Carol S. Davis

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

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.

Neuron-specific expression of CuZnSOD prevents the loss of muscle mass and function that occurs in homozygous CuZnSOD-knockout mice

Deletion of copper‐zinc superoxide dismutase (CuZnSOD) in Sod1–/– mice leads to accelerated loss of muscle mass and force during aging, but the losses do not occur with muscle‐specific deletion of CuZnSOD. To determine the role of motor neurons in the muscle decline, we generated transgenic Sod1–/– mice in which CuZnSOD was expressed under control of the synapsin 1 promoter (SynTgSod1–/– mice). SynTgSod1–/– mice expressed CuZnSOD in brain, spinal cord, and peripheral nerve, but not in other tissues. Sciatic nerve CuZnSOD content in SynTgSod1–/– mice was ~20% that of control mice, but no reduction in muscle mass or isometric force was observed in SynTg‐Sod1–/– mice compared with control animals, whereas muscles of age‐matched Sod1–/– mice displayed 30–40% reductions in mass and force. In addition, increased oxidative damage and adaptations in stress responses observed in muscles of Sod1–/– mice were absent in SynTgSod1–/– mice, and degeneration of neuromuscular junction (NMJ) structure and function occurred in Sod1–/– mice but not in SynTgSod1–/– mice. Our data demonstrate that specific CuZnSOD expression in neurons is sufficient to preserve NMJ and skeletal muscle structure and function in Sod1–/– mice and suggest that redox homeostasis in motor neurons plays a key role in initiating sarcopenia during aging.—Sakellariou, G. K., Davis, C. S., Shi, Y., Ivannikov, M. V., Zhang, Y., Vasilaki, A., Macleod, G. T., Richardson, A., Van Remmen, H., Jackson, M. J., McArdle, A., Brooks, S. V. Neuron‐specific expression of CuZnSOD prevents the loss of muscle mass and function that occurs in homozygous CuZnSOD‐knockout mice. FASEB J. 28, 1666‐1681 (2014). www.fasebj.org

Skeletal muscle weakness due to deficiency of CuZn-superoxide dismutase is associated with loss of functional innervation

An association between oxidative stress and muscle atrophy and weakness in vivo is supported by elevated oxidative damage and accelerated loss of muscle mass and force with aging in CuZn-superoxide dismutase-deficient (Sod1(-/-)) mice. The purpose was to determine the basis for low specific force (N/cm(2)) of gastrocnemius muscles in Sod1(-/-) mice and establish the extent to which structural and functional changes in muscles of Sod1(-/-) mice resemble those associated with normal aging. We tested the hypothesis that muscle weakness in Sod1(-/-) mice is due to functionally denervated fibers by comparing forces during nerve and direct muscle stimulation. No differences were observed for wild-type mice at any age in the forces generated in response to nerve and muscle stimulation. Nerve- and muscle-stimulated forces were also not different for 4-wk-old Sod1(-/-) mice, whereas, for 8- and 20-mo-old mice, forces during muscle stimulation were 16 and 30% greater, respectively, than those obtained using nerve stimulation. In addition to functional evidence of denervation with aging, fiber number was not different for Sod1(-/-) and wild-type mice at 4 wk, but 50% lower for Sod1(-/-) mice by 20 mo, and denervated motor end plates were prevalent in Sod1(-/-) mice at both 8 and 20 mo and in WT mice by 28 mo. The data suggest ongoing denervation in muscles of Sod1(-/-) mice that results in fiber loss and muscle atrophy. Moreover, the findings support using Sod1(-/-) mice to explore mechanistic links between oxidative stress and the progression of deficits in muscle structure and function.

