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1 The removal of defective mitochondria (known as mitophagy) has been implicated in the sarcopenia of aging muscle.
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Mitochondria play an important role in maintaining skeletal muscle function. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1‐Parkin independent manner. Finally, we confirmed in non‐muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPK‐dependent signaling. Interestingly, our data shows that TANK‐binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Although the PINK1‐Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read‐out of PTEN‐induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Here, we report that both carbonyl cyanide m‐chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by ~20%. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry‐green fluorescence protein‐mtFIS1 101-152). In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non‐native proteins in immortalized cell lines. Mitophagy is a key process regulating mitochondrial quality control.