Summary Molecular Mechanisms in Exercise-Induced Cardioprotection.

Molecular Mechanisms in Exercise-Induced Cardioprotection. It is generally accepted that regular exercise is an effective way for reducing cardiovascular morbidity and mortality [1]. Physical inactivity and obesity are also increasingly recognized as modifiable behavioral risk factors for a wide range of chronic diseases, including cardiovascular diseases. Furthermore, epidemiologic investigations indicate that the survival rate of heart attack victims is greater in physically active persons compared to sedentary counterparts [2]. Several large cohort studies have attempted to quantify the protective effect of physical activity on cardiovascular and all cause mortality. Nocon et al. [3] in a meta-analysis of 33 studies with 883,372 participants reported significant risk reductions for physically active participants. All-cause mortality was reduced by 33%, and cardiovascular mortality was associated with a 35% risk reduction. Exercise capacity or cardiorespiratory fitness is inversely related to cardiovascular and all-cause mortality, even after adjustments for other confounding factors [4–6]. The American College of Sports Medicine defines exercise as “Any and all activity involving generation of force by the activated muscle(s) that results in disruption of a homeostatic state”. Exercise is classified by the type, intensity, and duration of activity. Endurance exercise reflects prolonged and continuous periods of contractile activity (high repetition) against low resistance whereas resistance exercise (strength training) involves short periods of contractile activity (low repetition) against a high opposing resistance, while sprint exercise occurs during short periods of maximal (intense) repetitive contractile activity, where there is a short period of exercise against a low resistance, such as running a 100-m sprint race. However, sprint training can also be performed against high resistance, which results in a combination of resistance and endurance modalities, for example, running with added weights [7]. Although it is clear that exercise promotes a cardioprotective phenotype, a detailed understanding of the cellular mechanisms responsible for this cardioprotection remains incomplete. The cytoprotective mechanisms that have garnered most attention are inducible heat shock proteins and antioxidant enzymes, especially superoxide dismutase (SOD) [8–11]. However, there are numerous clues suggesting that other mechanisms may also be part of exercise-induced 2 Cardiology Research and Practice cardioprotective phenotype, and they may have significant roles in the protective process. Improving our understanding of the molecular basis for exercise-induced cardioprotection will play an important role in developing optimal exercise interventions for primary and secondary prophylaxis. The objective of this paper is to summarize the body of knowledge related to the molecular basis of cardioprotective effects of exercise and the evidence for other potential mediators. We mainly focused on adaptations within the cardiac muscles and will briefly examine coronary adaptations as well. However, it should be noted that other adaptive mechanisms in the blood, humoral and autonomic nervous system, muscles, and so forth can also affect this process. 2. Exercise-Induced Expression of HSPs in the Heart The ability of organisms to respond to various stressors was first described by Selye in 1936, who suggested a series of programmed events that help organisms to cope with the situation [12]. The heat shock response is a common cellular reaction to external stimuli such as ischemia [13], hypoxia [14], acidosis [15], oxidative stress [16], protein degradation [17], increased intracellular calcium [18], and energy depletion [19] (Table 1). Therefore, the terms “stress proteins” and “cellular stress response” reflect the array of stressors known to initiate heat shock protein (HSP) expression [20]. It is generally accepted that exercise increases the expression of cardiac HSPs. Locke et al. [21] performed a comprehensive study of the effects of acute exercise on myocardial expression of HSPs and showed that the expression of HSPs can begin within 30–60 min after an exercise bout in the rat myocardium. The mechanistic link between exercise and myocardial expression of HSPs is unclear. However, a variety of stresses associated with exercise, including heat stress and hypoxia, reduced intracellular pH, reactive oxygen and nitrogen species (ROS and RNS) production, and depletion of glucose and glycogen stores, increase in cytosolic calcium levels and cardiomyocyte stretching can all contribute to HSP elevation in cardiac muscle [22]. Several stress proteins such as HSP10, HSP40, HSP60, and HSP90 are thought to play cardioprotective roles [23, 24], with some detailed understanding of the role of the HSP70 family [25]. Production of HSP70 in the rat myocardium during treadmill running is mediated by an increase in the activation of heat shock transcription factor 1 (HSF 1), which then binds to the heat shock element of HSP70 genes and results in