Autophagy allows cell survival during starvation through the bulk degradation of proteins and organelles by lysosomal enzymes. However, the mechanisms responsible for the induction and regulation of the autophagy program are poorly understood. Here we show that the FoxO3 transcription factor, which plays a critical role in muscle atrophy, is necessary and sufficient for the induction of autophagy in skeletal muscle in vivo. Akt/PKB activation blocks FoxO3 activation and autophagy, and this effect is not prevented by rapamycin. FoxO3 controls the transcription of autophagy-related genes, including LC3 and Bnip3, and Bnip3 appears to mediate the effect of FoxO3 on autophagy. This effect is not prevented by proteasome inhibitors. Thus, FoxO3 controls the two major systems of protein breakdown in skeletal muscle, the ubiquitin-proteasomal and autophagic/lysosomal pathways, independently. These findings point to FoxO3 and Bnip3 as potential therapeutic targets in muscle wasting disorders and other degenerative and neoplastic diseases in which autophagy is involved.

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.

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.

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.

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.

Decreasing JunB expression causes muscle atrophy, whereas overexpression induces hypertrophy and blocks atrophy via myostatin inhibition and regulation of atrogin-1 and MuRF expression via FoxO3.

Regulated removal of proteins and organelles by autophagy-lysosome system is critical for muscle homeostasis. Excessive activation of autophagy-dependent degradation contributes to muscle atrophy and cachexia. Conversely, inhibition of autophagy causes accumulation of protein aggregates and abnormal organelles, leading to myofiber degeneration and myopathy. Defects in lysosomal function result in severe muscle disorders such as Pompe (glycogen storage disease type II (GSDII)) disease, characterized by an accumulation of autophagosomes. However, whether autophagy is detrimental or not in muscle function of Pompe patients is unclear. We studied infantile and late-onset GSDII patients and correlated impairment of autophagy with muscle wasting. We also monitored autophagy in patients who received recombinant a-glucosidase. Our data show that infantile and late-onset patients have different levels of autophagic flux, accumulation of p62-positive protein aggregates and expression of atrophy-related genes. Although the infantile patients show impaired autophagic function, the lateonset patients display an interesting correlation among autophagy impairment, atrophy and disease progression. Moreover, reactivation of autophagy in vitro contributes to acid a-glucosidase maturation in both healthy and diseased myotubes. Together, our data suggest that autophagy protects myofibers from disease progression and atrophy in late-onset patients.

Myotonic dystrophy (DM) is caused by a (CTG) n expansion in the 3 0 -untranslated region of DMPK gene. Mutant transcripts are retained in nuclear RNA foci, which sequester RNA binding proteins thereby misregulating the alternative splicing. Controversy still surrounds the pathogenesis of the DM1 muscle distress, characterized by myotonia, weakness and wasting with distal muscle atrophy. Eight primary human cell lines from adult-onset (DM1) and congenital (cDM1) patients, (CTG) n range 90-1800, were successfully differentiated into aneural-immature and contracting-innervated-mature myotubes. Morphological, immunohistochemical, RT-PCR and western blotting analyses of several markers of myogenesis indicated that in vitro differentiation-maturation of DM1 myotubes was comparable to age-matched controls. In all pathological muscle cells, (CTG) n expansions were confirmed by long PCR and RNA fluorescence in situ hybridization. Moreover, the DM1 myotubes showed the splicing alteration of insulin receptor and muscleblind-like 1 (MBNL1) genes associated with the DM1 phenotype. Considerable myotube loss and atrophy of 15-day-differentiated DM1 myotubes indicated activated catabolic pathways, as confirmed by the presence of apoptotic (caspase-3 activation, cytochrome c release, chromatin fragmentation) and autophagic (P62/LC3) markers. Z-VAD treatment significantly reduced the decrease in myonuclei number and in average width in 15-day-differentiated DM1 myotubes. We thus propose that the muscle wasting typical in DM1 is due to impairment of muscle mass maintenanceregeneration, through premature apoptotic-autophagic activation, rather than altered myogenesis. Myotonic dystrophy (DM) is a multi-systemic disorder caused by two different microsatellite expansions in non-coding regions. Together, these two mutations affect 1 out of 8000 individuals and represent the most common form of muscular dystrophy in adults. DM1 and DM2 have common symptoms such as myotonia, muscle weakness and early cataract development. 1,2 Although DM1 and DM2 initially affect different muscles (distal versus proximal), histological analysis of the muscular tissues shows common aspects such as central nucleation. The classic form of DM1 is characterized by muscle distress with myotonia, progressive muscle weakness and wasting. Atrophy has also been reported, occurring preferentially in type-1 fibers in DM1 and in type-2 in DM2. 3 DM1 but not DM2 also presents a congenital form (cDM1), characterized by a high neonatal mortality and symptoms such as hypotonia, mental retardation and respiratory distress. 4,5 DM1 is associated with an unstable (CTG) n trinucleotide expansion located in the 3 0 -untranslated (3 0 -UTR) region of the DM protein kinase (DMPK) gene on chromosome 19q13.3. The mutant DMPK transcript, containing the expanded (CTG) n sequence, accumulates in discrete nuclear foci able to sequester various nuclear factors such as RNAbinding proteins or splicing regulators, causing different and highly variable downstream deleterious effects. 2,6...