Divergent mitochondrial stressors elicit specific retrograde signaling pathways in muscle myotubes

Protein homeostasis is critical for mitochondrial function and is maintained by proteases and chaperones that respond to stress and mediate adaptive changes such as the mitochondrial unfolded protein response (UPRmt), the integrated stress response (ISR), and antioxidant signaling. However, the mechanisms by which stressors regulate these retrograde responses remains uncharacterized in muscle. Thus, we examined the effect of mitochondrial stressors on the activation of these pathways in myoblasts and differentiated myotubes. Cells were exposed to either 1) 2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO), a LonP1 protease inhibitor, 2) gamitrinib-triphenylphosphonium (GTPP), an HSP90 chaperone inhibitor, 3) carbonyl cyanide m-chlorophenyl hydrazone (CCCP), an energetic uncoupler, or 4) MitoBloCK-10 (MB-10), an inhibitor of protein import, and responses were compared with those induced by acute contractile activity (ACA). LonP1 inhibition activated activating transcription factor 4 (ATF4) and Nrf2 signaling, increased mitochondrial chaperones, and resulted in protein aggregation without elevating Reactive Oxygen Species (ROS). In contrast, blocking HSP90 led to increases in mitochondrial ROS and activation of C/EBP homologous protein (CHOP), indicating protein homeostasis-related stress with limited antioxidant signaling. ACA elicited responses similar to the inhibition of LonP1, including the activation of ATF4 and Nrf2, increased UPRmt markers, and a redox balance. Although CCCP and MB-10 both impaired protein import, they activated distinct downstream responses. CCCP resulted in ISR activation, whereas MB-10 induced Nrf2-mediated antioxidant responses. Together, these findings show that the type of mitochondrial stress determines the direction of the retrograde signaling pathways between protein homeostasis and redox signaling in muscle cells, and they provide insights on how muscle coordinates signaling pathways as part of mitochondrial adaptations to contractile activity.NEW & NOTEWORTHY This study investigates how different mitochondrial stressors activate distinct cellular signaling pathways in skeletal muscle cells. It examines how cells maintain a balance between protein homeostasis and oxidative stress when mitochondrial proteases, chaperones, and protein import are inhibited, and during acute contractile activity. The findings from this study provide key insights into mitochondrial protein homeostasis, stress signaling, and muscle adaptation mechanisms highlighting that downstream adaptive responses depend on the type of stressors.