CuZnSOD gene deletion targeted to skeletal muscle leads to loss of contractile force but does not cause muscle atrophy in adult mice

We have previously shown that deletion of CuZnSOD in mice (Sod1–/– mice) leads to accelerated loss of muscle mass and contractile force during aging. To dissect the relative roles of skeletal muscle and motor neurons in this process, we used a Cre‐Lox targeted approach to establish a skeletal muscle‐specific Sod1‐knockout (mKO) mouse to determine whether muscle‐specific CuZnSOD deletion is sufficient to cause muscle atrophy. Surprisingly, mKO mice maintain muscle masses at or above those of wild‐type control mice up to 18 mo of age. In contrast, maximum isometric specific force measured in gastrocnemius muscle is significantly reduced in the mKO mice. We found no detectable increases in global measures of oxidative stress or ROS production, no reduction in mitochondrial ATP production, and no induction of adaptive stress responses in muscle from mKO mice. However, Akt‐mTOR signaling is elevated and the number of muscle fibers with centrally located nuclei is increased in skeletal muscle from mKO mice, which suggests elevated regenerative pathways. Our data demonstrate that lack of CuZnSOD restricted to skeletal muscle does not lead to muscle atrophy but does cause muscle weakness in adult mice and suggest loss of CuZnSOD may potentiate muscle regenerative pathways.—Zhang, Y., Davis, C., Sakellariou, G.K., Shi, Y., Kayani, A.C., Pulliam, D., Bhattacharya, A., Richardson, A., Jackson, M.J., McArdle, A., Brooks, S.V., Van Remmen, H. CuZnSOD gene deletion targeted to skeletal muscle leads to loss of contractile force but does not cause muscle atrophy in adult mice. FASEB J. 27, 3536–3548 (2013). www.fasebj.org

Soleus muscle in glycosylation-deficient muscular dystrophy is protected from contraction-induced injury.

The glycosylation of dystroglycan is required for its function as a high-affinity laminin receptor, and loss of dystroglycan glycosylation results in congenital muscular dystrophy. The purpose of this study was to investigate the functional defects in slow- and fast-twitch muscles of glycosylation-deficient Large(myd) mice. While a partial alteration in glycosylation of dystroglycan in heterozygous Large(myd/+) mice was not sufficient to alter muscle function, homozygous Large(myd/myd) mice demonstrated a marked reduction in specific force in both soleus and extensor digitorum longus (EDL) muscles. Although EDL muscles from Large(myd/myd) mice were highly susceptible to lengthening contraction-induced injury, Large(myd/myd) soleus muscles surprisingly showed no greater force deficit compared with wild-type soleus muscles even after five lengthening contractions. Despite no increased susceptibility to injury, Large(myd/myd) soleus muscles showed loss of dystroglycan glycosylation and laminin binding activity and dystrophic pathology. Interestingly, we show that soleus muscles have a markedly higher sarcolemma expression of β(1)-containing integrins compared with EDL and gastrocnemius muscles. Therefore, we conclude that β(1)-containing integrins play an important role as matrix receptors in protecting muscles containing slow-twitch fibers from contraction-induced injury in the absence of dystroglycan function, and that contraction-induced injury appears to be a separable phenotype from the dystrophic pathology of muscular dystrophy.

Diaphragm muscle strip preparation for evaluation of gene therapies in mdx mice

1 Duchenne muscular dystrophy (DMD), a severe muscle wasting disease of young boys with an incidence of one in every 3000, results from a mutation in the gene that encodes dystrophin. The absence of dystrophin expression in skeletal muscles and heart results in the degeneration of muscle fibres and, consequently, severe muscle weakness and wasting. The mdx mouse discovered in 1984, with some adjustments for differences, has proven to be an invaluable model for scientific investigations of dystrophy. 2 The development of the diaphagm strip preparation provided an ideal experimental model for investigations of skeletal muscle impairments in structure and function induced by interactions of disease‐ and age‐related factors. Unlike the limb muscles of the mdx mouse, which show adaptive changes in structure and function, the diaphragm strip preparation reflects accurately the deterioration in muscle structure and function observed in boys with DMD. 3 The advent of sophisticated servo motors and force transducers interfaced with state‐of‐the‐art software packages to drive complex experimental designs during the 1990s greatly enhanced the capability of the mdx mouse and the diaphragm strip preparation to evaluate more accurately the impact of the disease on the structure–function relationships throughout the life span of the mouse. 4 Finally, during the 1990s and through the early years of the 21st century, many promising, sophisticated genetic techniques have been designed to ameliorate the devastating impact of muscular dystrophy on the structure and function of skeletal muscles. During this period of rapid development of promising genetic therapies, the combination of the mdx mouse and the diaphragm strip preparation has provided an ideal model for the evaluation of the success, or failure, of these genetic techniques to improve dystrophic muscle structure, function or both. With the 2 year life span of the mdx mouse, the impact of age‐related effects can be studied in this model.

