The Causes of Exercise Intolerance in Cardiovascular Diseases
Exercise intolerance is a key feature of most car- diovascular diseases. Its value for describing the stage of the disease is so important that standard- ized classification systems of exercise limitation (i.e. the New York Heart Association Classifica- tion) were developed more than half a century ago. While the symptoms of reduced maximal exercise capacity may be similar, the underlying mechanisms causing exercise limitations are fundamentally different between major disease entities:
In stenotic coronary artery disease (CAD) the patient is limited by the mismatch between myocardial perfusion and oxygen demand during physical exertion. The onset of relative myocardial ischemia determines the exercise capacity and is typically characterized by the development of angina pectoris. However, coronary stenoses are not the only source of myocardial ischemia – fre- quently they only become hemodynamically rele- vant in the presence of endothelial dysfunction, where an additional vasoconstriction can criti- cally diminish coronary blood flow in moder- ate stenoses. Alterations in coronary vascular endothelial function were first described in 1986 when Ludmer et al. observed a paradoxical vaso- constriction of atherosclerotic segments after infusing acetylcholine into the left coronary artery of patients with atypical chest pain.1 It has been shown that a paradoxical response to acetyl- choline is indicative of a greater vasoconstrictive
effect of both endogenous and exogenous cate- cholamines (i.e. norepinephrine and phenyle- phrine). Sympathetic activation and consecutive release of catecholamines occurs during physical activity, exposure to cold, or mental stress. In all these contexts paradoxical epicardial coronary vasoconstriction has been described, which makes endothelial dysfunction a likely pathome- chanism to explain stress- or exercise-induced angina pectoris in stable coronary artery disease.2 In chronic heart failure (CHF) exercise intoler- ance has traditionally been regarded as a conse- quence of either forward failure with inadequate rise in cardiac output during physical exertion or as secondary to backward failure with pulmonary congestion and dyspnea. However, this view was shattered in the 1980s when cardiologists found no correlation between the degree of left ventric- ular dysfunction (as measured by ejection frac- tion) and maximal exercise capacity.3 If central hemodynamics were not the principal determin- ing factor, alternative concepts needed to be gen- erated. The first focus was on systemic alterations in CHF: In the neurohormonal concept CHF was viewed as a clinical process that was initiated by myocardial dysfunction but then affected virtually any organ system as a consequence of activation of the renin–angiotensin–aldosterone system (RAAS) and augmented circulating cate- cholamines.4 This pathophysiological model of CHF is still valid today and has been extended by new findings of immune activation and inflamma- tion in CHF in recent years.5
Building on this model, peripheral changes associated with neurohumoral and inflammatory
12
The Molecular Base of Exercise
Rainer Hambrecht
67
activation have been systematically analyzed in the last decade. Characteristic changes of endothelial function, respiration, and skeletal muscle function – to name the factors most closely associated with exercise intolerance – have been found. Today we know that CHF causes a peripheral hypoperfusion due to impaired endothelium-dependent vasodilation,6 reduces the strength of respiratory muscles,7 and leads to profound morphologic,8,9 metabolic,10,11 and functional alterations in the skeletal muscles.12In the course of these scientific advances peripheral changes have become a new therapeutic target.
Over the last two decades the clinical applica- tion of physical exercise as a therapeutic strategy has developed from rehabilitation to exercise treatment of cardiovascular diseases. This shift in clinical application was accompanied by a more systematic research approach to the involved mechanisms and the objective clinical assessment of sport interventions using prospective random- ized clinical trials. This ongoing process estab- lished physical exercise as an evidence-based and guideline-oriented treatment option.
In this chapter some important molecular mechanisms involved in the training response to exercise will be discussed. Training mechanisms will be presented for two clinically important disease entities, which together represent the largest proportion of patients enrolled in rehabil- itation programs: stable coronary artery disease (CAD) and chronic heart failure (CHF).
Organ-Specific Adaptations to Exercise in Cardiovascular Diseases
Exercise Training in Coronary Artery Disease Effects of Exercise on the Vascular Endothelium in Atherosclerotic Disease
Despite the clear prognostic benefits of exercise training in reducing cardiovascular events, the underlying mechanisms have long remained obscure. Basically, regional myocardial hypoper- fusion in CAD results from a combination of four pathogenetic components: (A) vascular stenosis, (B) coronary vasomotion, (C) microrheology and hemostasis, and (D) mobilization of EPCs. All four
components may be affected by exercise training in stable CAD.
