal Stem Cells: A Randomized Controlled Trial.* Transplantation, 2015. **99**(8): p. 1681-90.

*Mesenchymal Stromal Cell-Based Therapy for Severe Osteoarthritis of the Knee: A Phase I Dose-Escalation Trial.* Stem Cells Transl Med, 2016. **5**(7): p. 847-56.

[169] Pers, Y.M., et al., *Adipose* 

[170] Jo, C.H., et al., *Intra-articular Injection of Mesenchymal Stem Cells for the Treatment of Osteoarthritis of the Knee: A 2-Year Follow-up Study.* Am J Sports Med, 2017. **45**(12): p. 2774-2783.

[171] Bastos, R., et al., *Intra-articular injections of expanded mesenchymal stem cells with and without addition of plateletrich plasma are safe and effective for knee osteoarthritis.* Knee Surg Sports Traumatol Arthrosc, 2018. **26**(11): p. 3342-3350.

[172] Emadedin, M., et al., *Intra-articular implantation of autologous bone marrowderived mesenchymal stromal cells to treat knee osteoarthritis: a randomized, tripleblind, placebo-controlled phase 1/2 clinical trial.* Cytotherapy, 2018. **20**(10):

[173] Khalifeh Soltani, S., et al., *Safety and efficacy of allogenic placental mesenchymal stem cells for treating knee osteoarthritis: a pilot study.* Cytotherapy,

[174] Jones, I.A., et al., *A randomized, controlled study to evaluate the efficacy of* 

p. 1238-1246.

2019. **21**(1): p. 54-63.

1390-405.

**19**(6): p. 902-7.

*ischemic hearts.* J Am Coll Cardiol, 2007.

[158] Deasy, B.M., et al., *A role for cell sex in stem cell-mediated skeletal muscle regeneration: female cells have higher muscle regeneration efficiency.* J Cell Biol,

[159] Lee, J.Y., et al., *Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing.* J Cell Biol, 2000. **150**(5): p. 1085-100.

[160] Matsumoto, T., et al., *The influence of sex on the chondrogenic potential of muscle-derived stem cells: implications for cartilage regeneration and repair.* Arthritis

[161] Scibetta, A.C., et al., *Characterization of the chondrogenic and osteogenic potential of male and female human muscle-derived stem cells: Implication for stem cell therapy.* J Orthop Res, 2019. **37**(6): p. 1339-1349.

Rheum, 2008. **58**(12): p. 3809-19.

[162] Corsi, K.A., et al., *Osteogenic potential of postnatal skeletal musclederived stem cells is influenced by donor sex.* J Bone Miner Res, 2007. **22**(10):

[163] Meszaros, L.B., et al., *Effect of host sex and sex hormones on muscle-derived stem cell-mediated bone formation and defect healing.* Tissue Eng Part A, 2012.

[164] Kuroda, R., et al., *Cartilage repair using bone morphogenetic protein 4 and muscle-derived stem cells.* Arthritis Rheum, 2006. **54**(2): p. 433-42.

[165] Matsumoto, T., et al., *Cartilage repair in a rat model of osteoarthritis through intraarticular transplantation of muscle-derived stem cells expressing bone morphogenetic protein 4 and soluble Flt-1.*

p. 1592-602.

**18**(17-18): p. 1751-9.

[157] Oshima, H., et al., *Differential myocardial infarct repair with muscle stem cells compared to myoblasts.* Mol Ther,

**50**(17): p. 1677-84.

2005. **12**(6): p. 1130-41.

2007. **177**(1): p. 73-86.

**54**

[175] Gupta, P.K., et al., *Efficacy and safety of adult human bone marrowderived, cultured, pooled, allogeneic mesenchymal stromal cells (Stempeucel®): preclinical and clinical trial in osteoarthritis of the knee joint.* Arthritis Res Ther, 2016. **18**(1): p. 301.

[176] Kuah, D., et al., *Safety, tolerability and efficacy of intra-articular Progenza in knee osteoarthritis: a randomized doubleblind placebo-controlled single ascending dose study.* J Transl Med, 2018. **16**(1): p. 49.

[177] Song, Y., et al., *Human adiposederived mesenchymal stem cells for osteoarthritis: a pilot study with long-term follow-up and repeated injections.* Regen Med, 2018. **13**(3): p. 295-307.

[178] Spasovski, D., et al., *Intra-articular injection of autologous adipose-derived mesenchymal stem cells in the treatment of knee osteoarthritis.* J Gene Med, 2018. **20**(1).

[179] Wyles, C.C., et al., *Mesenchymal stem cell therapy for osteoarthritis: current perspectives.* Stem Cells Cloning, 2015. **8**: p. 117-24.

[180] Sekiya, I., et al., *Arthroscopic Transplantation of Synovial Stem Cells Improves Clinical Outcomes in Knees With Cartilage Defects.* Clin Orthop Relat Res, 2015. **473**(7): p. 2316-26.

[181] Shimomura, K., et al., *First-in-Human Pilot Study of Implantation of a Scaffold-Free Tissue-Engineered Construct Generated From Autologous Synovial Mesenchymal Stem Cells for Repair of Knee Chondral Lesions.* Am J Sports Med, 2018. **46**(10): p. 2384-2393.

**57**

**Chapter 4**

Heart

**Abstract**

Strategies to Treat Pulmonary

Drugs without Damaging the

Hypertension Using Programmed

Cell Death-Inducing Anti-Cancer

*Yuichiro J. Suzuki, Yasmine F. Ibrahim, Vladyslava Rybka,* 

*Jaquantey R. Bowens, Adenike S. Falade and Nataliia V. Shults*

Pulmonary arterial hypertension (PAH) is a fatal disease without a cure. By the time patients are diagnosed with PAH, thickening of pulmonary arterial (PA) walls and the narrowing of vascular lumen have already developed due to the abnormal growth of pulmonary vascular cells, contributing to the elevated pulmonary vascular resistance and the right ventricle (RV) damage. Therefore, agents that eliminate excess pulmonary vascular wall cells have therapeutic potential, and the apoptosisbased therapy using anti-cancer drugs may be promising for the treatment of PAH. However, cell death agents could also exert adverse effects including cardiotoxicity, complicating the development of such therapies for PAH patients who already have the damaged heart. We tested the concept that programmed cell death-inducing anti-cancer drugs may reduce the PA wall thickening using rat models of PAH. We found that: (i) The treatment of PAH animals with anthracycline-, proteasome inhibitor- or Bcl-2 inhibitor-classes of anti-cancer drugs after the pulmonary vascular remodeling had already developed resulted in the reversal of PA wall thickening and opened up the lumen; (ii) These effects were accompanied by the apoptosis of PA wall cells in PAH rats, but not in normal healthy rats, suggesting the anti-cancer drugs selectively kill remodeled vascular cells; (iii) The RV affected by PAH was not further damaged by anthracyclines or proteasome inhibitors; (iv) While the left ventricle (LV) was damaged by these drugs, we identified cardioprotective agents that protect the heart against drug-induced cell death without affecting the efficacy to reverse the PA remodeling; and (v) docetaxel, not only reversed pulmonary vascular remodeling without exerting RV or LV toxicity, but also repaired the RV damage caused by PAH. Thus, the inclusion of programmed cell death-inducing

anti-cancer drugs should be considered for treating PAH patients.