increased levels of HSP70 mRNA [26]. In an attempt to provide additional insights, Melling et al. showed that protein kinase A (PKA), and not protein kinase C, plays a key role in exercise-induced regulation of HSP70 gene expression [27]. Activation of PKA mediates the suppression of an intermediary protein kinase, extracellular signal regulated kinases (ERK1/2), which phosphorylates and suppresses the activation of the HSF1. However, following exercise training, ERK1/2 regulates the transcriptional activation of several genes involved in cell growth and proliferation and has been shown to be associated with training-mediated myocardial hypertrophy. In another study, Melling et al. [28] reported increased expression of HSP70 following a single bout of exercise in the presence of elevated ERK1/2 in trained animals. These results suggest that training results in adaptations that allow for the simultaneous initiation of both proliferative and protective responses. In addition, HSP72 (a member of HSP70 family) augments myocardial antioxidant capacity [29]. During ischemia/reperfusion (I/R) insult, HSP72 preserves mitochondrial function and provides cellular protection against apoptosis [30, 31]. Moreover, by prevention of protein aggregation and denaturation, HSP72 also enhances recovery from acute cellular injury and so protects cells from subsequent injury [32]. Although exercise induces cardiac HSP72 expression (which is cardioprotective), there are some reports demonstrating that the beneficial effects of exercise on cardiac muscle is independent of HSPs formation [33]. Hamilton et al. postulated that exercise associated cardioprotection may depend, in part, on increases in myocardial content manganese superoxide dismutase (MnSOD), rather than HSP levels [34], while studies by Quindry et al. suggest that exercise-induced myocardial protection against I/R is unrelated to increases in cardiac levels of HSP10, HSP27, HSP40, HSP60, HSP72, HSP73, or HSP90 [35, 36]. One explanation for these findings is the activation of cardioprotective mechanisms through different pathways that then converge on common distal pathways, suggesting that exercise-induced cardioprotection could be achievable through multiple mechanisms. 3. Exercise and Myocardial Antioxidant Capacity Cells have evolved highly complex antioxidant systems (enzymic and nonenzymic), which work synergistically to protect cells and organ systems of the body against free radical-induced damage. The most efficient enzymatic antioxidants involve glutathione peroxidase, catalase, and superoxide dismutase. Nonenzymatic antioxidants include vitamins E and C and thiol antioxidants (glutathione, thioredoxin) [38]. Each of these antioxidants is capable of combining with reactive oxidants to produce other less reactive spices. Superoxide dismutase promotes the dismutation of the superoxide radical to form hydrogen peroxide (H2O2) and oxygen. While glutathione peroxidase (GPx) uses reduced glutathione (GSH), as a reducing equivalent, to reduce H2O2 to form oxidized glutathione and water. Furthermore, GSH can remove selected oxygen radicals directly and assist in the recycling of vitamin C and E (Table 2). Catalase converts H2O2 to water and oxygen. The newly identified peroxiredoxin family represents a group of peroxidases that also catalyze the reduction of H2O2. They include at least six isoforms in mammalian cells [39]. Among them, peroxiredoxin III is synthesized with a mitochondrial targeting sequence, as is MnSOD. The effects of exercise on myocardial antioxidant enzyme activities has been widely investigated but with variable conclusions. For example, while some studies report that exercise-induced increases in GPx activity [40, 41], others Cardiology Research and Practice 3 Table 1: Cellular location and function of selected heat shock proteins (HSPs) [22, 23, 32, 37]. Family Members Cellular location Proposed function (comment) Ubiquitin Ubiquitin Cytosol Protein degradation (ubiquitin levels in cells increase following cellular injury) HSP10 HSP10 Mitochondria Molecular chaperons, cofactor for HSP60 HSP27 HSP27, HSP26, and so forth Cytosol, nucleus Microfilament stabilization, antiapoptotic (variable in size and number in different organisms) HSP60 HSP60 Mitochondria Refolds proteins and prevents aggregation of denatured proteins, proapoptotic. HSP70 HSP72, HSP73, HSP75, HSP78 Cytosol, nucleus, mitochondria, endoplasmic reticulum Molecular chaperons, protein folding, and cytoprotection (HSP72 postulated to play an important role in myocardial protection against I/R injury). HSP90 HSP90, HSP100, Grp94 Cytosol, nucleus, ER May function as a molecular chaperone during maturation of steroid receptors and assists in the folding of newly synthesized peptides, protein translocation. suggest that myocardial GPx activity is not altered by exercise [42, 43]. Such equivocal results have also been reported for myocardial catalase, with increased [44, 45] and decreased [33, 43] levels. Although there is no clear explanation for these discrepancies, it is possible that the use of exercise protocols that differ in duration and intensity may be an