(A) Vascular Stenosis
The initial hypothesis that training would lead to a regression of coronary artery stenosis was not substantiated in the majority of patients. Only those with vigorous training programs were able to actually reverse the process of atherosclerosis.
However, training was effective in retarding disease progression.13
(B) Coronary Vasomotion
Coronary vasomotion is influenced by mechani- cal and agonist-mediated stimuli, both of which converge on endothelial nitric oxide synthesis/
release as the final common pathway. Endothelial dysfunction occurs as a result of decreased bioactive nitric oxide concentrations at vascular smooth muscle cells.
Nitric oxide concentrations can be affected by alterations at different steps of the NO pathway:
1. Availability of the precursor molecule L-arginine.
2. Alterations in NO synthesis rate as deter- mined by endothelial nitric oxide synthase (eNOS) conformational changes, expression, or genetic polymorphism.
3. Differences in NO breakdown velocity related to reactive oxidative species (ROS) once it is released (Figure 12-1).14
4. Finally, endothelial regeneration by circulat- ing bone-marrow derived endothelial progenitor cells (EPCs) is increasingly recognized as an important contributing factor for the pathogene- sis of endothelial dysfunction.
1. L-Arginine. The availability of L-arginine at the active site of eNOS depends on several factors: First, exogenous supply with L-arginine or endogenous synthesis. Second, intracellular accumulation of L-arginine, which depends on an active cytokine-regulated transmembraneous transport. Third, intracellular degradation of L-arginine. Fourth, nitric oxide synthesis from L-arginine may be blocked by its endogenous antagonist asymmetric dimethyl arginine (ADMA).15 Today, there is little doubt that a relative L-arginine deficiency is involved in
reduced NO generation. A rare genetic disorder (lysinuric protein intolerance – LPI) serves as a proof-of-concept model: In a patient with LPI markedly reduced L-arginine serum levels and endothelial dysfunction were described. A 30 min intravenous L-arginine infusion led to a dramatic improvement of endothelial function.16 Clinical intervention studies with oral L-arginine supple- mentation documented an improvement of endothelium-dependent vasodilation in several clinical situations like hypercholesterolemia, hypertension, diabetes, and chronic heart failure.17–19The effect of L-arginine supplementa- tion on vascular function is dose-dependent as evidenced by diverging results of low-dose and high-dose studies in patients with stable CAD:
While 9 g/day had no effect on vasomotion in patients with stable CAD,20 studies with either high-dose intracoronary L-arginine administra- tion or 21 g/day oral supplementation showed a significant improvement of endothelium- dependent vasodilation.21
In contrast to these encouraging results in pathologic situations, dietary L-arginine uptake was unrelated to the incidence of acute coronary events among 1981 men in the Kuopio Ischaemic Heart Disease Risk Factor Study.22
2. Endothelial Nitric Oxide Synthase. The activity of eNOS as the key enzyme for endothelial NO gen- eration can be modulated on several different levels: gene polymorphisms, mRNA expression, conformational changes, phosphorylation, and cofactor availability.
Among patients with overt atherosclerosis a number of gene polymorphisms of the eNOS gene have been described in recent years. Recent clinical trials suggest that the T768C and the Glu298Asp polymorphism may be associated with an increased risk for premature development of CAD.23,24 However, data are still controversial, with other studies failing to show an association between the Glu298Asp polymorphism and ather- osclerotic heart disease.25 Data from training studies with invasive measurement of endothelial function suggest that the improvement of vasomo- tion after exercise is attenuated or even abolished in eNOS promoter (T768C) polymorphisms.26 These data may have important implications for exercise-based rehabilitation strategies.
In a proatherosclerotic setting with high levels of oxidized low-density lipoprotein (oxLDL), elevated serum cytokines like TNF-α, and hypo- xia, eNOS expression is significantly reduced (reviewed in Harrison et al.27).
L-arginine
L-citrulline
vascular lumen
endothelial cell
extracellular space
vascular smooth muscle cell NO
peroxynitrite NO
eNOS
ecSOD ROS L-arg.
