Investigating the Importance of RAB7 in C2C12 Myoblast Differentiation
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Date
2021-06-01
Authors
Thoms, James
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Publisher
University of Waterloo
Abstract
Prior research has shown that autophagy and mitophagy are intimately linked to skeletal muscle differentiation and myogenesis. Recent studies show that the RAB7 cycle is crucial in multiple stages of autophagy and mitophagy; however, few studies examine the importance of RAB7 in muscle physiology. The objective of this study was to explore the significance of RAB7 in mammalian myoblast differentiation and myogenesis. The protein expression and localization patterns of 4 constituents of the RAB7 cycle (RAB5, CCZ1, RAB7, and RABGDI) were characterized over 5 days of differentiation. These experiments showed two major findings. Differentiation of C2C12 cells induced changes in protein content and localization of RAB5, CCZ1, RAB7, and RABGDI, meaning that differentiation and the RAB7 cycle are linked. Furthermore, RAB7 mostly localizes to mitochondrial-enriched fractions, suggesting that RAB7 is highly active and participates in mitochondria dynamics. Next, differentiating C2C12 cells were transfected with RAB7 siRNA or chronically treated with CID1067700. These experiments show three notable findings. RAB7 inhibition results in negligible changes to RAB5, CCZ1, and RABGDI content suggesting there is a compensatory RAB7-independent mechanism. Differentiation and myogenesis are affected by RAB7 inhibition as observed by dramatic decreases in MYH content and various morphological measures. These differentiation deficits were likely caused by defective autophagy and ubiquitin proteasome system (UPS), as given evidence by accumulating LC3-II, unstable SQSTM1, decreased proteasome activity, and potentially lessened autophagic flux. Overall, this is the first study to show that RAB7 is critical to mammalian myoblast differentiation and myogenesis, and that RAB7-mediated defects in differentiation are likely caused by faulty autophagy, the UPS, and the crosstalk between them.
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Keywords
differentiation, myogenesis, myoblast, autophagy, mitophagy, rab7, c2c12, muscle