Christopher L. Mendias

Inducible depletion of satellite cells in adult, sedentary mice impairs muscle regenerative capacity without affecting sarcopenia

A key determinant of geriatric frailty is sarcopenia, the age-associated loss of skeletal muscle mass and strength. Although the etiology of sarcopenia is unknown, the correlation during aging between the loss of activity of satellite cells, which are endogenous muscle stem cells, and impaired muscle regenerative capacity has led to the hypothesis that the loss of satellite cell activity is also a cause of sarcopenia. We tested this hypothesis in male sedentary mice by experimentally depleting satellite cells in young adult animals to a degree sufficient to impair regeneration throughout the rest of their lives. A detailed analysis of multiple muscles harvested at various time points during aging in different cohorts of these mice showed that the muscles were of normal size, despite low regenerative capacity, but did have increased fibrosis. These results suggest that lifelong reduction of satellite cells neither accelerated nor exacerbated sarcopenia and that satellite cells did not contribute to the maintenance of muscle size or fiber type composition during aging, but that their loss may contribute to age-related muscle fibrosis.

Tendons of myostatin-deficient mice are small, brittle, and hypocellular

Tendons play a significant role in the modulation of forces transmitted between bones and skeletal muscles and consequently protect muscle fibers from contraction-induced, or high-strain, injuries. Myostatin (GDF-8) is a negative regulator of muscle mass. Inhibition of myostatin not only increases the mass and maximum isometric force of muscles, but also increases the susceptibility of muscle fibers to contraction-induced injury. We hypothesized that myostatin would regulate the morphology and mechanical properties of tendons. The expression of myostatin and the myostatin receptors ACVR2B and ACVRB was detectable in tendons. Surprisingly, compared with wild type (MSTN+/+) mice, the tendons of myostatin-null mice (MSTN−/−) were smaller and had a decrease in fibroblast density and a decrease in the expression of type I collagen. Tendons of MSTN−/− mice also had a decrease in the expression of two genes that promote tendon fibroblast proliferation: scleraxis and tenomodulin. Treatment of tendon fibroblasts with myostatin activated the p38 MAPK and Smad2/3 signaling cascades, increased cell proliferation, and increased the expression of type I collagen, scleraxis, and tenomodulin. Compared with the tendons of MSTN+/+ mice, the mechanical properties of tibialis anterior tendons from MSTN−/− mice had a greater peak stress, a lower peak strain, and increased stiffness. We conclude that, in addition to the regulation of muscle mass and force, myostatin regulates the structure and function of tendon tissues.

Atrogin-1, MuRF-1, and sarcopenia.

Sarcopenia is one of the leading causes of disability in the elderly. Despite the growing prevalence of sarcopenia, the molecular mechanisms that control aging-related changes in muscle mass are not fully understood. The ubiquitin proteasome system is one of the major pathways that regulate muscle protein degradation, and this system plays a central role in controlling muscle size. Atrogin-1 and MuRF-1 are two E3 ubiquitin ligases that are important regulators of ubiquitin-mediated protein degradation in skeletal muscle. In this review, we will discuss: (i) aging-related changes to skeletal muscle structure and function; (ii) the regulation of protein synthesis and protein degradation by IGF-1, TGF-β, and myostatin, with emphasis on the control of atrogin-1 and MuRF-1 expression; and (iii) the potential for modulating atrogin-1 and MuRF-1 expression to treat or prevent sarcopenia.

Transforming growth factor-beta induces skeletal muscle atrophy and fibrosis through the induction of atrogin-1 and scleraxis.

Introduction: Transforming growth factor‐beta (TGF‐β) is a well‐known regulator of fibrosis and inflammation in many tissues. During embryonic development, TGF‐β signaling induces expression of the transcription factor scleraxis, which promotes fibroblast proliferation and collagen synthesis in tendons. In skeletal muscle, TGF‐β has been shown to induce atrophy and fibrosis, but the effect of TGF‐β on muscle contractility and the expression of scleraxis and atrogin‐1, an important regulator of muscle atrophy, were not known. Methods: We treated muscles from mice with TGF‐β and measured force production, scleraxis, procollagen Iα2, and atrogin‐1 protein levels. Results: TGF‐β decreased muscle fiber size and dramatically reduced maximum isometric force production. TGF‐β also induced scleraxis expression in muscle fibroblasts, and increased procollagen Iα2 and atrogin‐1 levels in muscles. Conclusion: These results provide new insight into the effect of TGF‐β on muscle contractility and the molecular mechanisms behind TGF‐β–mediated muscle atrophy and fibrosis. Muscle Nerve 45: 55–59, 2012

Physiological loading of tendons induces scleraxis expression in epitenon fibroblasts.