L-Arg. +
NAD(P)H oxidases Xanthine oxidase eNOS uncoupling
FIGURE12-1. Endothelial dysfunction develops as a consequence of several changes: L-arginine uptake and intracellular substrate avail- ability are reduced, eNOS expression is lowered, and extracellular inactivation of NO by reactive oxygen species (ROS) is increased. The major sources of ROS in the vascular wall are NAD(P)H oxidases, xanthine oxidase, and eNOS uncoupling. As a net effect, less NO reaches its target organ, the vascular smooth muscle cell.
eNOS phosphorylation plays an important role in short-term regulation of enzyme activity in response to flow conditions. Increased shear stress leads to an increased phosphorylation at Ser1177 with consecutive conformational changes leading to augmented NO generation.28
Finally, tetrahydrobiopterin (THB) is a neces- sary cofactor for NO production. However, in the absence of THB, eNOS uses molecular oxygen instead of L-arginine as a substrate, which leads to production of the toxic free radical superoxide O2−turning the enzyme “from good to evil.” This process is also referred to as eNOS uncoupling.
3. Nitric Oxide Breakdown. The integrity of en- dothelial function depends on an intricate balance between endothelial NO production and extracel- lular NO degradation. To cause vasodilation NO must reach the vascular smooth muscle cells by dif- fusion. In this way NO can be prematurely degraded in the presence of reactive oxygen species (OH, H2O2) leading to the formation of toxic per- oxynitrite (Figure 12-1), which in turn can induce endothelial cell damage and apoptosis by itself.
Several sources of ROS have been identified in endothelial dysfunction: (1) Adventitial NADPH oxidases, for example, produce quantities of superoxide high enough to affect endothelial function.29(2) Xanthine oxidase (XO) contributes to ROS generation especially in patients with CHF, where XO inhibition with oral or intra-arterial allopurinol led to significant improvements in endothelium-dependent vasodilation.30
The extracellular levels of ROS are also modu- lated by an antioxidative enzyme produced by the vascular smooth muscle cells: The extracellular superoxide dismutase (ecSOD) is induced by NO itself in a time- and dose-dependent fashion.31 It may be that despite constant or even slightly increased SOD the prevalence of large amounts of ROS in atherosclerosis leads to a mismatch between oxidative stress and antioxidative enzyme capacity resulting in decreased NO half-life.
4. Endothelial Progenitor Cell. The endothelium undergoes a constant process of cellular aging, apoptosis, and regeneration of endothelial cells.
Contrary to previous concepts, regeneration of endothelial cells in areas of diseased/damaged endothelium occurs not only from neighboring
endothelial cells by cell division but also from a pool of bone-marrow-derived circulating endothe- lial progenitor cells (EPCs). It has been docu- mented that these cells are reduced in the presence of established cardiovascular risk factors.32
EPCs are also involved in collateral formation.
In the past it was believed that sprouting of pre- existent vessels (i.e. angiogenesis) was the only way of forming new vessels in ischemic areas. In recent years it has become evident that EPCs are capable of forming entirely new vessels in a process termed vasculogenesis.
Exercise influences endothelial function on all four levels discussed above:
1. L-Arginine. Shear stress increases L-arginine uptake by endothelial cells by modulating the velocity of the endothelial transmembraneous transport system for L-arginine.33
2. Endothelial Nitric Oxide Synthase. Shear stress causes a dramatic increase in eNOS activity (up to 13 times basal levels) within only 60 minutes.34 This increase seems to be mediated by both short- term enhancement of eNOS activity and activa- tion of eNOS expression. Increases in eNOS expression were demonstrated in endothelial cell culture experiments after exposure to laminar shear stress35 and in animal studies of exercise training.36However, both alleles of the eNOS gene seem to be necessary to increase eNOS expression in response to exercise training: In mice heterozy- gotic for a loss of the eNOS gene no increased eNOS protein expression could be observed in the aorta whereas wild-type eNOS+/+mice had a 2.5 ± 0.4-fold increase.37
Several different phosphorylation sites exist within the eNOS enzyme – some of them inhibitory (threonine-495, serine-116), others stimulatory (serine-617, serine-635, and serine- 1177). Evidence from animal experiments confirmed that adenoviral transfection of a phosphomimetic serine-1179DeNOS (equivalent to the human serine-1177) to eNOS knockout mice completely restored endothelium-dependent vasodilation in response to acetylcholine whereas transfection with a non-phosphorylatable serine- 1179AeNOS resulted in significant endothelial dysfunction.38These findings underline the phys- iologic relevance of serine-1177 phosphorylation for NO generation.
