Examining the role of autophagy and mitophagy in regulating muscle differentiation

Abstract

Autophagy is a degradative process that is used to eliminate intracellular organelles and protein aggregates. Further, a selective form of autophagy, termed mitophagy, is used to specifically degrade mitochondria. Autophagy/mitophagy is important for eliminating damaged/dysfunctional mitochondria to limit ROS levels and apoptosis, and is also required during erythrocyte and myoblast differentiation. Moreover, recent studies have demonstrated that mitophagy is required to initiate mitochondrial biogenesis during myogenic differentiation. Previous work in our lab has demonstrated that autophagy-deficient myoblasts fail to differentiate, have increased mitochondrial dysfunction, and have elevated levels of apoptotic signaling. Therefore, the purpose of this thesis was to determine the role of autophagy- and mitophagy-related proteins during myogenic differentiation. Chapter 2 demonstrated that canonical mitophagy is disrupted in ATG7-deficient cells, but that mitochondria can still be degraded using an alternative mitophagy pathway. However, we also determined that mitochondrial damage was increased in ATG7-deficient cells, suggesting that targeted degradation of damaged mitochondria specifically is impaired in ATG7-deficient cells. Moreover, we found that increasing the expression of the mitophagy receptor protein BNIP3 was able to partially recover myogenesis in ATG7-deficient cells. Chapter 3 then explored the requirement for the mitophagy-related proteins BNIP3L/NIX and BNIP3 during myogenic differentiation, and found that a deficiency in either of these proteins was disruptive to myogenesis. Further, we demonstrated that bnip3-/- cells showed elevated levels of mitochondria-mediated apoptotic signaling, suggesting impairment in the elimination of dysfunctional mitochondria. Moreover, bnip3-/- cells had increased autophagy-related protein expression. Interestingly, we found that overexpression of ATG7 or treatment with the autophagy inducer rapamycin can disrupt myogenic differentiation in C2C12 myoblasts, suggesting that elevated autophagy might inhibit myogenesis. Additionally, Chapter 2 and Chapter 3 demonstrated that mitochondrial signaling and mitochondrial protein expression is reduced in both shAtg7 and bnip3-/- cells, suggesting impairment in mitochondrial remodelling during differentiation. Therefore, Chapter 4 examined whether upregulating mitochondrial biogenesis can compensate for a potential reduction in autophagy/mitophagy during differentiation. Interestingly, we found that treating ATG7- and BNIP3-deficient cells with SNP, a mitochondrial biogenesis inducer, caused increased mitochondrial biogenesis- and mitochondria-related protein expression, as well as an increase in differentiation and myotube formation. Overall, this thesis demonstrated that autophagy and mitophagy are important during myogenic differentiation, and that these processes must be tightly regulated in order to ensure that cell death is limited and differentiation can progress properly.