important underlying factor. The strongest evidence to directly link increases in myocardial antioxidants and exercise-induced cardioprotection implicates a contributory role for MnSOD. It is generally believed that even short-term endurance exercise results in a rapid increase in myocardial MnSOD activity [43, 46, 47] as shown in studies that employed antisense oligonucleotide techniques to silence MnSOD genes and so prevent exercise-induced increases in myocardial MnSOD activity [46, 48]. For example, Yamashita et al. [46] reported that inhibition of exerciseinduced increases in cardiac MnSOD abolished protection against myocardial infarction, findings that were confirmed by Hamilton et al. [49] who concluded that MnSOD plays a key role against I/R-induced cardiac arrhythmias. In contrast, Lennon et al. [50] reported that exercise-induced increases in cardiac MnSOD activity is not essential to achieve exercise-mediated protection against myocardial stunning. It is possible that mechanisms responsible for exercise-induced protection against myocardial stunning are different from those involved in myocardial arrhythmias and infarction [51] (Figure 1). 4. Additional Mechanisms Involved in Exercise-Induced Cardioprotection The induction of myocardial HSPs and upregulation of cardiac antioxidant enzymes are well-studied aspects of the response to exercise. Other related responses to exerciseinduced cardioprotection include anatomical and physiological changes in the coronary arteries, increased myocardial cyclooxygenase-2 (COX-2) activity, elevated endoplasmic reticulum stress proteins, nitric oxide production, increased autophagy, and improved function of sarcolemmal and/or mitochondrial ATP sensitive potassium (sarco KATP and/or mito KATP) channels [52] (Figure 2). Arrhythmia Stunning Myocardial infarction Time Ischemia intensity Figure 1: Sequence of pathologic events in ischemia reperfusion injury. 5. Myocardial Collateral Circulation Exercise-induced adaptation of the coronary circulation can be divided into two major processes: (a) angiogenesis, which is an expansion of the capillary network by the formation of new blood vessels and occurs at the level of capillaries and resistance arterioles, but not in large arteries and (b) arteriogenesis, which is as enlargement of existing vessels [53]. 5.1. Angiogenesis. It has been speculated that endurance exercise stimulates angiogenesis by either a division of preexisting endothelial cells or by bone marrow-derived endothelial progenitor cells and monocyte or macrophage derived angiogenic cells [54]. Some reports indicate that physical activity improves the mobilization of endothelial progenitor cells in healthy subjects and in patients with cardiovascular risk and coronary artery disease [55, 56]. Indeed, angiogenesis is regulated by a net balance between positive (angiogenic) and negative (angiostatic) regulators of blood vessel growth. A balance favoring predominantly positive regulators are an angiogenic phenotype whereas a shift favoring negative regulators is an angiostatic phenotype. Therefore, an impaired regulation of angiogenesis is often associated with the development of angiogenesis-dependent diseases such as atherosclerosis. 4 Cardiology Research and Practice Exercise Change in coronary circulation ↑ Cardiac HSPs ↑ COX II activity ↑ ER stress proteins ↑ Myocardial antioxidants ↑ Sarco and Mito KATP channels ↑ Autophagy Cardioprotection against I/R injury Figure 2: Proposed mechanisms for exercised-induced cardioprotection. Endostatin is an endogenous angiostatic factor identified originally in conditioned media of murine hemangioendothelioma cells [57, 58]. Many studies have shown that the proteolytic release of endostatin from collagen XVIII is mediated by proteases of many classes, such as cysteine proteases, matrix metalloproteases, and aspartic proteases [59, 60]. The potent antiangiogenic effects of endostatin are mediated via a combination of effects on endothelial cells, where endostatin inhibits cellular proliferation and migration and stimulates apoptosis [61, 62]. The biological effects of endostatin are mainly attributed to its antagonism of vascular endothelial growth factor (VEGF) signaling [63]. Angiogenesis has both beneficial and deleterious effects in atherosclerosis. While increased angiogenesis in cardiac tissue may be a favorable sign in the healing of the ischemic tissues [64], progressive angiogenesis in a primary atherosclerotic lesion could be a cause of plaque expansion [65, 66]. There are several studies showing that exercise induces a local angiogenic phenotype characterized by overexpression of VEGF in skeletal muscle [67] and heart [64]. This phenomenon can prevent ischemia in these tissues. Exercise can also exert beneficial effects against atherosclerosis by increasing circulating endostatin, which inhibits development of atherosclerotic plaque through blocking angiogenesis in the plaque tissue [68]. Meanwhile, Brixius et al. also reported that endurance activity improves angiogenesis by reducing endostatin plasma levels [69]. Even though the different exercise protocols in these experiments can explain these discrepant results, further studies are needed to elucidate the precise mechanisms. 