Muscle atrophy occurs in many pathological states and results primarily from accelerated protein degradation and activation of the ubiquitin-proteasome pathway. However, the importance of lysosomes in muscle atrophy has received little attention. Activation of FoxO transcription factors is essential for the atrophy induced by denervation or fasting, and activated FoxO3 by itself causes marked atrophy of muscles and myotubes. Here, we report that FoxO3 does so by stimulating overall protein degradation and coordinately activating both lysosomal and proteasomal pathways. Surprisingly, in C2C12 myotubes, most of this increased proteolysis is mediated by lysosomes. Activated FoxO3 stimulates lysosomal proteolysis in muscle (and other cell types) by activating autophagy. FoxO3 also induces the expression of many autophagy-related genes, which are induced similarly in mouse muscles atrophying due to denervation or fasting. These studies indicate that decreased IGF-1-PI3K-Akt signaling activates autophagy not only through mTOR but also more slowly by a transcription-dependent mechanism involving FoxO3.

Loss of myofibrillar proteins is a hallmark of atrophying muscle. Expression of muscle RING-finger 1 (MuRF1), a ubiquitin ligase, is markedly induced during atrophy, and MuRF1 deletion attenuates muscle wasting. We generated mice expressing a Ring-deletion mutant MuRF1, which binds but cannot ubiquitylate substrates. Mass spectrometry of the bound proteins in denervated muscle identified many myofibrillar components. Upon denervation or fasting, atrophying muscles show a loss of myosin-binding protein C (MyBP-C) and myosin light chains 1 and 2 (MyLC1 and MyLC2) from the myofibril, before any measurable decrease in myosin heavy chain (MyHC). Their selective loss requires MuRF1. MyHC is protected from ubiquitylation in myofibrils by associated proteins, but eventually undergoes MuRF1-dependent degradation. In contrast, MuRF1 ubiquitylates MyBP-C, MyLC1, and MyLC2, even in myofibrils. Because these proteins stabilize the thick filament, their selective ubiquitylation may facilitate thick filament disassembly. However, the thin filament components decreased by a mechanism not requiring MuRF1.

Overexpression of the transcriptional coactivator peroxisome proliferator-activated receptor ␥ coactivator 1␣ (PGC-1␣), like exercise, increases mitochondrial content and inhibits muscle atrophy. To understand these actions, we tested whether PGC-1␣ or its close homolog, PGC-1␤, influences muscle protein turnover. In myotubes, overexpression of either coactivator increased protein content by decreasing overall protein degradation without altering protein synthesis rates. Elevated PGC-1␣ or PGC-1␤ also prevented the acceleration of proteolysis induced by starvation or FoxO transcription factors and prevented the induction of autophagy and atrophy-specific ubiquitin ligases by a constitutively active FoxO3. In mouse muscles, overexpression of PGC-1␤ (like PGC-1␣) inhibited denervation atrophy, ubiquitin ligase induction, and transcription by NFB. However, increasing muscle PGC-1␣ levels pharmacologically by treatment of mice with 5-aminoimidazole-4-carboxamide 1-␤-D-ribofuranoside failed to block loss of muscle mass or induction of ubiquitin ligases upon denervation atrophy, although it prevented loss of mitochondria. This capacity of PGC-1␣ and PGC-1␤ to inhibit FoxO3 and NFB actions and proteolysis helps explain how exercise prevents muscle atrophy.The mass of a muscle and its functional capacity are determined by the balance between rates of protein synthesis and protein degradation. The rapid, debilitating loss of muscle that occurs upon inactivity, nerve damage, and in many systemic diseases (e.g. diabetes, cancer, sepsis, or renal failure) is characterized by an increased rate of protein degradation (1, 2) and coordinated changes in the expression of a set of atrophy-related genes, which have been termed "atrogenes" (3, 4). Many of these genes are induced by the FoxO family of transcription factors (5, 6), which is activated in atrophying muscles. In fact, activated FoxO3 alone stimulates overall protein degradation by both the ubiquitin-proteasome and the autophagic-lysosomal systems (7,8) and induces fiber atrophy (5). Two FoxOinduced genes are particularly important in enhancing proteolysis, the muscle-specific ubiquitin ligases, Atrogin1/MAFBx and MuRF1 (5, 9, 10), and muscles that lack either of these ligases show reduced fiber atrophy upon denervation (9). Another transcription factor that plays an essential role in muscle atrophy is NFB. Although activation of the NFB pathway is sufficient to induce muscle wasting (11, 12), its precise role and the factors that control its activity in muscle are still poorly understood.Despite appreciable recent progress in understanding the biochemical basis of atrophy, no pharmaceutical agents are available to inhibit this highly debilitating process. Exercise can protect against disuse atrophy as well as various systemic types of muscle wasting (13-15), but the mechanisms by which contractile activity reduces atrophy remain unclear. In principle, contractile activity may somehow enhance protein synthesis, suppress overall protein breakdown, and/or block the atrophy...

