Skeletal Muscle Protein Synthesis and Reduced Anabolic Hormones/Effectors

Skeletal muscle protein synthesis is controlled by mechanoresponsive pathways and paracrine growth factor signaling loops, such as the local insulin-like growth factor-1 (IGF-1) system, but also responds to systemic stimuli, including growth hormone (GH), systemic IGF-1, anabolic steroids, and others. The GH/IGF-1 axis plays a key role in skeletal muscle growth and differentiation. Low systemic IGF-1 levels have been associated with a reduced leg muscle cross-sectional area (CSA) and total muscle strength in HF patients. Catabolic syndromes, such as chronic inflammation, sepsis, or cancer, show an altered state of the GH/IGF-1 axis, most probably due to a peripheral IGF-1 deficiency because of an impaired IGF-1 response to GH but also abnormal intrahepatic responses to GH. This has been attributed, in part, to increased serum levels and the local expression of proinflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α).

In humans with advanced HF and animal models of ischemic cardiomyopathy, reduced local expression of IGF-1 was detected in skeletal muscle compared with controls, accompanied by an increased expression of the IGF-1 receptor in the presence of normal serum levels of IGF-1. The serum concentration of the proinflammatory cytokines IL-1β and IL-6 were not found to be significantly changed, whereas TNF-α showed a trend toward higher levels in HF. Notably, the local expression of both IL-1β and TNF-α is increased in chronic HF and can be reduced by aerobic exercise training. Furthermore, a decreased single muscle fiber CSA in HF has been linked to local expression of IL-1β and impaired expression levels of expression of IGF-1 in skeletal muscle. These results suggest that, despite normal serum levels of IGF-1 and proinflammatory cytokines, the local expression of IGF-I is substantially reduced in HF, indicating a reduction in local anabolic stimuli. Because decreased IGF-1 levels and a reduced muscle fiber CSA correlate with increased levels of IL-1β, these results point toward a cytokine-mediated local catabolic process that is mediated, in part, through reductions in anabolic hormone expression.

Local expression of IGF-1 in skeletal muscle is mainly regulated by two different mechanisms. The first is GH receptor–dependent, a mechanism which, at least in part, is impaired in HF-induced weight loss due to a peripheral GH resistance. In addition, skeletal muscle IGF-1 expression is modulated in response to alterations in muscle use, and the expression of IGF-1 increases significantly in response to work-overload and passive stretch. In contrast, TNF-α and other cytokines may decrease skeletal muscle expression of IGF-1.

In addition to its classical role to enhance protein synthesis, IGF-1 has antiapoptotic effects in various tissues, protecting against cytokine-mediated apoptosis. These findings suggest that IGF-1 may regulate cell survival by modulating both proapoptotic and antiapoptotic stimuli. Thus the increased rate of skeletal muscle apoptosis in HF may be explained by a decline in local IGF-1 expression. This is suggested by preclinical models, where stimulation of IGF-1 production via GH administration reduces atrophy via reductions in apoptosis.

In male HF patients, deficiencies in circulating total testosterone, dehydroepiandrosterone (DHEA), and IGF-1 are common and correlate with a poor prognosis. Testosterone maintains skeletal muscle mass by increasing fractional muscle protein synthesis. In addition, testosterone appears to stimulate IGF-1 expression, but the exact molecular pathways are incompletely understood. At supraphysiologic doses, testosterone appears to act through androgen receptor–independent mechanisms. In HF patients, serum levels of free testosterone and DHEA are decreased, and this decrease correlates with HF severity. Perhaps most importantly, replacement of testosterone improves exercise capacity, muscle strength, and metabolic function.

Finally, insulin resistance is a hallmark of advanced HF. This metabolic abnormality is more pronounced during acute decompensated HF and improves upon cardiac recompensation. Moreover, improved cardiac output through left ventricular assist device placement results in reduced insulin resistance and improved glucose homeostasis in advanced HF. The effects of insulin to promote skeletal muscle anabolism occur during the daily cycle of feeding and fasting, where it serves to promote postprandial anabolism by reducing skeletal muscle protein breakdown. Studies have suggested that HF patients experience impaired suppression of insulin’s effects to reduce protein breakdown in response to meal-associated stimuli and that this impaired response was related to the circulating IL-6 level. Thus tissue insulin sensitivity, possibly secondary to immune activation, may contribute to skeletal muscle atrophy.