5.2. Arteriogenesis. Exercise training increases the diameter of large arterioles, small arteries, and conduit arteries. Another important aspect of exercise-induced changes in capillarity is the onset and persistence of exercise-induced arteriogenesis. The induction of arteriogenesis is an important vascular adaptation [70], since arteriogenesis leads to the formation of large conductance arteries capable of compensating for the loss of function of occluded arteries. Animal studies and clinical observations provide evidence for a significant correlation between regular physical exercise and increased coronary artery lumen diameter [71, 72]. In one study, an 8-week training programme increased the contractile response to low doses of dobutamine in patients with chronic coronary artery disease and having a left ventricular ejection fraction below 40%. This implies that short-term exercise training can improve quality of life by improving left ventricular systolic function during mild to moderate physical activity in patients with ischemic cardiomyopathy [73]. Moreover, eight patients with coronary heart disease and exertional angina pectoris successfully completed an 11– 15 week programme of endurance exercise conditioning. Angina threshold was determined by upright bicycle ergometer exercise and by atrial pacing. The product of heart rate and arterial systolic blood pressure at the exercise angina threshold was higher after conditioning, suggesting that conditioning increased the maximum myocardial oxygen supply during exercise [74]. 6. Cyclooxygenase II and Exercise-Induced Cardioprotection The phenomenon of ischemic preconditioning whereby brief episodes of sublethal ischemia renders the myocardium resistant to subsequent ischemic stress occurs in two phases: (i) an early phase that starts within a few minutes after the initial ischemic stimulus, lasts for 2-3 h, and is due to adenosine and bradykinin release and (ii) a second phase, which begins 12–24 h later and lasts for 3-4 days [75, 76]. This later phase of ischemic preconditioning is caused by the simultaneous activation of multiple stress responsive signaling pathways, including COX-2 and the inducible form of nitric oxide synthase (iNOS), resulting in the heart developing a phenotype that confers sustained protection against both reversible (stunning) and irreversible (infarction) myocardial I/R injury [76]. Similar to ischemic stimuli, both short- (1–3 days) and long-term (weeks— months) exercise protocols are equally effective in conferring cardioprotection against I/R injury [77, 78]. Given the phenotypic similarities between ischemic and exercise preconditioning, and since COX-2 and iNOS are required to achieve the late phase of ischemic preconditioning-induced cardioprotection against both I/R stunning Cardiology Research and Practice 5 Table 2: Major antioxidants. Name Role Remarks Endogenous enzymes Superoxide dismutase (SOD) (a) mitochondrial (b) cytoplasmic (c) extracellular Dismutase superoxide to H2O2 (a) Contains manganese (MnSOD) (b) Contains copper and zinc (CuZnSOD) (c) Contains copper (CuSOD) Catalase Dismutase H2O2 to H2O Present in peroxisomes Glutathione peroxidase Removes H2O2 and lipid peroxides Selenoproteins (contains Se2+) Primarily in the cytosol also mitochondria Vitamin E Lipid peroxidation, scavenges superoxide, hydroxyl and lipid peroxide Fat soluble Nonenzymatic substance Vitamin C Scavenges superoxide, hydroxyl radicals and H2O2, contributes to regeneration of vit E. Neutralizes oxidants from stimulated neutrophils Water soluble β-carotene (provitamin A) Scavenges ·OH, O2 ·− and peroxy radicals Prevents oxidation of vitamin A Binds to transition metals Water soluble and infarction [76, 79], it had been postulated that COX2 activity, and subsequent prostanoids production, may also be essential for exercise-induced cardioprotection [80]. However, Nagy et al. reported that COX II inhibition did not prevent exercise-induced cardioprotection against ventricular arrhythmias after coronary occlusion in dogs [81]. Accordingly, studies by Quindry et al. [82, 83] revealed that exercise does not elevate cardiac COX-2 activity. Therefore, we conclude that different mechanisms may be responsible for cardioprotection afforded by ischemia preconditioning and aerobic exercise. 