The secreted factor TSK maintains slow-twitch myofiber integrity and exercise endurance and contributes to muscle regeneration
ABSTRACT: Mammalian skeletal muscle contains heterogenous myofibers with different contractile and metabolic properties that sustain muscle mass and endurance capacity. The transcriptional regulators that govern these myofiber gene programs have been elucidated. However, the hormonal cues that direct the specification of myofiber types and muscle endurance remain largely unknown. Here we uncover the secreted factor Tsukushi (TSK) as an extracellular signal that is required for maintaining muscle mass, strength, and endurance capacity, and contributes to muscle regeneration. Mice lacking TSK exhibited reduced grip strength and impaired exercise capacity. Muscle transcriptomic analysis revealed that TSK deficiency results in a remarkably selective impairment in the expression of myofibrillar genes characteristic of slow-twitch muscle fibers that is associated with abnormal neuromuscular junction formation. AAV-mediated overexpression of TSK failed to rescue these myofiber defects in adult mice, suggesting that the effects of TSK on myofibers are likely restricted to certain developmental stages. Finally, mice lacking TSK exhibited diminished muscle regeneration following cardiotoxin-induced muscle injury. These findings support a crucial role of TSK as a hormonal cue in the regulation of contractile gene expression, endurance capacity, and muscle regeneration. Overall design: RNA-seq on WT vs TSK KO mouse quadriceps
Project description:Muscle regeneration depends on satellite cells, myogenic stem cells that reside on the myofiber surface. Reduced numbers and/or decreased myogenic aptitude of these cells may impede proper maintenance and contribute to the age-associated decline in muscle mass and repair capacity. Endurance exercise was shown to improve muscle performance; however, the direct impact on satellite cells in aging was not yet thoroughly determined. Here, we focused on characterizing the effect of moderate-intensity endurance exercise on satellite cell, as possible means to attenuate adverse effects of aging. Young and old rats of both genders underwent 13 weeks of treadmill-running or remained sedentary.Gastrocnemius muscles were assessed for the effect of age, gender and exercise on satellite-cell numbers and myogenic capacity. Satellite cells were identified in freshly isolated myofibers based on Pax7 immunostaining (i.e., ex-vivo). The capacity of individual myofiber-associated cells to produce myogenic progeny was determined in clonal assays (in-vitro). We show an age-associated decrease in satellite-cell numbers and in the percent of myogenic clones in old sedentary rats. Upon exercise, there was an increase in myofibers that contain higher numbers of satellite cells in both young and old rats, and an increase in the percent of myogenic clones derived from old rats. Changes at the satellite cell level in old rats were accompanied with positive effects on the lean-to-fat Gast muscle composition and on spontaneous locomotion levels. The significance of these data is that they suggest that the endurance exercise-mediated boost in both satellite numbers and myogenic properties may improve myofiber maintenance in aging.
Project description:Impaired skeletal muscle energy metabolism is linked to the pathogenesis of insulin resistance and glucose intolerance in type 2 diabetes. The contractile and metabolic properties of myofibers exhibit a high degree of heterogeneity and plasticity. The regulatory circuitry underpinning skeletal muscle energy metabolism is critically linked to exercise endurance and systemic homeostasis. Recent work has identified the Baf60 subunits of the SWI/SNF chromatin-remodeling complex as powerful regulators of the metabolic gene programs. However, their role in integrating myofiber energy metabolism with exercise endurance and metabolic physiology remains largely unknown. In this study, we conditionally inactivated Baf60a, Baf60c, or both in mature skeletal myocytes to delineate their contribution to muscle bioenergetics and metabolic physiology. Our work revealed functional redundancy between Baf60a and Baf60c in maintaining oxidative and glycolytic metabolism in skeletal myofibers and exercise endurance. Unexpectedly, mice lacking these two factors in skeletal muscle were protected from diet-induced and age-associated metabolic disorders. Transcriptional profiling analysis identified the muscle thermogenic gene program and myokine secretion as key pathways that integrate myofiber metabolism with systemic energy balance. As such, Baf60 deficiency in skeletal muscle illustrates a surprising disconnect between exercise endurance and systemic metabolic homeostasis.
Project description:In response to physiological stimuli, skeletal muscle alters its myofiber composition to significantly affect muscle performance and metabolism. This process requires concerted regulation of myofiber-specific isoforms of sarcomeric and calcium regulatory proteins that couple action potentials to the generation of contractile force. Here, we identify Sox6 as a fast myofiber-enriched repressor of slow muscle gene expression in vivo. Mice lacking Sox6 specifically in skeletal muscle have an increased number of slow myofibers, elevated mitochondrial activity, and exhibit down-regulation of the fast myofiber gene program, resulting in enhanced muscular endurance. In addition, microarray profiling of Sox6 knockout muscle revealed extensive muscle fiber-type remodeling, and identified numerous genes that display distinctive fiber-type enrichment. Sox6 directly represses the transcription of slow myofiber-enriched genes by binding to conserved cis-regulatory elements. These results identify Sox6 as a robust regulator of muscle contractile phenotype and metabolism, and elucidate a mechanism by which functionally related muscle fiber-type specific gene isoforms are collectively controlled.