Skeletal Muscle Protein Breakdown and Increased Catabolic Hormones/Effectors

HF is a catabolic state, with increased levels of various catabolic hormones. Several authors have suggested a role for myostatin in muscle atrophy in patients with advanced HF. Myostatin is a local and circulating factor secreted from skeletal muscle with antianabolic and antihypertrophic actions. In fact, such a mechanism may be operable in patients, because circulating levels of myostatin, and other transforming growth factor (TGF) receptor ligands, such as activin, are increased in HF patients. Whatever might be the proximal hormonal/circulating effector, local skeletal muscle protein breakdown is mediated by several cellular systems that include lysosomal proteases, the adenosine triphosphate (ATP)–dependent ubiquitin-proteasome system, and the Ca ²⁺ -dependent calpain system ( Fig. 16.2 ). Specifically the ubiquitin–proteasome system has been implicated in the enhanced protein breakdown of atrophying skeletal muscle in a number of disease models, including chronic HF. Of particular interest is a group of ubiquitin-conjugating enzymes (E3-ligases) that target proteins for degradation by the proteasome. Through transcriptional screening, two E3-ligases, atrogin-1 (also called MFbx-1) and MURF-1 (muscle ring finger protein-1), have been identified to be highly induced in processes of muscular atrophy of different origin. Gomes and colleagues reported the identification and initial description of an increased expression of atrogin-1 (also called MAFbx-1), a muscle-specific E3-ligase, following starvation. Through a comparable analysis of genes regulated in atrophying muscle caused by different mechanisms, Bodine and colleagues identified the same gene (here called MAFbx-1) and, additionally, MURF-1, another E3-ligase. Through adenovirally mediated overexpression of these genes, their catabolic effects were demonstrated. Furthermore, animals with targeted deletion of MAFbx-1 or MURF-1 exhibit less muscular atrophy in response to denervation and hind limb suspension.

Regulation of muscle atrophy occurs through several highly conserved pathways of proteolytic changes in skeletal muscle. These are identical for various disease states, as well as disuse and immobilization. The main proteolytic pathway in skeletal muscle is the adenosine triphosphate (ATP)–dependent degradation of proteins through ubiquitination and action of the 26S proteasome. This pathway is transcriptionally regulated through FOXO transcription factors and expression of atrogenes, such as MURF-1 and atrogin-1. Activation of calcium-dependent calpains occurs due to increased levels of intracellular calcium released from the sarcoplasmic reticulum. Calpains are primarily responsible for destruction of tertiary structure and subsequent exposure of proteolytic cleavage sites. Finally, lysosomal protein degradation, or autophagy, is a highly regulated process that contributes to the destruction of cellular organelles. All these pathways likely contribute to skeletal muscle atrophy in advanced heart failure. IGF-1 , Insulin-like growth factor-1; IL-1β , interleukin-1β; ROS , reactive oxygen species; TNF-α , tumor necrosis factor-α.

Increased atrogin-1 has been reported in other animal models of muscular atrophy induced by immobilization, denervation, hind limb suspension, starvation, and sepsis. The induction of atrogin-1 expression prior to muscle weight loss in starvation suggests that the activation of this gene is involved in the development and progression of muscle protein loss. In support of this possibility, overexpression of atrogin-1 in C ₂ C ₁₂ myotubes induced atrophy in vitro, whereas muscular atrophy following denervation was prevented in animals with targeted deletion of atrogin-1. Intriguingly, infusion of the proinflammatory cytokine IL-1β induces the expression of atrogin-1 ⁴² and TNF-α increases the ubiquitin-conjugating capacity in myocytes, findings that again support the hypothesis of cytokines as putative mediators of muscular atrophy. Prior studies have shown transcriptional activation of E3 ligases in muscle of animals with left ventricular dysfunction and humans with advanced HF, whereas other reports did not show these changes. This might relate to the type of muscle injury and severity of HF. However, the exact mechanisms underlying the transcriptional activation of atrogin-1 by IL-1β remain to be elucidated.

Activation of the renin-angiotensin system results in vasoconstriction and elevated skeletal muscle concentration of angiotensin II (Ang II), which increases local oxidative stress, increases muscle proteolysis, and lowers skeletal muscle concentration of IGF-1. These mechanisms may accelerate protein degradation while decreasing protein synthesis. Loss of skeletal muscle mass contributes to muscle reflex alterations in HF. In normal subjects, muscle reflex activation helps to raise blood pressure and thereby maintain muscle perfusion during muscle acidosis. In HF, muscle reflex activation occurs at the onset of exercise, resulting in vasoconstriction and limited skeletal muscle perfusion.