pulmonary hypertension, vascular remodeling

**Keywords:** anti-cancer drugs, apoptosis, autophagy, heart, programmed cell death,

#### **Chapter 4**

## Strategies to Treat Pulmonary Hypertension Using Programmed Cell Death-Inducing Anti-Cancer Drugs without Damaging the Heart

*Yuichiro J. Suzuki, Yasmine F. Ibrahim, Vladyslava Rybka, Jaquantey R. Bowens, Adenike S. Falade and Nataliia V. Shults*

#### **Abstract**

Pulmonary arterial hypertension (PAH) is a fatal disease without a cure. By the time patients are diagnosed with PAH, thickening of pulmonary arterial (PA) walls and the narrowing of vascular lumen have already developed due to the abnormal growth of pulmonary vascular cells, contributing to the elevated pulmonary vascular resistance and the right ventricle (RV) damage. Therefore, agents that eliminate excess pulmonary vascular wall cells have therapeutic potential, and the apoptosisbased therapy using anti-cancer drugs may be promising for the treatment of PAH. However, cell death agents could also exert adverse effects including cardiotoxicity, complicating the development of such therapies for PAH patients who already have the damaged heart. We tested the concept that programmed cell death-inducing anti-cancer drugs may reduce the PA wall thickening using rat models of PAH. We found that: (i) The treatment of PAH animals with anthracycline-, proteasome inhibitor- or Bcl-2 inhibitor-classes of anti-cancer drugs after the pulmonary vascular remodeling had already developed resulted in the reversal of PA wall thickening and opened up the lumen; (ii) These effects were accompanied by the apoptosis of PA wall cells in PAH rats, but not in normal healthy rats, suggesting the anti-cancer drugs selectively kill remodeled vascular cells; (iii) The RV affected by PAH was not further damaged by anthracyclines or proteasome inhibitors; (iv) While the left ventricle (LV) was damaged by these drugs, we identified cardioprotective agents that protect the heart against drug-induced cell death without affecting the efficacy to reverse the PA remodeling; and (v) docetaxel, not only reversed pulmonary vascular remodeling without exerting RV or LV toxicity, but also repaired the RV damage caused by PAH. Thus, the inclusion of programmed cell death-inducing anti-cancer drugs should be considered for treating PAH patients.

**Keywords:** anti-cancer drugs, apoptosis, autophagy, heart, programmed cell death, pulmonary hypertension, vascular remodeling

#### **1. Introduction**

Pulmonary arterial hypertension (PAH) is a fatal disease that can affect both females and males of any age including children. If untreated, increased pulmonary vascular resistance results in right heart failure and kills patients within several years [1, 2]. Even with the currently available therapeutic drugs that are mainly vasodilators, the survival duration of the patients remains unacceptably short [3, 4]. It has been reported that the median survival for patients diagnosed with PAH is 2.8 years from the time of diagnosis (3-year survival: 48%) if untreated [5, 6]. Even with currently available therapies, only 58–75% of PAH patients survive for 3 years [7–10]. PAH is a progressive disease, and by the time patients are diagnosed, thickening of pulmonary artery (PA) walls and the narrowing of vascular lumen have already developed due to the abnormal growth of pulmonary vascular cells, contributing to the elevated pulmonary vascular resistance and the right ventricle (RV) damage. Therefore, agents that eliminate excess pulmonary vascular wall cells have therapeutic potential, and we hypothesize that the programmed cell death-based therapy using anti-cancer drugs would help treat PAH patients [11]. However, cell death agents could also exert adverse effects including cardiotoxicity, complicating the development of such therapies for PAH patients with the already damaged heart.

#### **2. Anti-cancer drugs reverse pulmonary vascular remodeling**

In our earlier study, we found that an anthracycline anti-cancer drug daunorubicin (DNR) is an effective agent that can cause apoptosis of cultured PA smooth muscle cells (PASMCs) [11, 12]. Based on these results, we hypothesized that the administration of DNR to rats would result in the reversal of pulmonary vascular remodeling. In these experiments, Sprague-Dawley (SD) rats were treated with chronic hypoxia (10% oxygen) for 2 weeks to promote the thickening of PA medial walls. After the PA wall thickening was developed, rats were injected with DNR and maintained in the hypoxia condition for 3 days. As shown in hematoxylin and eosin (H&E) stain images of **Figure 1A**, DNR effectively reduced the PA wall thickness [13]. Similarly, in this study, another class of anti-cancer drugs, proteasome inhibitors such as MG132 and bortezomib (**Figure 1B**) also reduced the PA wall thickening in the chronic hypoxia model of pulmonary hypertension (PH) in rats [13].

An animal model, in which SD rats are injected with SU5416 and exposed to hypoxia promoting severe PAH with pulmonary vascular lesions resembling those of humans [14], has become a gold standard to study PAH [15]. The experimental design often involves a single subcutaneous injection of SU5416, followed by subjecting the animals to chronic hypoxia for 3 weeks. Subsequently, the animals are kept in normoxia, and severe PAH and pulmonary vascular remodeling are progressively developed. We found that programmed cell death-inducing anticancer drugs reversed pulmonary vascular remodeling in this model of PAH as well. **Figure 1C** shows the results of our experiments, in which another proteasome inhibitor, carfilzomib (CFZ) injected 4 times over two weeks after the pulmonary vascular remodeling was developed effectively reduced the PA wall thickness in PAH rats [16]. Proteasome inhibition-dependent reversal of pulmonary vascular remodeling occurred through the reduction of both intimal and medial wall thickening, suggesting that both endothelial cells and smooth muscle cells (SMCs) can be affected by these anti-cancer drugs [13].

**59**

**Figure 1.**

*each other at P < 0.05.*

*Strategies to Treat Pulmonary Hypertension Using Programmed Cell Death-Inducing Anti…*

While anthracyclines such as DNR and proteasome inhibitors are effective inducers of apoptosis of PASMCs [11, 13, 16], these agents could exert other biologic actions. Thus, we tested the effects of a more 'pure' apoptosis inducer, navitoclax (ABT-263) that inhibits anti-apoptotic proteins Bcl-2 and Bcl-xL. We found that this drug also reversed PA remodeling in SD rats as well as in Fischer rats with PAH promoted by SU5416 + hypoxia (**Figure 1D**; [17]). The reversal of pulmonary vascular remodeling by navitoclax was also recently reported by van der Feen et al. [18] in a

*Effects of programmed cell death-inducing anti-cancer drugs on pulmonary vascular remodeling. (A & B) SD rats were treated with chronic hypoxia for 2 weeks to produce pulmonary vascular thickening and injected with DNR or bortezomib. Rats were then placed back in the hypoxic environment. Three days after the injection, lungs were harvested and H&E staining was performed (Adapted from Ibrahim et al. [13] with permission). (C & D) SD rats were subjected to SU5416/hypoxia to promote PAH. After pulmonary vascular remodeling was developed, rats were injected with CFZ or navitoclax twice a week for 2 weeks. Lungs were harvested and H&E staining was performed (Adapted from Wang et al. [16] and Rybka et al. [17] with permission). Bar graphs represent means ± SEM of % PA wall thickness. \* denotes that the values are significantly different from* 

These results provided important information, in live experimental animals, showing that programmed cell death-inducing anti-cancer drugs are capable of reversing pulmonary vascular remodeling in multiple models of PH. While this knowledge established a basis for exploring whether causing the death of pulmonary vascular cells clinically benefits PAH patients, it also generated many questions

different experimental model of PAH in rats.

that need to be addressed.