Scleraxis is a basic helix–loop–helix transcription factor that plays a central role in promoting fibroblast proliferation and matrix synthesis during the embryonic development of tendons. Mice with a targeted inactivation of scleraxis (Scx−/−) fail to properly form limb tendons, but the role that scleraxis has in regulating the growth and adaptation of tendons of adult organisms is unknown. To determine if scleraxis expression changes in response to a physiological growth stimulus to tendons, we subjected adult mice that express green fluorescent protein (GFP) under the control of the scleraxis promoter (ScxGFP) to a 6‐week‐treadmill training program designed to induce adaptive growth in Achilles tendons. Age matched sedentary ScxGFP mice were used as controls. Scleraxis expression was sparsely observed in the epitenon region of sedentary mice, but in response to treadmill training, scleraxis was robustly expressed in fibroblasts that appeared to be emerging from the epitenon and migrating into the superficial regions of tendon fascicles. Treadmill training also led to an increase in scleraxis, tenomodulin, and type I collagen gene expression as measured by qPCR. These results suggest that in addition to regulating the embryonic formation of limb tendons, scleraxis also appears to play an important role in the adaptation of adult tendons to physiological loading.

The Aging of Elite Male Athletes: Age-related Changes in Performance and Skeletal Muscle Structure and Function

Objective:The paper addresses the degree to which the attainment of the status as an elite athlete in different sports ameliorates the known age-related losses in skeletal muscle structure and function. Design:The retrospective design, based on comparisons of published data on former elite and masters athletes and data on control subjects, assessed the degree to which the attainment of elite and masters athlete status ameliorated the known age-related changes in skeletal muscle structure and function. Setting:Institutional. Participants:Elite male athletes. Interventions:Participation in selected individual and team sports. Main Outcome Measurements:Strength, power, VO2max, and performance. Results:For elite athletes in all sports, as for the general population, age-related muscle atrophy begins at about 50 years of age. Despite the loss of muscle mass, elite athletes who maintain an active lifestyle age gracefully with few health problems. Conversely, those who lapse into inactivity regress toward general population norms for fitness, weight control, and health problems. Elite athletes in the dual and team sports have careers that rarely extend into their 30s. Conclusions:Lifelong physical activity does not appear to have any impact on the loss in fiber number. The loss of fibers can be buffered to some degree by hypertrophy of fibers that remain. It is surprising that the performance of elite athletes in all sports appears to be impaired before the onset of the fiber loss. Even with major losses in physical capacity and muscle mass, the performance of elite and masters athletes is remarkable.

Role of cyclooxygenase-1 and -2 in satellite cell proliferation, differentiation, and fusion

Skeletal muscle satellite cells play an important role in muscle regeneration. Previous work has suggested that nonsteroidal anti‐inflammatory drugs may inhibit their activity. We cultured skeletal muscle satellite cells from 9‐month‐old Sprague‐Dawley rats and exposed them to naproxen sodium (a nonselective cyclooxygenase inhibitor), NS‐398 (a selective cyclooxygenase‐2 inhibitor), and SC‐560 (a selective cyclooxygenase‐1 inhibitor) for 96 h. Cyclooxygenase‐2 inhibition alone resulted in decreased satellite cell proliferation, and inhibition of both cyclooxygenase‐1 and cyclooxygenase‐2 resulted in decreased satellite cell differentiation and fusion. This study suggests that the cyclooxygenase enzymes appear to play an important part in satellite cell proliferation, differentiation, and fusion and that nonsteroidal anti‐inflammatory medication may have an adverse effect on muscle regeneration following injury. The use of a selective cyclooxygenase‐2 inhibitor over nonspecific cyclooxygenase inhibitors in the treatment of muscle injuries is not supported. Muscle Nerve 30: 497–500, 2004

Decreased specific force and power production of muscle fibers from myostatin-deficient mice are associated with a suppression of protein degradation.

