Mitophagy regulates mitochondrial network signaling, oxidative stress, and apoptosis during myoblast differentiation

Abstract

Macroautophagy/autophagy is a degradative process essential for various cellular processes. We previously demonstrated that autophagy-deficiency causes myoblast apoptosis and impairs myotube formation. In this study, we continued this work with particular emphasis on mitochondrial remodelling and stress/ apoptotic signaling. We found increased (p < 0.05) autophagic (e.g., altered LC3B levels, increased ATG7, decreased SQSTM1) and mitophagic (e.g., BNIP3 upregulation, mitochondrial localized GFP-LC3 puncta, and elevated mitochondrial LC3B-II) signaling during myoblast differentiation. shRNA-mediated knockdown of ATG7 (shAtg7) decreased these autophagic and mitophagic responses, while increasing CASP3 activity and ANXA5/annexin V staining in differentiating myoblasts; ultimately resulting in dramatically impaired myogenesis. Further confirming the importance of mitophagy in these responses, CRISPR-Cas9mediated knockout of Bnip3 (bnip3-/-) resulted in increased CASP3 activity and DNA fragmentation as well as impaired myoblast differentiation. In addition, shAtg7 myoblasts displayed greater endoplasmic reticulum (e.g., increased CAPN activity and HSPA) and mitochondrial (e.g., mPTP formation, reduced mitochondrial membrane potential, elevated mitochondrial 4-HNE) stress. shAtg7 and bnip3-/myoblasts also displayed altered mitochondria-associated signaling (e.g., PPARGC1A, DNM1L, OPA1) and protein content (e.g., SLC25A4, VDAC1, CYCS). Moreover, shAtg7 myoblasts displayed CYCS and AIFM1 release from mitochondria, and CASP9 activation. Similarly, bnip3-/myoblasts had significantly higher CASP9 activation during differentiation. Importantly, administration of a chemical inhibitor of CASP9 (Ac-LEHD-CHO) or dominant-negative CASP9 (ad-DNCASP9) partially recovered differentiation and myogenesis in shAtg7 myoblasts. Together, these data demonstrate an essential role for autophagy in protecting myoblasts from mitochondrial oxidative stress and apoptotic signaling during differentiation, as well as in the regulation of mitochondrial network remodelling and myogenesis.

Berberine suppresses influenza virus-triggered NLRP3 inflammasome activation in macrophages by inducing mitophagy and decreasing mitochondrial ROS

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MEKK3-MEK5-ERK5 Signaling Promotes Mitochondrial Degradation

“…It will be interesting to determine whether any of the previously identified roles for the ERK5 pathway in development, physiology, or disease represent an underlying role for mitochondrial degradation. For example, ERK5 signaling is well established to play an anti-apoptotic role, and mitochondria harbor several proapoptotic molecules including cytochrome c, Smac/DIABLO, and AIF (Adrain, Creagh, & Martin, 2001;Baechler, Bloemberg, & Quadrilatero, 2019;Nithianandarajah-Jones et al, 2012). It is possible that active ERK5 signaling antagonizes autophagy in part through elimination of pro-apoptotic molecules via delivery of mitochondria to lysosomes for degradation.…”

Low expression of ANT1 confers oncogenic properties to rhabdomyosarcoma tumor cells via modulating metabolism and death pathways

“…Whereas activation levels of ERK1/2 and mTOR do not appear to be modified ( Figure 2E), we observed that FOXO3 level, which has been demonstrated to impair muscle progenitors proliferation (31,32), is significantly decreased in CCL-136 Low (Figure 2E). During myogenesis, it was shown that the caspases, and notably caspase-3, activation is required at a 'sub-apoptotic' level to engage myoblasts in differentiation (33)(34)(35). Caspases are cysteine proteases executioners of apoptosis.…”