Investigating Fusion-Independent Roles of Muscle Progenitor Cells in Response to EPS-Induced Myotube Damage

Abstract

INTRODUCTION: Following damaging stimuli, skeletal muscle exhibits coordinated interplay between intra- and extra-cellular processes resulting in satellite cell (SC) recruitment. SCs are known to play a central role in muscle plasticity post-injury by differentiating into myoblasts (MBL) and fusing with damaged tissue to donate myonuclei. Yet, their role within skeletal muscle remodeling through paracrine signaling remains to be fully elucidated. Thus, the purpose of this project was two-fold: 1) develop an in vitro model of MBL intercellular communication following myotube damage and 2) to determine if MBL proximity alone is adequate for improving tissue repair and reducing cellular stress during recovery. METHODS: C2C12 myotubes were exposed to 1 hour of electrical pulse stimulation (EPS) with 15Hz pulse for 5s and 5Hz pulse for 5s, separated by a 5s break. Myotubes were then introduced to non-electrically stimulated (NS) MBL adhered to a porous cell insert to allow paracrine signaling and samples were collected at varying timepoints post-EPS. RESULTS: EPS induced Z line sarcomeric disorganization and creatine kinase release into the cell culture media, which was mitigated in MBL+ groups (p<0.05). A significant main effect of MBL exposure was observed in EPS myotubes where MBL+ myotubes had greater Hsp70 gene expression, calpain 3 protein and gene expression, and t-ACC, p-ACCSer79, t-ULK, p-ULKSer555 protein expression than MBL- myotubes when recovering from EPS (p<0.05). A main effect of time was observed where B-dystroglycan and p-mTORSer2448 protein expression decreased in the EPS myotubes, and myotube diameter only decreased in the MBL+ condition (p<0.05). CONCLUSION: MBL signaling to damaged myotubes is evident and may increase catabolic processes through upregulating contraction-mediated protease activity and autophagy, as well as increase ATP generation through oxidative phosphorylation during regeneration.