Autophagy Regulates the NOTCH Signaling Pathway During Skeletal Muscle Cell Differentiation

Abstract

Research has indicated a crucial role for autophagy during skeletal muscle differentiation. More so, the inhibition of autophagy using 3MA (3-methyladenine), CQ (chloroquine), and shRNA against Atg7 has been shown to impair myocyte fusion and differentiation. Thus far, research in skeletal muscle literature has primarily focused on the degradative system of autophagy, highlighting cell survival, and thereby overlooking the direct effects on numerous signaling processes, including the regulation of NOTCH signaling. The NOTCH signaling developmental pathway is implicated in a broad range of developmental processes, including cell fate, proliferation, and differentiation. Although, there is growing evidence of crosstalk between autophagy and NOTCH signaling in hemopoiesis, cardiogenesis, and neurogenesis, limited studies have investigated the role of autophagy in regulating NOTCH signaling during skeletal muscle cell differentiation. Therefore, in order to examine the role of NOTCH signaling, our laboratory has characterized NOTCH signaling during C2C12 myoblast differentiation. We have inhibited γ-secretase with DAPT treatment that decreased the production of NOTCH1 receptor and NOTCH1ICD intracellular domain (ICD) levels to discern whether NOTCH signaling is required in myogenesis. Furthermore, autophagy was induced with rapamycin (RAPA), and inhibited with CQ to manipulate NOTCH signaling and assess whether autophagy is an important regulator of NOTCH signaling. Additionally, using a genetic approach, C2C12 cells were stably transfected with shRNA against Atg7 (shAtg7) to assess differences in NOTCH1ICD levels during myogenic differentiation. We first confirmed that the downregulation of NOTCH1 and NOTCH1ICD occurs alongside increased autophagic flux during C2C12 myoblast differentiation. Furthermore, we found that induction of autophagy with RAPA was associated with decreased NOTCH1ICD levels, while inhibition of autophagy with CQ was associated with increased NOTCH1ICD levels in proliferating myoblasts. Similarly, shAtg7 cells showed 0.6-fold increase in NOTCH1ICD levels during differentiation. However, inhibition of γ-secretase with DAPT in shAtg7 cells decreased NOTCH1ICD levels and was associated with rescued myogenic differentiation. Collectively, our results indicate that autophagy may be an important regulator of NOTCH signaling, thus playing a critical role in both skeletal muscle cell maintenance and myogenesis.