Myofilament Contractile Adaptations

Alterations in skeletal muscle contractile function in HF may relate to changes in myofilament protein expression, their function, or both. Regarding the former, adult human skeletal muscle is composed of three fiber types, characterized primarily by the type of myosin isoform expressed. Table 16.1 details the structural and functional features of these types of muscle fibers. In general, most skeletal muscles contain a mixture of these fiber types, with myosin heavy chain (MHC) I and IIA being predominant and with very few fibers that express only the fastest MHC isoform (MHC IIX). This differs from rodents, which express a fourth fiber type MHC IIB that is faster and less oxidative than all the other fiber types. There are other contractile proteins whose expression varies by fiber types, but the functional character of each fiber is largely dictated by the type of myosin expressed.

Myofilament Protein Expression

One of the most often cited, skeletal muscle adaption to HF is the shift in fiber type toward a more fast-twitch, glycolytic phenotype, which is reflected by a change in expression of MHC toward a more fast-twitch isoform (IIA or IIX). The loss of slow-twitch, oxidative fibers has been demonstrated in animal models and humans and has long been held as a mechanism underlying the reduction in aerobic fitness. More recent studies have confirmed that this adaptation is also apparent in HFpEF patients. However, this switch toward a fast-twitch phenotype is apparent upon muscle disuse, raising the possibility that it may be a by-product of the physical inactivity that accompanies the HF syndrome. Early work that compared MHC expression between HF patients, healthy controls, and stroke patients confined to bed rest for more than 1 year as “inactive” controls argued against this notion. Whether patients who undergo muscle disuse secondary to loss of central neural activation represent the degree of activity restriction that occurs with HF, which is arguably more modest than being bedridden, is questionable. In fact, studies that have carefully matched HF patients to controls for fitness/activity level have observed no effect of HF to alter MHC isoform expression/fiber type.

Discordance between studies finding/not finding a shift in fiber type/MHC expression with HF may be explained by the advent of the use of angiotensin-converting enzyme inhibitors (ACEi) and angiotensin receptor blockers, as that these agents reverse the shift in fiber type back toward a more slow-twitch phenotype. However, more recent studies in human patients have observed this fiber type switch in patients taking an ACEi. In fact, studies conducted by the same laboratory in separate cohorts of human patients taking these medications have both observed and not observed changes in muscle fiber type, with the only difference between the two cohorts being the activity status of controls. This further highlights the potential modulating role of physical activity on fiber type characteristics in the HF population. Building on this notion, and considering that a similar switch in fiber type from a slow- to a fast-twitch phenotype is emblematic of muscle disuse, the effects of these medications might reflect their ability to improve symptomology and, in turn, increase physical activity in patients.

In addition to alterations in the expression of specific isoforms of MHC, recent studies have found evidence for a select reduction in the expression of myosin relative to other contractile proteins on the order of 15% to 20% ( Fig. 16.3 ). Mechanical assessments that reflect cross-bridge number were similarly reduced, suggesting that these results are not attributable to variation in biochemical extraction of myosin. Moreover, these reductions are similar in magnitude with what has been observed in animal models and have been shown to related to functional decrements. Importantly, this loss was not related to muscle disuse, as suggested by some studies, because patients were matched to controls for physical activity level. Similar patterns of myosin loss have been shown in other clinical conditions (e.g., chronic obstructive pulmonary disease [COPD], critical care myopathy ), suggesting this may be a common contractile protein adaptation related to some aspect of acute/chronic disease.

Effects of heart failure (HF) to decrease skeletal muscle contractile function. HF impairs intracellular Ca ²⁺ release, thereby diminishing activation of myofilament proteins and lessening contractile force (lower plateau of the force tracing). In addition, HF reduces myosin protein expression and myosin-actin cross-bridge function, which likely has the net effect of reducing contractile velocity (ascending limb of the force tracing). In addition, evidence suggests that HF increases relaxation time (descending limb of the force tracing), possibly secondary to reduced Ca ²⁺ reuptake into the sarcoplasmic reticulum (SR). Collectively, these adaptations likely reduce contractile force, velocity and, in turn, power output, leading to a reduced capacity for physical work. In addition, adaptations in myofilament function and Ca ²⁺ dynamics may contribute to increased fatiguability of skeletal muscle. FKBP, FK506 binding protein; RyR, ryanodine receptor; SERCA , sarcoplasmic reticulum Ca ²⁺ ATPase.

Excitation-Contraction Coupling Adaptations

Although alterations in myofilament protein function may explain diminished contractile velocity and, in turn, power production, they do not appear to explain reduced force production (i.e., muscle strength). This may be explained, instead, by impaired excitation-contraction coupling (ECC). More specifically, diminished calcium (Ca ²⁺ ) release from the sarcoplasmic reticulum leads to decreased myofilament activation and, in turn, force production (see Fig. 16.3 ). In addition, impaired ECC function may partially underlie the fatigue that HF patients report on exertion. In the next section, we review evidence that HF alters the ECC system. Because most of the components of the ECC system intrinsic to skeletal muscle cannot be assessed in humans, this discussion primarily relies on data from animal models.