*DOI: http://dx.doi.org/10.5772/intechopen.95264*

*Strategies to Treat Pulmonary Hypertension Using Programmed Cell Death-Inducing Anti… DOI: http://dx.doi.org/10.5772/intechopen.95264*

#### **Figure 1.**

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

patients with the already damaged heart.

Pulmonary arterial hypertension (PAH) is a fatal disease that can affect both females and males of any age including children. If untreated, increased pulmonary vascular resistance results in right heart failure and kills patients within several years [1, 2]. Even with the currently available therapeutic drugs that are mainly vasodilators, the survival duration of the patients remains unacceptably short [3, 4]. It has been reported that the median survival for patients diagnosed with PAH is 2.8 years from the time of diagnosis (3-year survival: 48%) if untreated [5, 6]. Even with currently available therapies, only 58–75% of PAH patients survive for 3 years [7–10]. PAH is a progressive disease, and by the time patients are diagnosed, thickening of pulmonary artery (PA) walls and the narrowing of vascular lumen have already developed due to the abnormal growth of pulmonary vascular cells, contributing to the elevated pulmonary vascular resistance and the right ventricle (RV) damage. Therefore, agents that eliminate excess pulmonary vascular wall cells have therapeutic potential, and we hypothesize that the programmed cell death-based therapy using anti-cancer drugs would help treat PAH patients [11]. However, cell death agents could also exert adverse effects including cardiotoxicity, complicating the development of such therapies for PAH

**2. Anti-cancer drugs reverse pulmonary vascular remodeling**

the chronic hypoxia model of pulmonary hypertension (PH) in rats [13].

can be affected by these anti-cancer drugs [13].

An animal model, in which SD rats are injected with SU5416 and exposed to hypoxia promoting severe PAH with pulmonary vascular lesions resembling those of humans [14], has become a gold standard to study PAH [15]. The experimental design often involves a single subcutaneous injection of SU5416, followed by subjecting the animals to chronic hypoxia for 3 weeks. Subsequently, the animals are kept in normoxia, and severe PAH and pulmonary vascular remodeling are progressively developed. We found that programmed cell death-inducing anticancer drugs reversed pulmonary vascular remodeling in this model of PAH as well. **Figure 1C** shows the results of our experiments, in which another proteasome inhibitor, carfilzomib (CFZ) injected 4 times over two weeks after the pulmonary vascular remodeling was developed effectively reduced the PA wall thickness in PAH rats [16]. Proteasome inhibition-dependent reversal of pulmonary vascular remodeling occurred through the reduction of both intimal and medial wall thickening, suggesting that both endothelial cells and smooth muscle cells (SMCs)

In our earlier study, we found that an anthracycline anti-cancer drug daunorubicin (DNR) is an effective agent that can cause apoptosis of cultured PA smooth muscle cells (PASMCs) [11, 12]. Based on these results, we hypothesized that the administration of DNR to rats would result in the reversal of pulmonary vascular remodeling. In these experiments, Sprague-Dawley (SD) rats were treated with chronic hypoxia (10% oxygen) for 2 weeks to promote the thickening of PA medial walls. After the PA wall thickening was developed, rats were injected with DNR and maintained in the hypoxia condition for 3 days. As shown in hematoxylin and eosin (H&E) stain images of **Figure 1A**, DNR effectively reduced the PA wall thickness [13]. Similarly, in this study, another class of anti-cancer drugs, proteasome inhibitors such as MG132 and bortezomib (**Figure 1B**) also reduced the PA wall thickening in

**1. Introduction**

**58**

*Effects of programmed cell death-inducing anti-cancer drugs on pulmonary vascular remodeling. (A & B) SD rats were treated with chronic hypoxia for 2 weeks to produce pulmonary vascular thickening and injected with DNR or bortezomib. Rats were then placed back in the hypoxic environment. Three days after the injection, lungs were harvested and H&E staining was performed (Adapted from Ibrahim et al. [13] with permission). (C & D) SD rats were subjected to SU5416/hypoxia to promote PAH. After pulmonary vascular remodeling was developed, rats were injected with CFZ or navitoclax twice a week for 2 weeks. Lungs were harvested and H&E staining was performed (Adapted from Wang et al. [16] and Rybka et al. [17] with permission). Bar graphs represent means ± SEM of % PA wall thickness. \* denotes that the values are significantly different from each other at P < 0.05.*

While anthracyclines such as DNR and proteasome inhibitors are effective inducers of apoptosis of PASMCs [11, 13, 16], these agents could exert other biologic actions. Thus, we tested the effects of a more 'pure' apoptosis inducer, navitoclax (ABT-263) that inhibits anti-apoptotic proteins Bcl-2 and Bcl-xL. We found that this drug also reversed PA remodeling in SD rats as well as in Fischer rats with PAH promoted by SU5416 + hypoxia (**Figure 1D**; [17]). The reversal of pulmonary vascular remodeling by navitoclax was also recently reported by van der Feen et al. [18] in a different experimental model of PAH in rats.

These results provided important information, in live experimental animals, showing that programmed cell death-inducing anti-cancer drugs are capable of reversing pulmonary vascular remodeling in multiple models of PH. While this knowledge established a basis for exploring whether causing the death of pulmonary vascular cells clinically benefits PAH patients, it also generated many questions that need to be addressed.

#### **3. Susceptibility of normal and diseased cells toward apoptosis-inducing anti-cancer drugs**

One question is whether both the proliferative synthetic phenotype and the differentiated contractile phenotype of PASMCs are killed by these drugs. It is preferable that only abnormally grown cells are killed, as it is important to preserve contractile SMCs that are needed for the pulmonary circulatory system to function.

The examination of PAs from rats treated with DNR by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, which detects apoptotic cells, demonstrated that only remodeled PAs of rats with PH exhibited apoptotic cells, but not healthy control rats (**Figure 2A**; [13]). Similar results were obtained in the analysis of cleaved caspase-3 as an indication of the occurrence of apoptotic cells by Western blotting. As shown in **Figure 2B**, PAs from rats treated with chronic hypoxia to promote PH and subsequently treated with DNR exhibited significantly higher levels of cleaved caspase-3 compared to healthy rats injected

#### **Figure 2.**

*Effects of programmed cell death-inducing anti-cancer drugs on apoptosis. (A & B) SD rats were treated with chronic hypoxia for 2 weeks to produce pulmonary vascular thickening and injected with DNR. Rats were then placed back in the hypoxic environment. Three days after the injection, lungs were harvested and TUNEL staining and Western blotting with the cleaved caspase-3 antibody were performed to monitor apoptosis (Adapted from Ibrahim et al. [13] with permission). (C & D) SD rats were subjected to SU5416/hypoxia to promote PAH. After pulmonary vascular remodeling was developed, rats were injected with CFZ twice a week for 2 weeks. Lungs were harvested and TUNEL staining and Western blotting with the cleaved caspase-3 antibody were performed to monitor apoptosis (Adapted from Wang et al. [16] with permission). Bar graphs represent means ± SEM. \* denotes that the values are significantly different from each other at P < 0.05.*

**61**

muscle functions.

