The size of skeletal muscle cells is precisely regulated by intracellular signaling networks that determine the balance between overall rates of protein synthesis and degradation. Myofiber growth and protein synthesis are stimulated by the IGF-1/Akt/mammalian target of rapamycin (mTOR) pathway. In this study, we show that the transcription factor JunB is also a major determinant of whether adult muscles grow or atrophy. We found that in atrophying myotubes, JunB is excluded from the nucleus and that decreasing JunB expression by RNA interference in adult muscles causes atrophy. Furthermore, JunB overexpression induces hypertrophy without affecting satellite cell proliferation and stimulated protein synthesis independently of the Akt/mTOR pathway. When JunB is transfected into denervated muscles, fiber atrophy is prevented. JunB blocks FoxO3 binding to atrogin-1 and MuRF-1 promoters and thus reduces protein breakdown. Therefore, JunB is important not only in dividing populations but also in adult muscle, where it is required for the maintenance of muscle size and can induce rapid hypertrophy and block atrophy.
Mitochondria are crucial organelles in the production of energy and in the control of signalling cascades. A machinery of pro-fusion and fission proteins regulates their morphology and subcellular localization. In muscle this results in an orderly pattern of intermyofibrillar and subsarcolemmal mitochondria. Muscular atrophy is a genetically controlled process involving the activation of the autophagy-lysosome and the ubiquitin-proteasome systems. Whether and how the mitochondria are involved in muscular atrophy is unknown. Here, we show that the mitochondria are removed through autophagy system and that changes in mitochondrial network occur in atrophying muscles. Expression of the fission machinery is per se sufficient to cause muscle wasting in adult animals, by triggering organelle dysfunction and AMPK activation. Conversely, inhibition of the mitochondrial fission inhibits muscle loss during fasting and after FoxO3 overexpression. Mitochondrial-dependent muscle atrophy requires AMPK activation as inhibition of AMPK restores muscle size in myofibres with altered mitochondria. Thus, disruption of the mitochondrial network is an essential amplificatory loop of the muscular atrophy programme.
Autophagy is required for cellular survival and for the clearance of damaged proteins and altered organelles. Excessive autophagy activation contributes to muscle loss in different catabolic conditions. However, the function of basal autophagy for homeostasis of skeletal muscle was unknown. To clarify this issue we have generated conditional and inducible knockout mice for the critical gene Atg7, to block autophagy specifically in skeletal muscle. Atg7 null muscles reveal an unexpected phenotype which is characterized by muscle atrophy, weakness and features of myofiber degeneration. Morphological, biochemical and molecular analyses of our autophagy knockout mice show the presence of protein aggregates, abnormal mitochondria, accumulation of membrane bodies, sarcoplasmic reticulum distension, vacuolization, oxidative stress and apoptosis. Moreover, autophagy inhibition does not protect skeletal muscles from atrophy during denervation and fasting, but instead promotes greater muscle loss. In conclusion, autophagy plays a critical role for myofiber maintenance and its activation is crucial to avoid accumulation of toxic proteins and dysfunctional organelles that, in the end, would lead to atrophy and weakness.
A better understanding of the signaling pathways that control muscle growth is required to identify appropriate countermeasures to prevent or reverse the loss of muscle mass and force induced by aging, disuse, or neuromuscular diseases. However, two major issues in this field have not yet been fully addressed. The first concerns the pathways involved in leading to physiological changes in muscle size. Muscle hypertrophy based on perturbations of specific signaling pathways is either characterized by impaired force generation, e.g., myostatin knockout, or incompletely studied from the physiological point of view, e.g., IGF-1 overexpression. A second issue is whether satellite cell proliferation and incorporation into growing muscle fibers is required for a functional hypertrophy. To address these issues, we used an inducible transgenic model of muscle hypertrophy by short-term Akt activation in adult skeletal muscle. In this model, Akt activation for 3 wk was followed by marked hypertrophy ( approximately 50% of muscle mass) and by increased force generation, as determined in vivo by ankle plantar flexor stimulation, ex vivo in intact isolated diaphragm strips, and in single-skinned muscle fibers. No changes in fiber-type distribution and resistance to fatigue were detectable. Bromodeoxyuridine incorporation experiments showed that Akt-dependent muscle hypertrophy was accompanied by proliferation of interstitial cells but not by satellite cell activation and new myonuclei incorporation, pointing to an increase in myonuclear domain size. We can conclude that during a fast hypertrophic growth myonuclear domain can increase without compromising muscle performance.
The ubiquitin-proteasome and autophagy-lysosome pathways are the two major routes for protein and organelle clearance. In skeletal muscle, both systems are under FoxO regulation and their excessive activation induces severe muscle loss. Although altered autophagy has been observed in various myopathies, the specific role of autophagy in skeletal muscle has not been determined by loss-of-function approaches. Here, we report that muscle-specific deletion of a crucial autophagy gene, Atg7, resulted in profound muscle atrophy and age-dependent decrease in force. Atg7 null muscles showed accumulation of abnormal mitochondria, sarcoplasmic reticulum distension, disorganization of sarcomere, and formation of aberrant concentric membranous structures. Autophagy inhibition exacerbated muscle loss during denervation and fasting. Thus, autophagy flux is important to preserve muscle mass and to maintain myofiber integrity. Our results suggest that inhibition/alteration of autophagy can contribute to myofiber degeneration and weakness in muscle disorders characterized by accumulation of abnormal mitochondria and inclusions.
The autophagy-lysosome system is essential for muscle cell homeostasis and its dysfunction has been linked to muscle disorders that are typically distinguished by massive autophagic buildup. Among them, glycogen storage disease type II (GSDII) is characterized by the presence of large glycogen-filled lysosomes in the skeletal muscle, due to a defect in the lysosomal enzyme acid ?-glucosidase (GAA). The accumulation of autophagosomes is believed to be detrimental for myofiber function. However, the role of autophagy in the pathogenesis of GSDII is still unclear. To address this issue we monitored autophagy in muscle biopsies and myotubes of early and late-onset GSDII patients at different time points of disease progression. Moreover we also analyzed muscles from patients treated with enzyme replacement therapy (ERT). Our data suggest that autophagy is a protective mechanism that is required for myofiber survival in late-onset forms of GSDII. Importantly, our findings suggest that a normal autophagy flux is important for a correct maturation of GAA and for the uptake of recombinant human GAA. In conclusion, autophagy failure plays an important role in GSDII disease progression, and the development of new drugs to restore the autophagic flux should be considered to improve ERT efficacy.
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