Proteasome-dependent Activation of Mammalian Target of Rapamycin Complex 1 (mTORC1) Is Essential for Autophagy Suppression and Muscle Remodeling Following Denervation

Drastic protein degradation occurs during muscle atrophy induced by denervation, fasting, immobility, and various systemic diseases. Although the ubiquitin-proteasome system is highly up-regulated in denervated muscles, the involvement of autophagy and protein synthesis has been controversial. Here, we report that autophagy is rather suppressed in denervated muscles even under autophagy-inducible starvation conditions. This is due to a constitutive activation of mammalian target of rapamycin complex 1 (mTORC1). We further reveal that denervation-induced mTORC1 activation is dependent on the proteasome, which is likely mediated by amino acids generated from proteasomal degradation. Protein synthesis and ribosome biogenesis are paradoxically increased in denervated muscles in an mTORC1-dependent manner, and mTORC1 activation plays an anabolic role against denervation-induced muscle atrophy. These results suggest that denervation induces not only muscle degradation but also adaptive muscle response in a proteasome- and mTORC1-dependent manner.     

Fasting-related autophagic response in slow- and fast-twitch skeletal muscle

This study investigated regulation of autophagy in slow-twitch soleus and fast-twitch plantaris muscles in fasting-related atrophy. Male Fischer-344 rats were subjected to fasting for 1, 2, or 3 days. Greater weight loss was observed in plantaris muscle than in soleus muscle in response to fasting. Western blot analysis demonstrated that LC3-II, a marker protein for macroautophagy, was expressed at a notably higher level in plantaris than in soleus muscle, and that the expression level was fasting duration-dependent. To identify factors related to LC3-II enhancement, autophagy-related signals were examined in both types of muscle. Phosphorylated mTOR was reduced in plantaris but not in soleus muscle. FOXO3a and ER stress signals were unchanged in both muscle types during fasting. These findings suggest that preferential atrophy of fast-twitch muscle is associated with induction of autophagy during fasting and that differences in autophagy regulation are attributable to differential signal regulation in soleus and plantaris muscle.     

Signalling pathways that mediate skeletal muscle hypertrophy and atrophy

Atrophy of skeletal muscle is a serious consequence of numerous diseases, including cancer and AIDS. Successful treatments for skeletal muscle atrophy could either block protein degradation pathways activated during atrophy or stimulate protein synthesis pathways induced during skeletal muscle hypertrophy. This perspective will focus on the signalling pathways that control skeletal muscle atrophy and hypertrophy, including the recently identified ubiquitin ligases muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx), as a basis to develop targets for pharmacologic intervention in muscle disease.     

Bnip3-mediated mitochondrial autophagy is independent of the mitochondrial permeability transition pore

Bnip3 is a pro-apoptotic BH3-only protein which is associated with mitochondrial dysfunction and cell death. Bnip3 is also a potent inducer of autophagy in many cells. In this study, we have investigated the mechanism by which Bnip3 induces autophagy in adult cardiac myocytes. Overexpression of Bnip3 induced extensive autophagy in adult cardiac myocytes. Fluorescent microscopy studies and ultrastructural analysis revealed selective degradation of mitochondria by autophagy in myocytes overexpressing Bnip3. Oxidative stress and increased levels of intracellular Ca²⁺ have been reported by others to induce autophagy, but Bnip3-induced autophagy was not abolished by antioxidant treatment or the Ca²⁺ chelator BAPTA-AM. We also investigated the role of the mitochondrial permeability transition pore (mPTP) in Bnip3-induced autophagy. Although the mPTP has previously been implicated in the induction of autophagy and selective removal of damaged mitochondria by autophagosomes, mitochondria sequestered by autophagosomes in Bnip3-treated cardiac myocytes had not undergone permeability transition, and treatment with the mPTP inhibitor cyclosporine A did not inhibit mitochondrial autophagy in cardiac myocytes. Moreover, cyclophilin D (cypD) is an essential component of the mPTP and Bnip3 induced autophagy to the same extent in embryonic fibroblasts isolated from wild-type and cypD-deficient mice. These results support a model where Bnip3 induces selective removal of the mitochondria in cardiac myocytes, and that Bnip3 triggers induction of autophagy independent of Ca²⁺, ROS generation, and mPTP opening.     

Quantitation of "autophagic flux" in mature skeletal muscle

Reliable and quantitative assays to measure in vivo autophagy are essential. Currently, there are varied methods for monitoring autophagy; however, it is a challenge to measure "autophagic flux" in an in vivo model system. Conversion and subsequent degradation of the microtubule-associated protein light chain 3 (MAP1-LC3/LC3) to the autophagosome associated LC3II isoform can be evaluated by immunoblot. However, static levels of endogenous LC3II protein may render possible misinterpretations since LC3II levels can increase, decrease or remain unchanged in the setting of autophagic induction. Therefore, it is necessary to measure LC3II protein levels in the presence and absence of lysomotropic agents that block the degradation of LC3II, a technique aptly named the "autophagometer". In order to measure autophagic flux in mouse skeletal muscle, we treated animals with the microtubule depolarizing agent colchicine. Two days of 0.4 mg/kg/day intraperitoneal colchicine blocked autophagosome maturation to autolysosomes and increased LC3II protein levels in mouse skeletal muscle by >100%. The addition of an autophagic stimulus such as dietary restriction or rapamycin led to an additional increase in LC3II above that seen with colchicine alone. Moreover, this increase was not apparent in the absence of a "colchicine block." Using this assay, we evaluated the autophagic response in skeletal muscle upon denervation induced atrophy. Our studies highlight the feasibility of performing an "in vivo autophagometer" study using colchicine in skeletal muscle.     

Mitochondrial autophagy by Bnip3 involves Drp1-mediated mitochondrial fission and recruitment of Parkin in cardiac myocytes

The Bcl2/adenovirus E1B 19-kDa interacting protein 3 (Bnip3) is an atypical BH3-only protein that is associated with mitochondrial dysfunction and cell death. Bnip3 is also a potent inducer of mitochondrial autophagy, and in this study we have investigated the mechanisms by which Bnip3 induces autophagy in cardiac myocytes. We found that Bnip3 induced mitochondrial translocation of dynamin-related protein 1 (Drp1), a protein involved in mitochondrial fission in adult myocytes. Drp1-mediated mitochondrial fission correlated with increased autophagy, and inhibition of Drp1 reduced Bnip3-mediated autophagy. Overexpression of Drp1K38E, a dominant negative of Drp1, or mitofusin 1 prevented mitochondrial fission and autophagy by Bnip3. Also, inhibition of mitochondrial fission or autophagy resulted in increased death of myocytes overexpressing Bnip3. Moreover, Bnip3 promoted translocation of the E3 ubiquitin ligase Parkin to mitochondria, which was prevented in the presence of a Drp1 inhibitor. Interestingly, induction of autophagy by Bnip3 was reduced in Parkin-deficient myocytes. Thus our data suggest that induction of autophagy in response to Bnip3 is a protective response activated by the cell that involves Drp1-mediated mitochondrial fission and recruitment of Parkin.