New insights into molecular changes in skeletal muscle aging and disease: Differential alternative splicing and senescence.

Progressive loss of muscle mass and function due to muscle fiber atrophy and loss in the elderly and chronically ill is now defined as sarcopenia. It is a major contributor to loss of independence, disability, need of long-term care as well as overall mortality. Sarcopenia is a heterogenous disease and underlying mechanisms are not completely understood. Here, we newly identified and used Tmem158, alongside Cdkn1a, as relevant senescence and denervation markers (SDMs), associated with muscle fiber atrophy. Subsequent application of laser capture microdissection (LCM) and RNA analyses revealed age- and disease-associated differences in gene expression and alternative splicing patterns in a rodent sarcopenia model. Of note, genes exhibiting such differential alternative splicing (DAS) are mainly involved in the contractile function of the muscle. Many of these splicing events are also found in a mouse model for myotonic dystrophy type 1 (DM1), underscoring the premature aging phenotype of this disease. We propose to add differential alternative splicing to the hallmarks of aging.

Partial Inhibition of mTORC1 in Aged Rats Counteracts the Decline in Muscle Mass and Reverses Molecular Signaling Associated with Sarcopenia.

There is a lack of pharmacological interventions available for sarcopenia, a progressive age-associated loss of muscle mass, leading to a decline in mobility and quality of life. We found mTORC1 (mammalian target of rapamycin complex 1), a well-established positive modulator of muscle mass, to be surprisingly hyperactivated in sarcopenic muscle. Furthermore, partial inhibition of the mTORC1 pathway counteracted sarcopenia, as determined by observing an increase in muscle mass and fiber type cross-sectional area in select muscle groups, again surprising because mTORC1 signaling has been shown to be required for skeletal muscle mass gains in some models of hypertrophy. Additionally, several genes related to senescence were downregulated and gene expression indicators of neuromuscular junction denervation were diminished using a low dose of a "rapalog" (a pharmacological agent related to rapamycin). Therefore, partial mTORC1 inhibition may delay the progression of sarcopenia by directly and indirectly modulating multiple age-associated pathways, implicating mTORC1 as a therapeutic target to treat sarcopenia.

Defective autophagy and mTORC1 signaling in myotubularin null mice.

Autophagy is a vesicular trafficking pathway that regulates the degradation of aggregated proteins and damaged organelles. Initiation of autophagy requires several multiprotein signaling complexes, such as the ULK1 kinase complex and the Vps34 lipid kinase complex, which generates phosphatidylinositol 3-phosphate [PtdIns(3)P] on the forming autophagosomal membrane. Alterations in autophagy have been reported for various diseases, including myopathies. Here we show that skeletal muscle autophagy is compromised in mice deficient in the X-linked myotubular myopathy (XLMTM)-associated PtdIns(3)P phosphatase myotubularin (MTM1). Mtm1-deficient muscle displays several cellular abnormalities, including a profound increase in ubiquitin aggregates and abnormal mitochondria. Further, we show that Mtm1 deficiency is accompanied by activation of mTORC1 signaling, which persists even following starvation. In vivo pharmacological inhibition of mTOR is sufficient to normalize aberrant autophagy and improve muscle phenotypes in Mtm1 null mice. These results suggest that aberrant mTORC1 signaling and impaired autophagy are consequences of the loss of Mtm1 and may play a primary role in disease pathogenesis.

Genomic and proteomic profiling reveals reduced mitochondrial function and disruption of the neuromuscular junction driving rat sarcopenia.

Molecular mechanisms underlying sarcopenia, the age-related loss of skeletal muscle mass and function, remain unclear. To identify molecular changes that correlated best with sarcopenia and might contribute to its pathogenesis, we determined global gene expression profiles in muscles of rats aged 6, 12, 18, 21, 24, and 27 months. These rats exhibit sarcopenia beginning at 21 months. Correlation of the gene expression versus muscle mass or age changes, and functional annotation analysis identified gene signatures of sarcopenia distinct from gene signatures of aging. Specifically, mitochondrial energy metabolism (e.g., tricarboxylic acid cycle and oxidative phosphorylation) pathway genes were the most downregulated and most significantly correlated with sarcopenia. Also, perturbed were genes/pathways associated with neuromuscular junction patency (providing molecular evidence of sarcopenia-related functional denervation and neuromuscular junction remodeling), protein degradation, and inflammation. Proteomic analysis of samples at 6, 18, and 27 months confirmed the depletion of mitochondrial energy metabolism proteins and neuromuscular junction proteins. Together, these findings suggest that therapeutic approaches that simultaneously stimulate mitochondrogenesis and reduce muscle proteolysis and inflammation have potential for treating sarcopenia.