**Figure 3.**

**4. Role of autophagic cell death**

nuclear protein 1 (TP53INP1) in this mechanism [16].

*Strategies to Treat Pulmonary Hypertension Using Programmed Cell Death-Inducing Anti…*

with DNR [13]. These results revealed that unwanted abnormally grown pulmonary

*Effects of genestein on proliferating and differentiated human PASMC apoptosis. Proliferating human PASMCs purchased from Cell Applications grown in Human SMC Growth Medium and differentiated PASMCs produced using the Human SMC Differentiation Medium were treated with genistein (50* μ*M). Apoptotic cells* 

Results shown in **Figure 2C** and **D** demonstrated that this increased susceptibility of pulmonary vascular cells in PH animals can also be seen with another anti-cancer drug. CFZ also caused the apoptosis in PAs of rats with PAH induced by SU5416/hypoxia, while no apoptosis signals were observed in control healthy rats treated with CFZ as monitored by TUNEL assay (**Figure 2C**) and Western blotting

We hypothesized that anti-cancer drugs preferentially kill the proliferating phenotype of SMCs over differentiated SMCs. Our experiments using cultured PASMCs showed that only proliferating SMCs, but not differentiated SMCs, were killed by DNR [13]. **Figure 3** shows similar experimental results when proliferating and differentiated human PASMCs were treated with genistein, a naturally occurring isoflavone. DePsipher Mitochondrial Potential assay (Trevigen, Gaithersburg, MD, USA) showed that green fluorescent apoptotic cells were only observed when proliferating PASMCs were treated with genistein, while differentiated PASMCs produced by using the Differentiation Medium (Cell Applications, Inc., San Diego, CA, USA) were resistant to be killed by the same concentration of genistein. These results demonstrate that proliferating PASMCs are more susceptible to undergo apoptosis compared to differentiated PASMCs, suggesting that apoptosis-inducing drugs eliminated unwanted proliferating PASMCs while preserving the contractile phenotypic cells with

One interesting observation we came across in relation to the mechanism of PASMC killing by anthracycline- and proteasome inhibitor-classes of anti-cancer drug is that, in addition, to apoptosis, another programmed cell death mechanism, namely autophagic cell death is also involved. We initially found that autophagy of the cells is increased in PAs of PH rats treated with DNR [13]. Similar results were observed in cultured proliferating human PASMCs when cells were treated with DNR. Further, DNR-induced cell killing was attenuated when an autophagy mediator, LC3B, was knocked down [13]. CFZ-induced cell killing was also found to involve autophagy, and we further identified the role of tumor protein p53-inducible

vascular cells can preferentially be killed by this anti-cancer drug.

*were identified by green fluorescence produced using the DePsipher Mitochondrial Potential assay.*

using the cleaved caspase-3 antibody (**Figure 2D**) [16].

*DOI: http://dx.doi.org/10.5772/intechopen.95264*

*Strategies to Treat Pulmonary Hypertension Using Programmed Cell Death-Inducing Anti… DOI: http://dx.doi.org/10.5772/intechopen.95264*

#### **Figure 3.**

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

**anti-cancer drugs**

**3. Susceptibility of normal and diseased cells toward apoptosis-inducing** 

One question is whether both the proliferative synthetic phenotype and the differentiated contractile phenotype of PASMCs are killed by these drugs. It is preferable that only abnormally grown cells are killed, as it is important to preserve contractile SMCs that are needed for the pulmonary circulatory system to function. The examination of PAs from rats treated with DNR by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, which detects apoptotic cells, demonstrated that only remodeled PAs of rats with PH exhibited apoptotic cells, but not healthy control rats (**Figure 2A**; [13]). Similar results were obtained in the analysis of cleaved caspase-3 as an indication of the occurrence of apoptotic cells by Western blotting. As shown in **Figure 2B**, PAs from rats treated with chronic hypoxia to promote PH and subsequently treated with DNR exhibited significantly higher levels of cleaved caspase-3 compared to healthy rats injected

*Effects of programmed cell death-inducing anti-cancer drugs on apoptosis. (A & B) SD rats were treated with chronic hypoxia for 2 weeks to produce pulmonary vascular thickening and injected with DNR. Rats were then placed back in the hypoxic environment. Three days after the injection, lungs were harvested and TUNEL staining and Western blotting with the cleaved caspase-3 antibody were performed to monitor apoptosis (Adapted from Ibrahim et al. [13] with permission). (C & D) SD rats were subjected to SU5416/hypoxia to promote PAH. After pulmonary vascular remodeling was developed, rats were injected with CFZ twice a week for 2 weeks. Lungs were harvested and TUNEL staining and Western blotting with the cleaved caspase-3 antibody were performed to monitor apoptosis (Adapted from Wang et al. [16] with permission). Bar graphs represent means ± SEM. \* denotes that the values are significantly different from each other at P < 0.05.*

**60**

**Figure 2.**

*Effects of genestein on proliferating and differentiated human PASMC apoptosis. Proliferating human PASMCs purchased from Cell Applications grown in Human SMC Growth Medium and differentiated PASMCs produced using the Human SMC Differentiation Medium were treated with genistein (50* μ*M). Apoptotic cells were identified by green fluorescence produced using the DePsipher Mitochondrial Potential assay.*

with DNR [13]. These results revealed that unwanted abnormally grown pulmonary vascular cells can preferentially be killed by this anti-cancer drug.

Results shown in **Figure 2C** and **D** demonstrated that this increased susceptibility of pulmonary vascular cells in PH animals can also be seen with another anti-cancer drug. CFZ also caused the apoptosis in PAs of rats with PAH induced by SU5416/hypoxia, while no apoptosis signals were observed in control healthy rats treated with CFZ as monitored by TUNEL assay (**Figure 2C**) and Western blotting using the cleaved caspase-3 antibody (**Figure 2D**) [16].

We hypothesized that anti-cancer drugs preferentially kill the proliferating phenotype of SMCs over differentiated SMCs. Our experiments using cultured PASMCs showed that only proliferating SMCs, but not differentiated SMCs, were killed by DNR [13]. **Figure 3** shows similar experimental results when proliferating and differentiated human PASMCs were treated with genistein, a naturally occurring isoflavone. DePsipher Mitochondrial Potential assay (Trevigen, Gaithersburg, MD, USA) showed that green fluorescent apoptotic cells were only observed when proliferating PASMCs were treated with genistein, while differentiated PASMCs produced by using the Differentiation Medium (Cell Applications, Inc., San Diego, CA, USA) were resistant to be killed by the same concentration of genistein. These results demonstrate that proliferating PASMCs are more susceptible to undergo apoptosis compared to differentiated PASMCs, suggesting that apoptosis-inducing drugs eliminated unwanted proliferating PASMCs while preserving the contractile phenotypic cells with muscle functions.