Most of the research in this field has focused on Ca ²⁺ release and reuptake from the sarcoplasmic reticulum. Early work in the rat coronary artery ligation model of HF showed that reduced skeletal muscle (extensor digitorum longus; fast-twitch muscle) tension was accompanied by decreased intracellular Ca ²⁺ release. In addition, HF animals were associated with accelerated fatigue rates, as assessed by the decrease in tension following repeated tetanic contractions. Other studies have further suggested that Ca ²⁺ uptake may also be impaired in HF, based on diminished expression of the sarcoplasmic reticulum Ca ²⁺ ATPase (SERCA). These seminal results stimulated research in this area because such decrements in Ca ²⁺ release and reuptake could underlie greater fatiguability in skeletal muscle in HF.

Studies that included slow- (soleus) and fast-twitch muscles/isolated muscle fibers of rats using the same coronary artery ligation model of HF showed conflicting results. Under nonfatigued conditions, there were no decrements in contractile strength with HF, although there was evidence for slowed relaxation times. Interestingly, contrary to the previously mentioned reports of a loss of SERCA protein expression, this study found no effect of HF on SERCA expression, despite the presence of its resultant physiological phenotype (i.e., slowed relaxation). Moreover, there were no prominent differences between HF and control animals during fatiguing contractions in tension development or intracellular Ca ²⁺ levels, although force relaxation was markedly slowed in the slow-twitch soleus muscle. Follow-up studies by this same group, where contractile properties of the soleus muscle were assessed in situ using a protocol that more closely simulated submaximal contractile activity, observed a reduced relaxation rate in HF animals, which was also accompanied by increased fatigability. Subsequent studies in isolated single muscle fibers have further suggested that increased fatigability in HF animals was not associated with impaired Ca ²⁺ release. Instead, the authors assert that impairments in myofilament protein function may develop with fatiguing contraction to produce the reduction in tension. This could potentially be explained by reduced cross-bridge kinetics detailed previously, because maneuvers that increase the rate of cross-bridge cycling reduce skeletal muscle fatigue.

More recent studies, also conducted in the rat coronary ligation model of HF, have further supported impaired sarcoplasmic reticulum (SR) Ca ²⁺ release. Ward and colleagues showed a transient reduction in the intracellular Ca ²⁺ in HF animals. Ca ²⁺ sparks, which are spontaneous, localized Ca ²⁺ release events that play an important role in dictating aggregate intracellular Ca ²⁺ release, have smaller amplitude, slower temporal kinetics, and greater spatial spread. In these same studies, the sarcoplasmic reticulum Ca ²⁺ release channel, the ryanodine receptor, was hyperphosphorylated and had less associated FKBP12 (also known as calstabin). Further work showed that protein kinase A–mediated phosphorylation of the ryanodine receptor causes dissociation of FKBP12, which normally functions to inhibit the channel. This conspiration of changes in ryanodine receptor biochemistry is associated with a “leaky” channel phenotype that has been found in cardiomyocytes in models of HF and is thought to contribute to disease progression. This is significant because it links the well-known heightened sympathetic nervous system activity in the HF syndrome to the skeletal muscle contractile phenotype. In fact, this leaky channel phenotype may be important for exercise intolerance in HF because it has been suggested to contribute to fatigue in humans.

Whether similar alterations in intracellular Ca ²⁺ dynamics are present in humans with HF is unknown. Using isolated sarcoplasmic reticulum vesicles, recent studies have found no alterations in Ca ²⁺ release, uptake, or leak in patients compared with controls. In addition, contrary to animal models, some studies have suggested an upregulation of Ca ²⁺ uptake in patients with HF compared with controls, although some studies suggest downregulation of the expression of these proteins. Thus the balance of available evidence, although quite small, suggests that the phenotype of ECC in human HF may not be adequately reflected in available animal models. This could be due to the fact that measurements in human samples are necessarily different than in animal models. Alternatively, discordant results may be explained by the fact that patients are treated with pharmacologic agents that counteract some of the pathologic Ca ²⁺ regulatory alterations (e.g., beta-blockers may prevent ryanodine receptor hyperphosphorylation and the resulting calcium release abnormalities). Further work is required to clearly define the ECC phenotype in human HF to determine its contribution to reduced skeletal muscle contractile strength.