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Peroxisomes are indispensable organelles for lipid metabolism in humans, and their biogenesis has been assumed to be under regulation by peroxisome proliferator-activated receptors (PPARs). However, recent studies in hepatocytes suggest that the mitochondrial proliferator PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1α) also acts as an upstream transcriptional regulator for enhancing peroxisomal abundance and associated activity. It is unknown whether the regulatory mechanism(s) for enhancing peroxisomal function is through the same node as mitochondrial biogenesis in human skeletal muscle (HSkM) and whether fatty acid oxidation (FAO) is affected. Primary myotubes from vastus lateralis biopsies from lean donors (BMI = 24.0 ± 0.6 kg/m; = 6) were exposed to adenovirus encoding human PGC-1α or GFP control. Peroxisomal biogenesis proteins (peroxins) and genes () responsible for proliferation and functions were assessed by Western blotting and real-time qRT-PCR, respectively. [1-C]palmitic acid and [1-C]lignoceric acid (exclusive peroxisomal-specific substrate) were used to assess mitochondrial oxidation of peroxisomal-derived metabolites. After overexpression of PGC-1α, ) peroxisomal membrane protein 70 kDa (PMP70), PEX19, and mitochondrial citrate synthetase protein content were significantly elevated ( < 0.05), ), , key, and peroxisomal β-oxidation mRNA expression levels were significantly upregulated ( < 0.05), and ) a concomitant increase in lignoceric acid oxidation by both peroxisomal and mitochondrial activity was observed ( < 0.05). These novel findings demonstrate that, in addition to the proliferative effect on mitochondria, PGC-1α can induce peroxisomal activity and accompanying elevations in long-chain and very-long-chain fatty acid oxidation by a peroxisomal-mitochondrial functional cooperation, as observed in HSkM cells.

The biophysical environment of membrane phospholipids affects structure, function, and stability of membrane-bound proteins. 1,2 Obesity can disrupt membrane lipids, and in particular, alter the activity of sarco/endoplasmic reticulum (ER/SR) Ca 2+ -ATPase (SERCA) to affect cellular Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Objective Reduced skeletal muscle mitochondrial function may be a contributing mechanism to the myopathy and activity based limitations that typically plague peripheral arterial disease (PAD) patients. 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. Methods Genetically ischemia protected (C57BL/6) and susceptible (BALB/c) mice underwent either unilateral subacute hindlimb ischemia (SLI) or myotoxic injury (CTX) for 28 days. Limbs were monitored for blood flow and tissue oxygen saturation (SO2) and tissue was collected for the assessment of histology, muscle contractile force, gene expression, mitochondrial content, and respiratory function. Results Despite similar tissue SO2 and mitochondrial content between strains, BALB/c mice suffered persistent ischemic myofiber atrophy (55.3% of C57BL/6) and muscle contractile deficits (~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 d7, 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. Non-ischemic CTX injury revealed similar recovery of myofiber area, contractile force, mitochondrial respiratory capacity and calcium retention between strains. Conclusions 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.