#### **4. Role of autophagic cell death**

One interesting observation we came across in relation to the mechanism of PASMC killing by anthracycline- and proteasome inhibitor-classes of anti-cancer drug is that, in addition, to apoptosis, another programmed cell death mechanism, namely autophagic cell death is also involved. We initially found that autophagy of the cells is increased in PAs of PH rats treated with DNR [13]. Similar results were observed in cultured proliferating human PASMCs when cells were treated with DNR. Further, DNR-induced cell killing was attenuated when an autophagy mediator, LC3B, was knocked down [13]. CFZ-induced cell killing was also found to involve autophagy, and we further identified the role of tumor protein p53-inducible nuclear protein 1 (TP53INP1) in this mechanism [16].

#### **5. The ability of the remodeled RV to cope with program cell death-inducing drugs**

Drugs that promote programmed cell death are effective anti-cancer drugs, however, they also exert serious potentially life-threatening complications [19]. Cardiotoxicity is a major complication that accompanies the use of anti-cancer drugs especially anthracyclines. Since PAH patients already have the weakened heart, the use of these anti-cancer drugs would be considered to be contraindications. However, we found that the RV affected by PAH is remarkably resistant to drug-induced myocardial cell killing. As we characterized the RV of PAH rats injected with DNR to reverse PA remodeling as described above, we found that DNR administration to PAH rats did not influence the RV contractility or the RV structure [13]. This study also found that DNR did not promote apoptosis of cardiomyocytes in hypertrophied RV in rats with PH (**Figure 4A**; [13]). Similarly, CFZ that was found to effectively reverse PA remodeling did not cause apoptosis in the RV in SU5416/hypoxia model of PAH in rats (**Figure 4B**; [16]). These are highly significant findings revealing that the RV affected by PAH is resistant to DNR and CFZ, drugs that are known induce cardiotoxicity and cardiomyocyte killing in the normal heart, providing evidence that the clinical use of these anti-cancer drugs in PAH patients may not be contraindications.

By contrast, bortezomib was found to promote apoptosis in both RV and left ventricle (LV) of rats with PH induced by monocrotaline [20]. Also, navitoclax (ABT-263; an inhibitor of anti-apoptotic proteins, Bcl-2 and Bcl-xL), not only

#### **Figure 4.**

*Effects of programmed cell death-inducing anti-cancer drugs on the RV affected by PAH. (A) SD rats were treated with chronic hypoxia for 2 weeks to produce pulmonary vascular thickening and injected with DNR. Rats were then placed back in the hypoxic environment. Three days after the injection, RV tissues were harvested and Western blotting with the cleaved caspase-3 antibody was performed to monitor apoptosis (Adapted from Ibrahim et al. [13] with permission). (B) SD rats were subjected to SU5416/hypoxia to promote PAH. After pulmonary vascular remodeling was developed, rats were injected with CFZ twice a week for 2 weeks. RV tissues were harvested and Western blotting with the cleaved caspase-3 antibody were performed to monitor apoptosis (Adapted from Wang et al. [16] with permission). Bar graphs represent means ± SEM. All the values were not significantly different from each other at P < 0.05.*

**63**

clax (**Figure 5C**).

**Figure 5.**

[22, 23] could be more promising.

of these drugs to reverse PA remodeling.

**by anti-cancer drugs**

*Strategies to Treat Pulmonary Hypertension Using Programmed Cell Death-Inducing Anti…*

caused apoptosis in the remodeled PA [17], but also promoted apoptosis in RV myocytes in PAH rats. **Figure 5** shows the transmission electron microscopy images of normal SD rat RV myocytes (**Figure 5A**) and RV myocytes from PAH SD rats treated with navitoclax exhibiting signs of apoptosis (**Figure 5B**). The nuclei PAH rats treated with navitoclax underwent the fragmentation with dramatic changes in the nuclear chromatin with the segregated heterochromatin that distributed preferentially within the nuclear envelope as sharply defined clumped bodies (**Figure 5B**, red arrowheads). The quantification of apoptotic nuclei revealed that the most of RV myocytes in PAH rats became apoptotic when treated with navito-

*microscopy studies were performed as described in Shults et al. [32].*

*Navitoclax promotes cardiomyocyte apoptosis in the RV affected by PAH. (A) The normal RV structure of RV cardiomyocytes as monitored by transmission electron microscopy. Magnification ×16,000. (B) SD rats were subjected to SU5416/hypoxia to promote PAH. After pulmonary vascular remodeling was developed, rats were injected with navitoclax twice a week for 2 weeks as described in Rybka et al. [17]. RV tissues were harvested and a transmission electron microscope was used to analyze the apoptotic nuclei. Bar graphs represent means ± SEM. \* denotes that the values are significantly different from each other at P < 0.05. Transmission electron* 

These results suggest that, while three classes of anti-cancer drugs have so far been found to be effective in reversing PA remodeling, hypertrophied RV myocytes are only resistant to DNR and CFZ, while Bcl-2/Bcl-xL inhibition seems target downstream of apoptotic pathway thus escapes from the resistance to cardiomyocyte killing. Whether the RV damaging effects of bortezomib in PAH rats [20] is specific to the model induced by monocrotaline that can exert non-specific pathophysiologic actions need further investigations, however, the data so far do not support the use of bortezomib in the PAH treatment. CFZ that is considered to be a safe alternative to bortezomib in cancer therapy [21] and is a more selective and irreversible inhibitor of the chymotrypsin-like activity of the 20S proteasome

Our laboratory previously found a cell-signaling pathway for the downregulation of Bcl-xL/Bcl-2 that results in the apoptosis of cardiomyocytes [24]. This pathway was found as a consequence of our laboratory cloning the promoter region of the GATA4 transcription factor that regulates gene transcription of Bcl-xL and Bcl-2. We found that CBF/NF-Y binding to the CCAAT box of the Gata4 promoter is inhibited by DNR through the activation of p53 in cardiomyocytes [24], but not in PASMCs [13]. Thus, we hypothesized that p53 inhibitors would protect the heart against cardiotoxicity induced by anti-cancer drugs without affecting the efficacy

**6. Cardioprotective agents to cope with LV myocyte death** 

*DOI: http://dx.doi.org/10.5772/intechopen.95264*

*Strategies to Treat Pulmonary Hypertension Using Programmed Cell Death-Inducing Anti… DOI: http://dx.doi.org/10.5772/intechopen.95264*

#### **Figure 5.**

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

**death-inducing drugs**

PAH patients may not be contraindications.