Myostatin (MSTN) is a member of the transforming growth factor-β superfamily of cytokines and is a negative regulator of skeletal muscle mass. Compared with MSTN(+/+) mice, the extensor digitorum longus muscles of MSTN(-/-) mice exhibit hypertrophy, hyperplasia, and greater maximum isometric force production (F(o)), but decreased specific maximum isometric force (sF(o); F(o) normalized by muscle cross-sectional area). The reason for the reduction in sF(o) was not known. Studies in myotubes indicate that inhibiting myostatin may increase muscle mass by decreasing the expression of the E3 ubiquitin ligase atrogin-1, which could impact the force-generating capacity and size of muscle fibers. To gain a greater understanding of the influence of myostatin on muscle contractility, we determined the impact of myostatin deficiency on the contractility of permeabilized muscle fibers and on the levels of atrogin-1 and ubiquitinated myosin heavy chain in whole muscle. We hypothesized that single fibers from MSTN(-/-) mice have a greater F(o), but no difference in sF(o), and a decrease in atrogin-1 and ubiquitin-tagged myosin heavy chain levels. The results indicated that fibers from MSTN(-/-) mice have a greater cross-sectional area, but do not have a greater F(o) and have a sF(o) that is significantly lower than fibers from MSTN(+/+) mice. The extensor digitorum longus muscles from MSTN(-/-) mice also have reduced levels of atrogin-1 and ubiquitinated myosin heavy chain. These findings suggest that myostatin inhibition in otherwise healthy muscle increases the size of muscle fibers and decreases atrogin-1 levels, but does not increase the force production of individual muscle fibers.

Rotator cuff tear reduces muscle fiber specific force production and induces macrophage accumulation and autophagy

Full‐thickness tears to the rotator cuff can cause severe pain and disability. Untreated tears progress in size and are associated with muscle atrophy and an infiltration of fat to the area, a condition known as “fatty degeneration.” To improve the treatment of rotator cuff tears, a greater understanding of the changes in the contractile properties of muscle fibers and the molecular regulation of fatty degeneration is essential. Using a rat model of rotator cuff injury, we measured the force generating capacity of individual muscle fibers and determined changes in muscle fiber type distribution that develop after a full thickness rotator cuff tear. We also measured the expression of mRNA and miRNA transcripts involved in muscle atrophy, lipid accumulation, and matrix synthesis. We hypothesized that a decrease in specific force of rotator cuff muscle fibers, an accumulation of type IIb fibers, and an upregulation in fibrogenic, adipogenic, and inflammatory gene expression occur in torn rotator cuff muscles. Thirty days following rotator cuff tear, we observed a reduction in muscle fiber force production, an induction of fibrogenic, adipogenic, and autophagocytic mRNA and miRNA molecules, and a dramatic accumulation of macrophages in areas of fat accumulation.

Intrinsic stiffness of extracellular matrix increases with age in skeletal muscles of mice

Advanced age is associated with increases in muscle passive stiffness, but the contributors to the changes remain unclear. Our purpose was to determine the relative contributions of muscle fibers and extracellular matrix (ECM) to muscle passive stiffness in both adult and old animals. Passive mechanical properties were determined for isolated individual muscle fibers and bundles of muscle fibers that included their associated ECM, obtained from tibialis anterior muscles of adult (8-12 mo old) and old (28-30 mo old) mice. Maximum tangent moduli of individual muscle fibers from adult and old muscles were not different at any sarcomere length tested. In contrast, the moduli of bundles of fibers from old mice was more than twofold greater than that of fiber bundles from adult muscles at sarcomere lengths >2.5 μm. Because ECM mechanical behavior is determined by the composition and arrangement of its molecular constituents, we also examined the effect of aging on ECM collagen characteristics. With aging, muscle ECM hydroxyproline content increased twofold and advanced glycation end-product protein adducts increased threefold, whereas collagen fibril orientation and total ECM area were not different between muscles from adult and old mice. Taken together, these findings indicate that the ECM of tibialis anterior muscles from old mice has a higher modulus than the ECM of adult muscles, likely driven by an accumulation of densely packed extensively crosslinked collagen.