We are only beginning to understand how the complex phosphorylation status of the eNOS enzyme is affected by exercise training. Increases in eNOS phosphorylation at serine-1177 have recently been documented in patients with CAD after 4 weeks of ergometer training in left internal mammary artery rings.28 Based on the data from this clinical study and other in vitro experi- ments shear-stress induced phosphorylation is mediated via phosphoinositide-3 (PI3) kinase and AKT (protein kinase B) in a calcium- independent manner and is maintained over a longer period of time (days) as compared to the short-term calcium-dependent changes in phosphorylation associated with exposure to bradykinin (for review refer to Fleming and Busse39).
3. Nitric Oxide Breakdown. It has long been unclear why exercise training, which increases total oxygen uptake (VO2) and in turn production of ROS, can yet improve endothelial function.
As mentioned above, it has just recently been shown that endothelium-derived NO increases expression of ecSOD in vascular smooth muscle cells.31 In the same publication the authors demonstrated that exercise training increased both eNOS and ecSOD in wild-type mice whereas ecSOD remained unchanged in mice lacking eNOS. This suggests that the effect of training on ecSOD is mediated via endothelium-derived NO.
ROS production is also affected by exercise:
After 4 weeks of ergometer training the activity of NAD(P)H oxidase – a key ROS-generating enzyme in the vascular wall – and the expression of its components were significantly reduced (Figure 12-2).40
4. Endothelial Progenitor Cells. A growing number of studies address the issue of how exercise – both acute and chronic – may affect circulating EPC levels. In the first such study, Adams documented a significant increase in circulating EPCs after a single maximal exercise test in patients with stenotic CAD and exercise-induced myocardial ischemia. Healthy subjects and non-ischemic CAD-patients showed no such increase.41 The effects of chronic endurance exercise were investi- gated in a second study by Laufs et al. who described an NO dependence of EPC increases in a transgenic animal model: eNOS−/−mice showed lower EPC numbers at baseline and a significantly attenuated increase of EPC in response to physical activity.42 Wild-type mice, on the other hand, showed a significant, nearly 3-fold increase in cir- culating EPCs after running training. In a prospec- tive randomized training study in patients with peripheral arterial occlusive disease (PAOD) with and without prior revascularization, we were able to document that only ischemic training induced a significant increase in circulating EPCs while non- ischemic training in patients post successful per- cutaneous angioplasty had no effect.
NO Generation
• eNOS expression
• eNOS activity
• ADMA
Oxidative Stress
• ROS generation
• NAD(P)H oxidase
• ecSOD activity
Oxidative Stress NO Generation
• ROS generation
• NAD(P)H oxidase
• ecSOD activity
• eNOS expression
• eNOS activity
• ADMA Training
FIGURE12-2. Imbalance between reduced NO generation and increased NO degradation as a result of higher oxidative stress in the vas- cular wall in atherosclerotic coronary artery disease. Exercise training and vascular shear stress augment endothelial NO production by increasing eNOS expression and activity; on the other hand, the local generation of ROS is reduced by exercise due to lower activity of ROS-producing enzymes like NAD(P)H oxidase and better antioxidative protection. As a result, endothelial function is improved.
(C) Microrheology and Hemostasis
Exercise training affects both functional and morphologic aspects of the microvascular bed:
Resistance vessel sensitivity and maximal respon- siveness to adenosine is improved and total vascular bed cross-sectional area increased by up to 37% after 16 weeks. While acute bouts of exer- cise may have thrombogenic side-effects, with platelet number and activity being increased,
chronic exercise training has been shown to attenuate this potentiation of platelet function, to increase platelet cGMP content, and to suppress coagulability.
Exercise Training in Chronic Heart Failure
Exercise training in CHF is a nonspecific inter- vention which simultaneously affects several organ systems (Figure 12-3). As the focus of this
FIGURE12-3. Sites of action of exercise training in CHF patients.
chapter is on molecular mechanisms rather than on functional changes, training effects on ventila- tion will be omitted.