**5. The ability of the remodeled RV to cope with program cell** 

Drugs that promote programmed cell death are effective anti-cancer drugs, however, they also exert serious potentially life-threatening complications [19]. Cardiotoxicity is a major complication that accompanies the use of anti-cancer drugs especially anthracyclines. Since PAH patients already have the weakened heart, the use of these anti-cancer drugs would be considered to be contraindications. However, we found that the RV affected by PAH is remarkably resistant to drug-induced myocardial cell killing. As we characterized the RV of PAH rats injected with DNR to reverse PA remodeling as described above, we found that DNR administration to PAH rats did not influence the RV contractility or the RV structure [13]. This study also found that DNR did not promote apoptosis of cardiomyocytes in hypertrophied RV in rats with PH (**Figure 4A**; [13]). Similarly, CFZ that was found to effectively reverse PA remodeling did not cause apoptosis in the RV in SU5416/hypoxia model of PAH in rats (**Figure 4B**; [16]). These are highly significant findings revealing that the RV affected by PAH is resistant to DNR and CFZ, drugs that are known induce cardiotoxicity and cardiomyocyte killing in the normal heart, providing evidence that the clinical use of these anti-cancer drugs in

By contrast, bortezomib was found to promote apoptosis in both RV and left ventricle (LV) of rats with PH induced by monocrotaline [20]. Also, navitoclax (ABT-263; an inhibitor of anti-apoptotic proteins, Bcl-2 and Bcl-xL), not only

*Effects of programmed cell death-inducing anti-cancer drugs on the RV affected by PAH. (A) SD rats were treated with chronic hypoxia for 2 weeks to produce pulmonary vascular thickening and injected with DNR. Rats were then placed back in the hypoxic environment. Three days after the injection, RV tissues were harvested and Western blotting with the cleaved caspase-3 antibody was performed to monitor apoptosis (Adapted from Ibrahim et al. [13] with permission). (B) SD rats were subjected to SU5416/hypoxia to promote PAH. After pulmonary vascular remodeling was developed, rats were injected with CFZ twice a week for 2 weeks. RV tissues were harvested and Western blotting with the cleaved caspase-3 antibody were performed to monitor apoptosis (Adapted from Wang et al. [16] with permission). Bar graphs represent means ± SEM. All* 

*the values were not significantly different from each other at P < 0.05.*

**62**

**Figure 4.**

*Navitoclax promotes cardiomyocyte apoptosis in the RV affected by PAH. (A) The normal RV structure of RV cardiomyocytes as monitored by transmission electron microscopy. Magnification ×16,000. (B) SD rats were subjected to SU5416/hypoxia to promote PAH. After pulmonary vascular remodeling was developed, rats were injected with navitoclax twice a week for 2 weeks as described in Rybka et al. [17]. RV tissues were harvested and a transmission electron microscope was used to analyze the apoptotic nuclei. Bar graphs represent means ± SEM. \* denotes that the values are significantly different from each other at P < 0.05. Transmission electron microscopy studies were performed as described in Shults et al. [32].*

caused apoptosis in the remodeled PA [17], but also promoted apoptosis in RV myocytes in PAH rats. **Figure 5** shows the transmission electron microscopy images of normal SD rat RV myocytes (**Figure 5A**) and RV myocytes from PAH SD rats treated with navitoclax exhibiting signs of apoptosis (**Figure 5B**). The nuclei PAH rats treated with navitoclax underwent the fragmentation with dramatic changes in the nuclear chromatin with the segregated heterochromatin that distributed preferentially within the nuclear envelope as sharply defined clumped bodies (**Figure 5B**, red arrowheads). The quantification of apoptotic nuclei revealed that the most of RV myocytes in PAH rats became apoptotic when treated with navitoclax (**Figure 5C**).

These results suggest that, while three classes of anti-cancer drugs have so far been found to be effective in reversing PA remodeling, hypertrophied RV myocytes are only resistant to DNR and CFZ, while Bcl-2/Bcl-xL inhibition seems target downstream of apoptotic pathway thus escapes from the resistance to cardiomyocyte killing. Whether the RV damaging effects of bortezomib in PAH rats [20] is specific to the model induced by monocrotaline that can exert non-specific pathophysiologic actions need further investigations, however, the data so far do not support the use of bortezomib in the PAH treatment. CFZ that is considered to be a safe alternative to bortezomib in cancer therapy [21] and is a more selective and irreversible inhibitor of the chymotrypsin-like activity of the 20S proteasome [22, 23] could be more promising.

#### **6. Cardioprotective agents to cope with LV myocyte death by anti-cancer drugs**

Our laboratory previously found a cell-signaling pathway for the downregulation of Bcl-xL/Bcl-2 that results in the apoptosis of cardiomyocytes [24]. This pathway was found as a consequence of our laboratory cloning the promoter region of the GATA4 transcription factor that regulates gene transcription of Bcl-xL and Bcl-2. We found that CBF/NF-Y binding to the CCAAT box of the Gata4 promoter is inhibited by DNR through the activation of p53 in cardiomyocytes [24], but not in PASMCs [13]. Thus, we hypothesized that p53 inhibitors would protect the heart against cardiotoxicity induced by anti-cancer drugs without affecting the efficacy of these drugs to reverse PA remodeling.

#### **Figure 6.**

*Dexrazoxane (DEX) and pifithrin-*α *(PFT-*α*) protects the LV from CFZ-induced apoptosis without affecting the efficacy of CFZ in reversing PA remodeling. (A) PAH rats (SU5416/hypoxia) were divided into 4 groups. DEX or PFT-*α *was injected intraperitoneally along with CFZ, twice a week for two weeks. Rats were then sacrificed 3 days after the last injection. LV tissues were homogenized, and subjected to Western blotting for the cleaved capase-3 formation. The bar graph represents means ± SEM. \* denotes that the values are significantly different from each other at P < 0.05. (B) The reduction of remodeled PA thickness induced by CFZ was not affected by DEX or PFT-*α *by analyzing H&E staining. The bar graph represents means ± SEM. \* denotes that the values are significantly different from the PAH value at P < 0.05. (Adapted from Wang et al. [16] with permission).*

In our study of CFZ as described above, we found that this proteasome inhibitor is effective in reversing PA remodeling and that the RV affected by PAH is resistant to CFZ toxicity [16]. However, as expected from the earlier cancer studies, CFZ did cause the cardiomyocyte apoptosis in the LV of PAH rats (**Figure 6A**). As a support for our hypothesis, this CFZ-induced apoptosis of LV cardiomyocytes was inhibited by a p53 inhibitor, pifithrin-α in PAH rats (**Figure 6A**), while this cardioprotective agent did not interfere with CFZ reducing the PA wall thickening (**Figure 6B**). Interestingly, we found that a clinically used cardioprotective drug, dexrazoxane, also protected that LV of PAH rats from CFZ toxicity without affecting the reversal of PA remodeling. Further investigations are needed to determine whether these actions of dexrazoxane involve p53. Nevertheless, these results suggest including dexrazoxane or a p53 inhibitor to protect the LV against drug-induced damage while treating PAH patients with anti-cancer drugs.