7. Exercise-Induced Cardioprotection by Endoplasmic Reticulum Stress Proteins Another candidate mechanism explaining exercise-induced cardioprotection is through a family of cardioprotective protein collectively called endoplasmic reticulum (ER) stress proteins. During a cardiac I/R insult, the ER helps cellular homeostasis by maintaining intracellular calcium regulation and protein folding [84]. Malfunction of the ER during an I/R event can lead to both mitochondrial-dependent and mitochondrial-independent cell death due to disruption of calcium homeostasis and/or impaired protein folding [85]. The two most important ER stress proteins are Grp78 and Grp94 which belong to HSP families and are overexpressed in cultured cardiomyocytes during oxidative stress and calcium overload [86]. Martindale et al. suggested that the unfolded protein response (UPR) is activated in the heart during I/R, where formation of “Activation of Transcription Factor 6 (ATF6)” induces the expression of proteins such as Grp78 and Grp94, which in turn reduces I/R-induced necrosis and apoptosis in the heart [87]. Since overexpression of these ER stress proteins provide ER protection during an I/R insult, it may be that these proteins contribute to exerciseinduced cardioprotection. However, studies by Murlasits et al. demonstrate that at least short-term exercise training does not elevate ER stress proteins, and therefore, short-term exercise-induced cardioprotection may not be linked to ER stress adaptation [88]. 8. Nitric Oxide and Exercise-Induced Cardioprotection Nitric oxide plays a fundamental role in protecting the heart against I/R injury. Specifically, it plays a critical bifunctional role as a trigger and a mediator of late phase of preconditioning. Increased production of NO by eNOS is an important trigger in the late phase of ischemia and exerciseinduced preconditioning, and enhanced NO production by iNOS is mandatory in the mediation of the antistunning and antiinfarct actions of late preconditioning [89]. In other words, the heart responds to ischemia by using two types NOS isoforms in a harmonized manner; it uses preexisting eNOS for a prompt burst of NO production, while at the same time, there is upregulation of iNOS to switch to a defensive phenotype by a sustained enhancement of NO biosynthesis. The exact cardioprotective mechanisms of NO during I/R is a subject of much debate; however, hypothesized mechanisms include inhibition of calcium influx into myocytes [90], antagonism of β-adrenergic stimulation [91], reduction in cardiac oxygen consumption [92, 93], and actions on sarco KATP channels [94]. Although there are many studies on the cardioprotective effect of nitric oxide in I/R-induced preconditioning, there are less investigations evaluating the protection afforded by nitric oxide in exercise-induced cardioprotection. Exercise increases the expression and activity of eNOS, likely due 6 Cardiology Research and Practice to changes shear stress, which modulates the production of NO [95–97]. Besides its vascular and metabolic effects (vasodilation, altered carbohydrate metabolism) [98], NO possess a number of physiological properties which makes it a cardioprotective molecule in the setting of myocardial I/R injury. For example, NO reversibly inhibits mitochondrial respiration [99] and apoptosis [100]. This effect results in a reduction in mitochondrial-induced cardiac injury in the failing human heart. Babai et al. showed that even a single period of exercise affords delayed protection against I/Rinduced ventricular tachycardia and other ischemic changes that are mediated by NO [101]. Further evidence for a central role of eNOS in exercise physiology comes from experiments with eNOS deficient mice (eNOS−/−). Running capacity in these animals is 50%–60% less than age-matched wild-type mice [102, 103]. de Waard et al. found that the beneficial effects of exercise after MI on LV remodeling and dysfunction depends critically on endogenous eNOS. They observed that the lack of one eNOS allele is sufficient to negate all beneficial effects of exercise, strongly suggesting that the beneficial effects of exercise depends on full eNOS expression [104]. Taylor et al. in an in vitro setting showed that exercise can render a cardioprotective phenotype against I/R insult even in the presence of blockade of all NOS isoforms with L-NAME, leading them to conclude that acute exercise-induced preconditioning is not mediated by NOS (or either increases in HSP 72 or other antioxidant enzymes) [105]. These discrepant findings can be accounted for by considering the role of nitrite and nitrates in the physiology of the cardiovascular system. For example, it has been shown that nitrite (and nitrates) acts as a storage reservoir on NO so that under pathological conditions such as ischemia and hypoxia, nitrite can readily be reduced to NO [106, 107]. The beneficial effect of nitrite therapy (which generates NO-independent of eNOS) has been shown in animal models of cardiac I/R injury [108, 109]. Nitrite levels are increased following exercise in both rodents and humans [110, 111]. Since nitrite accumulated in the heart can provide cardioprotection during an ischemic event by being reduced to NO, it is likely that NO produced from endothelial cells during exercise can be oxidized to nitrite, transported in the plasma to then affect the heart or vasculature. In the case of myocardial ischemia, nitrite can be reduced back to NO and exert cardioprotection in an NO-dependent manner [112]. This hypothesis potentially could be applied to all the experiments in which exercise-induced cardioprotection was not abolished in the presence of an inhibitor of NOS [105, 113]. To add to the importance of NO in exercise physiology is its ability to activate KATP channels through cGMP-PKG signaling [114, 115] and to increase expression of HSP70 [116] during exercise