Neurohormonal Adaptations
An aerobic training program in CHF patients reg- ularly reduces the resting heart rate, which indi- cates a reduction in sympathoadrenergic drive.
This has also been confirmed for serum cate- cholamine levels: Coats et al. showed a 16% reduc- tion in radiolabeled norepinephrine secretion after 8 weeks of training. This reduction in adren- ergic tone was accompanied by increase in heart rate variability.43In addition to the reduction in circulating catecholamines Braith et al. described a 25–30% reduction in angiotensin II, aldosterone, arginine vasopeptide, and atrial natriuretic pep- tide following 4 months of walking training in CHF patients.44
Vascular Adaptations
Exercise has a profound impact on peripheral endothelial dysfunction in CHF: It can improve basal nitric oxide production and enhance endothelium-dependent peripheral vasodila- tion.45 There seems to be a close correlation between the increase in endothelium-dependent dilation and the change in peak VO2(r= 0.64, P <
0.005), suggesting that peripheral hypoperfusion might play a contributory role to exercise intoler- ance in CHF. Data from animal experiments suggest that exercise can upregulate endothelial nitric oxide synthase (eNOS)36 and reduce NO breakdown by attenuating oxidative stress.46 Cardiac Adaptations
In a recently published randomized clinical trial47 it was confirmed that exercise has no negative impact on cardiac function. On the contrary, after 6 months of a regular aerobic training program, a small but significant improvement of left ventric- ular ejection fraction was observed accompanied by a reduction in left ventricular end-diastolic diameter.47
Skeletal Muscle Adaptations
CHF causes profound alterations in skeletal muscle morphology, metabolism, and function,
which are not just a consequence of decondition- ing but represent intrinsic changes induced by the systemic neurohumoral and inflammatory response in CHF.
Aerobic Energy Metabolism
All aspects of skeletal muscle characteristics can be positively influenced by training: On the ultrastructural level, the volume density of cytochrome-C positive mitochondria is increased,48 permitting an enhanced oxidative phosphorylation. These changes are also reflected by a fiber type shift from anaerobic fast type II to aerobic slow type I fibers in skeletal muscle biop- sies after 6 months of training.48
Anabolic–Catabolic Balance
As suggested by the association between low serum insulin-like growth factor I (IGF-I) levels and loss of lean muscle mass, patients with CHF suffer from a clinically relevant imbalance between anabolic and catabolic influences on the peripheral muscle.49In skeletal muscle biopsies of non-cachectic patients with CHF, the local IGF-I expression was substantially reduced despite normal growth hormone (GH) and IGF-I serum concentrations.50 It has been previously shown that a state of GH resistance may develop in cardiac cachexia. Exercise training has the poten- tial to increase local IGF-I expression and to reverse muscle catabolism.
Cytokines and Local Inflammation
Cytokine levels are elevated not only in the serum of patients with advanced CHF but also in skele- tal muscle biopsies of patients with stable moder- ate heart failure where TNF-α, IFN-γ, and IL-1β are potent activators of iNOS expression.51 The intracellular accumulation of NO is high enough to inhibit key enzymes of the oxidative phospho- rylation.52In vitro experiments documented that NO can thus attenuate the contractile perfor- mance of the skeletal muscle, a finding which puts cytokine production and iNOS expression in perspective for the development of exercise intolerance in patients with severe heart failure.
Regular exercise training can significantly reduce local cytokine expression and improve aerobic metabolism.53
Future Perspectives
Exercise training affects virtually every human organ system and we are just beginning to under- stand the complexity of the mechanisms involved.
Two new frontiers are currently being investigated with great enthusiasm: (1) In ischemic heart disease the potential role of bone-marrow-derived EPCs in the focus of several studies involving either interventional administration or endoge- nous mobilization by exercise. (2) After aerobic endurance training has been successfully estab- lished in CHF, researchers are now assessing the potential benefits of adding resistance exercise to achieve greater increases in muscle mass.
Despite the advances of the last years our understanding of the complex molecular path- ways activated by physical exercise is still in its infancy. A coordinated basic research approach is needed to shed more light on the physiological adaptations to exercise. Thus we will be able to recruit beneficial training effects for clinical applications on the basis of a better pathophysio- logical understanding as well as on empirical knowledge.
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