#### **7. Docetaxel as a fascinating drug that reduces pulmonary vascular wall thickening and repairs the damaged right ventricle**

Since experiments described above provided results that support the use of anti-cancer drugs to reverse pulmonary vascular remodeling, we further searched for other drugs that could be useful. In an effort to find effective drugs that preferentially kill proliferating PASMCs, we screened various drugs [25]. We found that docetaxel (a taxane class of anti-cancer drugs that stabilizes and inhibits microtubules) effectively killed proliferating human PASMCs, but not differentiated human PASMCs in culture [25]. As we tested docetaxel for reversing pulmonary vascular remodeling in the SU5416/hypoxia model of PAH, we found that this drug indeed was effective in reducing thickened pulmonary vascular walls (**Figure 7A**). Effects were similar to anthracycline-, proteasome inhibitor-, and Bcl-2/Bcl-xL

**65**

**Figure 7.**

*Strategies to Treat Pulmonary Hypertension Using Programmed Cell Death-Inducing Anti…*

inhibitor-classes of drugs. As described above, we found that DNR and CFZ did not have adverse effects on the hypertrophied RV in PAH rats while Bcl-2/Bcl-xL inhibition resulted in the apoptosis of RV myocytes. Docetaxel also did not exhibit adverse effects on the hypertrophied RV in PAH rats. Moreover, this drug repaired damaged RV caused by PAH. In SU5416/hypoxia model of PAH, the RV was found to have significant cardiac fibrosis as shown in the blue stain of Masson's trichrome staining in **Figure 7B**. Remarkably, these fibrotic lesions were eliminated by the

These results suggest that docetaxel is an effective drug that can reverse pulmonary vascular remodeling and at the same time it can also repair the damaged RV caused by PAH at least in SD rats treated with SU5416/hypoxia. Another taxane drug, paclitaxel has also been shown to attenuate pulmonary vascular remodeling in rodent models of PAH induced by monocrotaline or SU5416/hypoxia [26–30]. However, the ability of paclitaxel to repair the RV in PAH animals has not been reported. It is interesting to note that paclitaxel has been shown to improve cardiac function during ischemia in isolated rat and rabbit hearts [31], reinforcing the idea

*Docetaxel reverses pulmonary vascular remodeling and cardiac fibrosis in the RV in PAH rats. SD rats were subjected to SU5416/hypoxia to promote PAH. After pulmonary vascular remodeling was developed, rats were injected with DTX twice a week for 2 weeks. (A) Lungs were harvested and H&E staining was performed. The bar graph represents means ± SEM of % PA wall thickness. \* denotes that the values are significantly different from each other at P < 0.05. (B) Heart tissues were harvested and Masson's trichrome staining was performed to monitor fibrosis. The bar graph represents means ± SEM of % fiborsis in the RV. \* denotes that the values are significantly different from the PAH value at P < 0.05. (Adapted from Ibrahim et al. [13] with permission).*

*DOI: http://dx.doi.org/10.5772/intechopen.95264*

treatment of PAH rats with docetaxel (**Figure 7B**; [25]).

that taxanes have the capacity to promote cardiac repair.

#### *Strategies to Treat Pulmonary Hypertension Using Programmed Cell Death-Inducing Anti… DOI: http://dx.doi.org/10.5772/intechopen.95264*

inhibitor-classes of drugs. As described above, we found that DNR and CFZ did not have adverse effects on the hypertrophied RV in PAH rats while Bcl-2/Bcl-xL inhibition resulted in the apoptosis of RV myocytes. Docetaxel also did not exhibit adverse effects on the hypertrophied RV in PAH rats. Moreover, this drug repaired damaged RV caused by PAH. In SU5416/hypoxia model of PAH, the RV was found to have significant cardiac fibrosis as shown in the blue stain of Masson's trichrome staining in **Figure 7B**. Remarkably, these fibrotic lesions were eliminated by the treatment of PAH rats with docetaxel (**Figure 7B**; [25]).

These results suggest that docetaxel is an effective drug that can reverse pulmonary vascular remodeling and at the same time it can also repair the damaged RV caused by PAH at least in SD rats treated with SU5416/hypoxia. Another taxane drug, paclitaxel has also been shown to attenuate pulmonary vascular remodeling in rodent models of PAH induced by monocrotaline or SU5416/hypoxia [26–30]. However, the ability of paclitaxel to repair the RV in PAH animals has not been reported. It is interesting to note that paclitaxel has been shown to improve cardiac function during ischemia in isolated rat and rabbit hearts [31], reinforcing the idea that taxanes have the capacity to promote cardiac repair.

#### **Figure 7.**

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

In our study of CFZ as described above, we found that this proteasome inhibitor is effective in reversing PA remodeling and that the RV affected by PAH is resistant to CFZ toxicity [16]. However, as expected from the earlier cancer studies, CFZ did cause the cardiomyocyte apoptosis in the LV of PAH rats (**Figure 6A**). As a support for our hypothesis, this CFZ-induced apoptosis of LV cardiomyocytes was inhibited by a p53 inhibitor, pifithrin-α in PAH rats (**Figure 6A**), while this cardioprotective agent did not interfere with CFZ reducing the PA wall thickening (**Figure 6B**). Interestingly, we found that a clinically used cardioprotective drug, dexrazoxane, also protected that LV of PAH rats from CFZ toxicity without affecting the reversal of PA remodeling. Further investigations are needed to determine whether these actions of dexrazoxane involve p53. Nevertheless, these results suggest including dexrazoxane or a p53 inhibitor to protect the LV against drug-induced damage while

*Dexrazoxane (DEX) and pifithrin-*α *(PFT-*α*) protects the LV from CFZ-induced apoptosis without affecting the efficacy of CFZ in reversing PA remodeling. (A) PAH rats (SU5416/hypoxia) were divided into 4 groups. DEX or PFT-*α *was injected intraperitoneally along with CFZ, twice a week for two weeks. Rats were then sacrificed 3 days after the last injection. LV tissues were homogenized, and subjected to Western blotting for the cleaved capase-3 formation. The bar graph represents means ± SEM. \* denotes that the values are significantly different from each other at P < 0.05. (B) The reduction of remodeled PA thickness induced by CFZ was not affected by DEX or PFT-*α *by analyzing H&E staining. The bar graph represents means ± SEM. \* denotes that the values are significantly different from the PAH value at P < 0.05. (Adapted from Wang et al. [16] with* 

**7. Docetaxel as a fascinating drug that reduces pulmonary vascular** 

Since experiments described above provided results that support the use of anti-cancer drugs to reverse pulmonary vascular remodeling, we further searched for other drugs that could be useful. In an effort to find effective drugs that preferentially kill proliferating PASMCs, we screened various drugs [25]. We found that docetaxel (a taxane class of anti-cancer drugs that stabilizes and inhibits microtubules) effectively killed proliferating human PASMCs, but not differentiated human PASMCs in culture [25]. As we tested docetaxel for reversing pulmonary vascular remodeling in the SU5416/hypoxia model of PAH, we found that this drug indeed was effective in reducing thickened pulmonary vascular walls (**Figure 7A**). Effects were similar to anthracycline-, proteasome inhibitor-, and Bcl-2/Bcl-xL

**wall thickening and repairs the damaged right ventricle**

treating PAH patients with anti-cancer drugs.

