Determining the role of ATF4 in the regulation of mitochondrial remodeling during myotube differentiation and contractile activity.

ATF4 is a transcriptional regulator that is selectively induced in response to cellular stress conditions, such as exercise, through the activation of the integrated stress response (ISR). Specifically, in the context of mitochondrial-specific stress, ATF4 is induced as a key component of the mitochondrial unfolded protein response (UPR ) and is suggested to upregulate various organellar chaperones and proteases that both preserve, and promote, mitochondrial function. In response to the stress brought about by contractile activity, ATF4 has been implicated in regulating skeletal muscle health by mediating the various signaling events associated with mitochondrial quality control (MQC), including i) mitochondrial biogenesis (expansion), ii) the mitophagy-lysosomal clearance of damaged and thus potentially harmful organelles, or iii) by activating the mitochondrial unfolded protein response (UPR ) as an intermediate response to acute cellular stress. However, it remains to be determined whether ATF4 is necessary for mitochondrial adaptations in skeletal muscle. Therefore, our aim was to determine whether ATF4 is required for the maintenance of mitochondrial function and adaptation following an acute 3h bout of contractile activity (ACA), or after repeated bouts (4 days; CCA) in C2C12 myotubes in which ATF4 was either overexpressed (OE) or knocked down (KD) via lentiviral transduction of plasmids containing the ATF4 open reading frame, or siRNA, respectively. Knockdown of ATF4 promoted elongated myotube formation following 5 days of differentiation, whereas ATF4 OE contributed to the opposite effect in which shorter myotubes were observed relative to the control condition. Induction of PGC-1α mRNA following ACA in ATF4 KD myotubes was attenuated, suggesting diminished drive for mitochondrial biogenesis in the absence of ATF4. The mRNA expression of ATF5, a downstream target of ATF4, was induced both in response to ACA as well as with ATF4 OE, and was reduced in the absence of ATF4. Mitophagy flux, measured by mitochondrial-localized LC3-II, was upregulated in ATF4 OE and KD myotubes basally, and was augmented in both control and ATF4 OE cells following ACA, but not in the absence of ATF4. Furthermore, ATF4 OE revealed decrements in mitochondrial content indicated by 20%, and 40%, reductions in VDAC and COX I protein expression relative to control, while 1.2-1.5-fold increases in these markers were observed when ATF4 was knocked down. However, following CCA, COX I and VDAC protein content were increased 3-4-fold in ATF4 OE cells, while there was no observable increase in mitochondrial content in ATF4 KD myotubes. Together, these data highlight a potential role of ATF4 in regulating basal mitochondrial content and further suggest that ATF4 may be required for contractile activity-induced increases in mitochondrial content.