An HMGA2-IGF2BP2 axis regulates myoblast proliferation and myogenesis.

A group of genes that are highly and specifically expressed in proliferating skeletal myoblasts during myogenesis was identified. Expression of one of these genes, Hmga2, increases coincident with satellite cell activation, and later its expression significantly declines correlating with fusion of myoblasts into myotubes. Hmga2 knockout mice exhibit impaired muscle development and reduced myoblast proliferation, while overexpression of HMGA2 promotes myoblast growth. This perturbation in proliferation can be explained by the finding that HMGA2 directly regulates the RNA-binding protein IGF2BP2. Add-back of IGF2BP2 rescues the phenotype. IGF2BP2 in turn binds to and controls the translation of a set of mRNAs, including c-myc, Sp1, and Igf1r. These data demonstrate that the HMGA2-IGF2BP2 axis functions as a key regulator of satellite cell activation and therefore skeletal muscle development.

The SCF-Fbxo40 complex induces IRS1 ubiquitination in skeletal muscle, limiting IGF1 signaling.

Insulin-like growth factor 1 (IGF1) induces skeletal muscle hypertrophy by activating the IGF1R/IRS1/PI3K/Akt pathway. However the effect of IGF1 in differentiated muscle is limited by IRS1 ubiquitination and proteasome-mediated breakdown. In skeletal muscle, IGF1R activation sensitizes IRS1 to degradation, and a screen for the responsible E3 ligase identified Fbxo40 as mediating this rapid turnover of IRS1, since IRS1 loss can be rescued by knockdown of Fbxo40. In biochemical assays, an SCF E3 ligase complex containing Fbxo40 directly ubiquitinates IRS1, and this activity is enhanced by increased tyrosine phosphorylation of IRS1. Fbxo40 is muscle specific in expression and is upregulated during differentiation. Knockdown of Fbxo40 induces dramatic hypertrophy of myofibers. Mice null for Fbxo40 have increased levels of IRS1 and demonstrate enhanced body and muscle size during the growth phase associated with elevated IGF1 levels. These findings establish an important means of restraining IGF1's effects on skeletal muscle.

Voluntary running, skeletal muscle gene expression, and signaling inversely regulated by orchidectomy and testosterone replacement.

Declines in skeletal muscle size and strength, often seen with chronic wasting diseases, prolonged or high-dose glucocorticoid therapy, and the natural aging process in mammals, are usually associated with reduced physical activity and testosterone levels. However, it is not clear whether the decline in testosterone and activity are causally related. Using a mouse model, we found that removal of endogenous testosterone by orchidectomy results in an almost complete cessation in voluntary wheel running but only a small decline in muscle mass. Testosterone replacement restored running behavior and muscle mass to normal levels. Orchidectomy also suppressed the IGF-I/Akt pathway, activated the atrophy-inducing E3 ligases MuRF1 and MAFBx, and suppressed several energy metabolism pathways, and all of these effects were reversed by testosterone replacement. The study also delineated a distinct, previously unidentified set of genes that is inversely regulated by orchidectomy and testosterone treatment. These data demonstrate the necessity of testosterone for both speed and endurance of voluntary wheel running in mice and suggest a potential mechanism for declined activity in humans where androgens are deficient.

FGFR1 inhibits skeletal muscle atrophy associated with hindlimb suspension.

BACKGROUND: Skeletal muscle atrophy can occur under many different conditions, including prolonged disuse or immobilization, cachexia, cushingoid conditions, secondary to surgery, or with advanced age. The mechanisms by which unloading of muscle is sensed and translated into signals controlling tissue reduction remains a major question in the field of musculoskeletal research. While the fibroblast growth factors (FGFs) and their receptors are synthesized by, and intimately involved in, embryonic skeletal muscle growth and repair, their role maintaining adult muscle status has not been examined. METHODS: We examined the effects of ectopic expression of FGFR1 during disuse-mediated skeletal muscle atrophy, utilizing hindlimb suspension and DNA electroporation in mice. RESULTS: We found skeletal muscle FGF4 and FGFR1 mRNA expression to be modified by hind limb suspension,. In addition, we found FGFR1 protein localized in muscle fibers within atrophying mouse muscle which appeared to be resistant to atrophy. Electroporation and ectopic expression of FGFR1 significantly inhibited the decrease in muscle fiber area within skeletal muscles of mice undergoing suspension induced muscle atrophy. Ectopic FGFR1 expression in muscle also significantly stimulated protein synthesis in muscle fibers, and increased protein degradation in weight bearing muscle fibers. CONCLUSION: These results support the theory that FGF signaling can play a role in regulation of postnatal skeletal muscle maintenance, and could offer potentially novel and efficient therapeutic options for attenuating muscle atrophy during aging, illness and spaceflight.