**64**

**Figure 6.**

*permission).*

*Docetaxel reverses pulmonary vascular remodeling and cardiac fibrosis in the RV in PAH rats. SD rats were subjected to SU5416/hypoxia to promote PAH. After pulmonary vascular remodeling was developed, rats were injected with DTX twice a week for 2 weeks. (A) Lungs were harvested and H&E staining was performed. The bar graph represents means ± SEM of % PA wall thickness. \* denotes that the values are significantly different from each other at P < 0.05. (B) Heart tissues were harvested and Masson's trichrome staining was performed to monitor fibrosis. The bar graph represents means ± SEM of % fiborsis in the RV. \* denotes that the values are significantly different from the PAH value at P < 0.05. (Adapted from Ibrahim et al. [13] with permission).*

### **8. Conclusions**

We tested the concept that cell death-inducing anti-cancer drugs may reduce the PA wall thickening using rat models of PAH. We found that: (1) The treatment of PAH rats with anthracycline-, proteasome inhibitor- or Bcl-2/Bcl-xL inhibitorclasses of drugs after pulmonary vascular remodeling had occurred resulted in the reversal of pulmonary vascular remodeling and opened up the lumen; (2) These effects were accompanied by the apoptosis of PA wall cells in PAH rats, but these drugs did not promote apoptosis in normal healthy rats, suggesting the anti-cancer drugs selectively kill remodeled vascular cells; (3) DNR, an anthracycline, and CFZ, a proteasome inhibitor, did not adversely affect the hypertrophied RV of PAH rats. (4) While the LV was damaged by CFZ, we identified cardioprotective agents (dexrazoxane and pifithrin-alpha) that can protect the heart against drug-induced cell death without affecting the efficacy of the drugs to reduce PA remodeling; (5) Docetaxel, a taxane class of anti-cancer drugs, not only reversed pulmonary vascular remodeling without exerting RV or LV toxicity, but also repaired the RV damaged caused by PAH. These findings from our laboratory as well as reports by other laboratories on the topic of the effects of programmed cell death-inducing anti-cancer drugs on remodeled PA and the RV affected by PAH in experimental animals are summarized in **Table 1**.

These results demonstrate that certain anti-cancer drugs effectively and selectively cause programmed cell death of abnormally grown cells in the remodeled pulmonary vasculature without adversely affecting the RV in rat models of PAH. Thus, the inclusion of programmed cell death-induced anti-cancer drugs may be promising for treating PAH patients. Human clinical trials of PAH treatment that test the effectiveness of these anti-cancer drugs as mono-therapies or combination therapies along with cardioprotective agents described here as well as already available vasodilators are warranted.


**Table 1.**

*Abilities of various anti-cancer drugs to affect PA and RV remodeling.*

#### **Acknowledgements**

This work was supported in part by the NIH (grant numbers R01HL072844, R21AI142649, R03AG059554, and R03AA026516) to Y.J.S. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

**67**

Egypt

**Author details**

Yuichiro J. Suzuki1

Adenike S. Falade1

Center, Washington, DC, USA

\*, Yasmine F. Ibrahim<sup>2</sup>

and Nataliia V. Shults1

\*Address all correspondence to: ys82@georgetown.edu

provided the original work is properly cited.

, Vladyslava Rybka1

1 Department of Pharmacology and Physiology, Georgetown University Medical

2 Department of Pharmacology, Minia University Faculty of Medicine, Minia,

© 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,

, Jaquantey R. Bowens1

,

*Strategies to Treat Pulmonary Hypertension Using Programmed Cell Death-Inducing Anti…*

*DOI: http://dx.doi.org/10.5772/intechopen.95264*

#### **Conflict of interest**

None.

*Strategies to Treat Pulmonary Hypertension Using Programmed Cell Death-Inducing Anti… DOI: http://dx.doi.org/10.5772/intechopen.95264*

### **Author details**

*Muscle Cell and Tissue - Novel Molecular Targets and Current Advances*

We tested the concept that cell death-inducing anti-cancer drugs may reduce the PA wall thickening using rat models of PAH. We found that: (1) The treatment of PAH rats with anthracycline-, proteasome inhibitor- or Bcl-2/Bcl-xL inhibitorclasses of drugs after pulmonary vascular remodeling had occurred resulted in the reversal of pulmonary vascular remodeling and opened up the lumen; (2) These effects were accompanied by the apoptosis of PA wall cells in PAH rats, but these drugs did not promote apoptosis in normal healthy rats, suggesting the anti-cancer drugs selectively kill remodeled vascular cells; (3) DNR, an anthracycline, and CFZ, a proteasome inhibitor, did not adversely affect the hypertrophied RV of PAH rats. (4) While the LV was damaged by CFZ, we identified cardioprotective agents (dexrazoxane and pifithrin-alpha) that can protect the heart against drug-induced cell death without affecting the efficacy of the drugs to reduce PA remodeling; (5) Docetaxel, a taxane class of anti-cancer drugs, not only reversed pulmonary vascular remodeling without exerting RV or LV toxicity, but also repaired the RV damaged caused by PAH. These findings from our laboratory as well as reports by other laboratories on the topic of the effects of programmed cell death-inducing anti-cancer drugs on remodeled PA and the RV affected by PAH in experimental

These results demonstrate that certain anti-cancer drugs effectively and selectively cause programmed cell death of abnormally grown cells in the remodeled pulmonary vasculature without adversely affecting the RV in rat models of PAH. Thus, the inclusion of programmed cell death-induced anti-cancer drugs may be promising for treating PAH patients. Human clinical trials of PAH treatment that test the effectiveness of these anti-cancer drugs as mono-therapies or combination therapies along with cardioprotective agents described here as well as already avail-

Daunorubicin, DNR (Anthracycline) Yes No effects Carfilzomib, CFZ (Proteasome inhibitor) Yes No effects Bortezomib (Proteasome inhibitor) Yes Apoptosis Navitoclax, ABT-263 (Bcl-2/Bcl-xL inhibitor) Yes Apoptosis Docetaxel, DTX (Taxane; Microtubule inhibitor) Yes Repairs Paclitaxel (Taxane; Microtubule inhibitor) Yes Unknown

**Reduces remodeled PA Affects remodeled RV**

This work was supported in part by the NIH (grant numbers R01HL072844, R21AI142649, R03AG059554, and R03AA026516) to Y.J.S. The content is solely the responsibility of the authors and does not necessarily represent the official views of

**8. Conclusions**

animals are summarized in **Table 1**.

able vasodilators are warranted.

**Acknowledgements**

*Abilities of various anti-cancer drugs to affect PA and RV remodeling.*

**Conflict of interest**

**66**

the NIH.

**Table 1.**

None.

Yuichiro J. Suzuki1 \*, Yasmine F. Ibrahim<sup>2</sup> , Vladyslava Rybka1 , Jaquantey R. Bowens1 , Adenike S. Falade1 and Nataliia V. Shults1

1 Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington, DC, USA

2 Department of Pharmacology, Minia University Faculty of Medicine, Minia, Egypt

\*Address all correspondence to: ys82@georgetown.edu

© 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.

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Section 2

Hypothenar Muscles

Section 2