Neuron specific reduction in CuZnSOD is not sufficient to initiate a full sarcopenia phenotype

Our previous studies showed that adult (8 month) mice lacking CuZn-superoxide dismutase (CuZnSOD, Sod1KO mice) have neuromuscular changes resulting in dramatic accelerated muscle atrophy and weakness that mimics age-related sarcopenia. We have further shown that loss of CuZnSOD targeted to skeletal muscle alone results in only mild weakness and no muscle atrophy. In this study, we targeted deletion of CuZnSOD specifically to neurons (nSod1KO mice) and determined the effect on muscle mass and weakness. The nSod1KO mice show a significant loss of CuZnSOD activity and protein level in brain and spinal cord but not in muscle tissue. The masses of the gastrocnemius, tibialis anterior and extensor digitorum longus (EDL) muscles were not reduced in nSod1KO compared to wild type mice, even at 20 months of age, although the quadriceps and soleus muscles showed small but statistically significant reductions in mass in the nSod1KO mice. Maximum isometric specific force was reduced by 8–10% in the gastrocnemius and EDL muscle of nSod1KO mice, while soleus was not affected. Muscle mitochondrial ROS generation and oxidative stress measured by levels of reactive oxygen/nitrogen species (RONS) regulatory enzymes, protein nitration and F2-isoprostane levels were not increased in muscle from the nSod1KO mice. Although we did not find evidence of denervation in the nSod1KO mice, neuromuscular junction morphology was altered and the expression of genes associated with denervation acetylcholine receptor subunit alpha (AChRα), the transcription factor, Runx1 and GADD45α) was increased, supporting a role for neuronal loss of CuZnSOD initiating alterations at the neuromuscular junction. These results and our previous studies support the concept that CuZnSOD deficits in either the motor neuron or muscle alone are not sufficient to initiate a full sarcopenic phenotype and that deficits in both tissues are required to recapitulate the loss of muscle observed in Sod1KO mice.

Haploinsufficiency of myostatin protects against aging-related declines in muscle function and enhances the longevity of mice.

The molecular mechanisms behind aging‐related declines in muscle function are not well understood, but the growth factor myostatin (MSTN) appears to play an important role in this process. Additionally, epidemiological studies have identified a positive correlation between skeletal muscle mass and longevity. Given the role of myostatin in regulating muscle size, and the correlation between muscle mass and longevity, we tested the hypotheses that the deficiency of myostatin would protect oldest‐old mice (28–30 months old) from an aging‐related loss in muscle size and contractility, and would extend the maximum lifespan of mice. We found that MSTN+/− and MSTN−/− mice were protected from aging‐related declines in muscle mass and contractility. While no differences were detected between MSTN+/+ and MSTN−/− mice, MSTN+/− mice had an approximately 15% increase in maximal lifespan. These results suggest that targeting myostatin may protect against aging‐related changes in skeletal muscle and contribute to enhanced longevity.

Changes in skeletal muscle and tendon structure and function following genetic inactivation of myostatin in rats

Myostatin is an important regulator of muscle mass and a potential therapeutic target for the treatment of diseases and injuries that result in muscle atrophy. Targeted genetic mutations of myostatin have been generated in mice, and spontaneous loss‐of‐function mutations have been reported in several species. The impact of myostatin deficiency on the structure and function of muscles has been well described for mice, but not for other species. We report the creation of a genetic model of myostatin deficiency in rats using zinc finger nuclease technology. The main findings of the study are that genetic inactivation of myostatin in rats results in increases in muscle mass without a deleterious impact on the specific force production and tendon mechanical properties. The increases in mass occur through a combination of fibre hypertrophy, hyperplasia and activation of the insulin‐like growth factor‐1 pathway, with no substantial changes in atrophy‐related pathways. This large rodent model has enabled us to identify that the chronic loss of myostatin is void of the negative consequences to muscle fibres and extracellular matrix observed in mouse models. Furthermore, the greatest impact of myostatin in the regulation of muscle mass may not be to induce atrophy directly, but rather to block hypertrophy signalling.