Recent research reveals that dysfunction and subsequent loss of mitochondria (mitophagy) is a potent inducer of skeletal muscle wasting. However, the molecular mechanisms that govern the deregulation of mitochondrial function during muscle wasting are unclear. In this report, we show that different muscle-wasting stimuli upregulated mitochondrial E3 ubiquitin protein ligase 1 (Mul1), through a mechanism involving FoxO1/3 transcription factors. Overexpression of Mul1 in skeletal muscles and myoblast cultures was sufficient for the induction of mitophagy. Consistently, Mul1 suppression not only protected against mitophagy but also partially rescued the muscle wasting observed in response to muscle-wasting stimuli. In addition, upregulation of Mul1, while increasing mitochondrial fission, resulted in ubiquitination and degradation of the mitochondrial fusion protein Mfn2. Collectively, these data explain the molecular basis for the loss of mitochondrial number during muscle wasting.
Atrogin-1, a muscle-specific E3 ligase, targets MyoD for degradation through the ubiquitin-proteasome-mediated system. Myostatin, a member of the transforming growth factor-β superfamily, potently inhibits myogenesis by lowering MyoD levels. While atrogin-1 is upregulated by myostatin, it is currently unknown whether atrogin-1 plays a role in mediating myostatin signaling to regulate myogenesis. In this report, we have confirmed that atrogin-1 increasingly interacts with MyoD upon recombinant human myostatin (hMstn) treatment. The absence of atrogin-1, however, led to elevated MyoD levels and permitted the differentiation of atrogin-1(-/-) primary myoblast cultures despite the presence of exogenous myostatin. Furthermore, inactivation of atrogin-1 rescued myoblasts from growth inhibition by hMstn. Therefore, these results highlight the central role of atrogin-1 in regulating myostatin signaling during myogenesis. Currently, there are only two known targets of atrogin-1. Thus, we next characterized the associated proteins of atrogin-1 in control and hMstn-treated C2C12 cell cultures by stably expressing tagged atrogin-1 in myoblasts and myotubes, and sequencing the coimmunoprecipitated proteome. We found that atrogin-1 putatively interacts with sarcomeric proteins, transcriptional factors, metabolic enzymes, components of translation, and spliceosome formation. In addition, we also identified that desmin and vimentin, two components of the intermediate filament in muscle, directly interacted with and were degraded by atrogin-1 in response to hMstn. In summary, the muscle wasting effects of the myostatin-atrogin-1 axis are not only limited to the degradation of MyoD and eukaryotic translation initiation factor 3 subunit f, but also encompass several proteins that are involved in a wide variety of cellular activities in the muscle.
Humoral and tumoral factors collectively promote cancer-induced skeletal muscle wasting by increasing protein degradation. Although several humoral proteins, namely TNFα (tumour necrosis factor α) and IL (interleukin)-6, have been shown to induce skeletal muscle wasting, there is a lack of information regarding the tumoral factors that contribute to the atrophy of muscle during cancer cachexia. Therefore, in the present study, we have characterized the secretome of C26 colon cancer cells to identify the tumoral factors involved in cancer-induced skeletal muscle wasting. In the present study, we show that myostatin, a procachectic TGFβ (transforming growth factor β) superfamily member, is abundantly secreted by C26 cells. Consistent with myostatin signalling during cachexia, treating differentiated C2C12 myotubes with C26 CM (conditioned medium) resulted in myotubular atrophy due to the up-regulation of muscle-specific E3 ligases, atrogin-1 and MuRF1 (muscle RING-finger protein 1), and enhanced activity of the ubiquitin–proteasome pathway. Furthermore, the C26 CM also activated ActRIIB (activin receptor type II B)/Smad and NF-κB (nuclear factor κB) signalling, and reduced the activity of the IGF-I (insulin-like growth factor 1)/PI3K (phosphoinositide 3-kinase)/Akt pathway, three salient molecular features of myostatin action in skeletal muscles. Antagonists to myostatin prevented C26 CM-induced wasting in muscle cell cultures, further confirming that tumoral myostatin may be a key contributor in the pathogenesis of cancer cachexia. Finally, we show that treatment with C26 CM induced the autophagy–lysosome pathway and reduced the number of mitochondria in myotubes. These two previously unreported observations were recapitulated in skeletal muscles collected from C26 tumour-bearing mice.
The red box highlights the region used for image preparation. Please note that the cropped image was reflected along the y-axis. Molecular weight markers are recorded on the image. Please note that the top half of this immunoblot was used to detect for p-FoxO3a (Figure 7.3.2). * Significantly different to Mstn +/+ ::Apc Min/+ skeletal muscle weight (p<0.05) ** Significantly different to Mstn +/+ :: Apc Min/+ skeletal muscle weight (p<0.01) *** Significantly different to Mstn +/+ :: Apc Min/+ skeletal muscle weight (p<0.001) Significance was assessed by one-way ANOVA followed by Tukey's post-hoc test
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