Project description:Skeletal muscle physiology and the mechanisms of muscle diseases can be effectively studied by an in-vitro tissue model produced by muscle tissue engineering. Engineered human cell-based tissues are required more than ever because of the advantages they bring as tissue models in research studies. This study reports on a production method of a human skeletal myofiber sheet that demonstrates biomimetic properties including the aligned structure of myofibers, basement membrane-like structure of the extracellular matrix, and unidirectional contractile ability. The contractile ability and drug responsibility shown in this study indicate that this engineered muscle tissue has potential as a human cell-based tissue model for clinically relevant in-vitro studies in muscle physiology and drug discovery. Moreover, this engineered tissue can be used to better understand the relationships between mechanical stress and myogenesis, including muscle growth and regeneration. In this study, periodic exercise induced by continuous electrical pulse stimulation enhanced the contractile ability of the engineered myofibers and the secretion of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) from the exercising myofibers. Since the physiology of skeletal muscle is directly related to mechanical stress, these features point to application as a tissue model and platform for future biological studies of skeletal muscle including muscle metabolism, muscle atrophy and muscle regeneration.
Project description:We performed gene expression profiling by microarray using RNA extracted from the tibialis anterior of control and Hira myofiber-specific conditional knockout mice at 6 months of age. Hira is a histone chaperone responsible for replication-independent incorporation of histone variant H3.3 at actively transcribed loci. Conditional knockout of Hira in myofibers improved strength and endurance, caused hypertrophy and regeneration, and increased the percentage of type I fibers. These results illustrate the physiological consequences of disrupting Hira-mediated chromatin assembly in myofibers. Overall design: 4 animals per group (control and Hira conditional knockout) hybridized in triplicate. RNA was extracted from the tibialis anterior muscle.
Project description:OBJECTIVE:The purpose of this side product of another unpublished research project, was to address the effects of a training program on skeletal muscle adaptations of people with myotonic dystrophy type 1 (DM1), under a multifaceted perspective. The objective of this study was to look at training induced muscular adaptations by evaluating changes in muscle strength, myofiber cross-sectional area (CSA), proportion of myofiber types and with indirect markers of muscle growth [proportion of centrally nucleated fibers (CNF) and density of neutrophils and macrophages]. Two men with DM1 underwent a 12-week strength/endurance training program (18 sessions). Two muscle biopsies were obtained pre- and post-training program. RESULTS:Muscular adaptations occurred only in Patient 1, who attended 72% of the training sessions compared to 39% for Patient 2. These adaptations included increase in the CSA of type I and II myofibers and changes in their proportion. No changes were observed in the percentage of CNF, infiltration of neutrophils and macrophages and muscle strength. These results illustrate the capacity of skeletal muscle cells to undergo adaptations linked to muscle growth in DM1 patients. Also, these adaptations seem to be dependent on the attendance. Trial registration Clinicaltrials.gov NCT04001920 retrospectively registered on June 26th, 2019.
Project description:Sarcopenia, the loss of muscle mass and strength during normal aging, involves coordinate changes in skeletal myofibers and the cells that contact them, including satellite cells and motor neurons. Here we show that the protein O-fucosyltransferase 1 gene (Pofut1), which encodes a glycosyltransferase required for NotchR-mediated cell-cell signaling, has reduced expression in aging skeletal muscle. Moreover, premature postnatal deletion of Pofut1 in skeletal myofibers can induce aging-related phenotypes in cis within skeletal myofibers and in trans within satellite cells and within motor neurons via the neuromuscular junction. Changed phenotypes include reduced skeletal muscle size and strength, decreased myofiber size, increased slow fiber (type 1) density, increased muscle degeneration and regeneration in aged muscles, decreased satellite cell self-renewal and regenerative potential, and increased neuromuscular fragmentation and occasional denervation. Pofut1 deletion in skeletal myofibers reduced NotchR signaling in young adult muscles, but this effect was lost with age. Increasing muscle NotchR signaling also reduced muscle size. Gene expression studies point to regulation of cell cycle genes, muscle myosins, NotchR and Wnt pathway genes, and connective tissue growth factor by Pofut1 in skeletal muscle, with additional effects on ? dystroglycan glycosylation.
Project description:Reduced skeletal muscle mitochondrial function might be a contributing mechanism to the myopathy and activity based limitations that typically plague patients with peripheral arterial disease (PAD). We hypothesized that mitochondrial dysfunction, myofiber atrophy, and muscle contractile deficits are inherently determined by the genetic background of regenerating ischemic mouse skeletal muscle, similar to how patient genetics affect the distribution of disease severity with clinical PAD.Genetically ischemia protected (C57BL/6) and susceptible (BALB/c) mice underwent either unilateral subacute hind limb ischemia (SLI) or myotoxic injury (cardiotoxin) for 28 days. Limbs were monitored for blood flow and tissue oxygen saturation and tissue was collected for the assessment of histology, muscle contractile force, gene expression, mitochondrial content, and respiratory function.Despite similar tissue O2 saturation and mitochondrial content between strains, BALB/c mice suffered persistent ischemic myofiber atrophy (55.3% of C57BL/6) and muscle contractile deficits (approximately 25% of C57BL/6 across multiple stimulation frequencies). SLI also reduced BALB/c mitochondrial respiratory capacity, assessed in either isolated mitochondria (58.3% of C57BL/6 at SLI on day (d)7, 59.1% of C57BL/6 at SLI d28 across multiple conditions) or permeabilized myofibers (38.9% of C57BL/6 at SLI d7; 76.2% of C57BL/6 at SLI d28 across multiple conditions). SLI also resulted in decreased calcium retention capacity (56.0% of C57BL/6) in BALB/c mitochondria. Nonischemic cardiotoxin injury revealed similar recovery of myofiber area, contractile force, mitochondrial respiratory capacity, and calcium retention between strains.Ischemia-susceptible BALB/c mice suffered persistent muscle atrophy, impaired muscle function, and mitochondrial respiratory deficits during SLI. Interestingly, parental strain susceptibility to myopathy appears specific to regenerative insults including an ischemic component. Our findings indicate that the functional deficits that plague PAD patients could include mitochondrial respiratory deficits genetically inherent to the regenerating muscle myofibers.
Project description:Skeletal muscle is dynamic, adapting to environmental needs, continuously maintained, and capable of extensive regeneration. These hallmarks diminish with age, resulting in a loss of muscle mass, reduced regenerative capacity, and decreased functionality. Although the mechanisms responsible for this decline are unclear, complex changes within the local and systemic environment that lead to a reduction in regenerative capacity of skeletal muscle stem cells, termed satellite cells, are believed to be responsible. We demonstrate that engraftment of myofiber-associated satellite cells, coupled with an induced muscle injury, markedly alters the environment of young adult host muscle, eliciting a near-lifelong enhancement in muscle mass, stem cell number, and force generation. The abrogation of age-related atrophy appears to arise from an increased regenerative capacity of the donor stem cells, which expand to occupy both myonuclei in myofibers and the satellite cell niche. Further, these cells have extensive self-renewal capabilities, as demonstrated by serial transplantation. These near-lifelong, physiological changes suggest an approach for the amelioration of muscle atrophy and diminished function that arise with aging through myofiber-associated satellite cell transplantation.
Project description:Vascular endothelial growth factor (VEGF) is exercise responsive, pro-angiogenic, and expressed in several muscle cell types. We hypothesized that in adult mice, VEGF generated within skeletal myofibers (and not other cells within muscle) is necessary for the angiogenic response to exercise training. This was tested in adult conditional, skeletal myofiber-specific VEGF gene-deleted mice (skmVEGF-/-), with VEGF levels reduced by >80%. After 8 wk of daily treadmill training, speed and endurance were unaltered in skmVEGF-/- mice, but increased by 18% and 99% (P < 0.01), respectively, in controls trained at identical absolute speed, incline, and duration. In vitro, isolated soleus and extensor digitorum longus contractile function was not impaired in skmVEGF-/- mice. However, training-induced angiogenesis was inhibited in plantaris (wild type, 38%, skmVEGF-/- 18%, P < 0.01), and gastrocnemius (wild type, 43%, P < 0.01; skmVEGF-/-, 7%, not significant). Capillarity was maintained (different from VEGF gene deletion targeted to multiple cell types) in untrained skmVEGF-/- mice. Arteriogenesis (smooth muscle actin+, artery number, and diameter) and remodeling [vimentin+, 5'-bromodeoxycytidine (BrdU)+, and F4/80+ cells] occurred in skmVEGF-/- mice, even in the absence of training. skmVEGF-/- mice also displayed a limited oxidative enzyme [citrate synthase and ?-hydroxyacyl CoA dehydrogenase (?-HAD)] training response; ?-HAD activity levels were elevated in the untrained state. These data suggest that myofiber expressed VEGF is necessary for training responses in capillarity and oxidative capacity and for improved running speed and endurance.