Mitocytosis, mitophagy, and apoptosis coordinately drive mitochondrial clearance to regulate myogenic differentiation

Skeletal muscle is a high-energy-consuming tissue whose development and function critically depend on mitochondrial homeostasis. Mitochondrial quality control involves multiple clearance mechanisms, including mitocytosis, mitophagy, and apoptosis. However, how these pathways are coordinated during myogenic differentiation remains systematically unexplained.

This study aimed to investigate the sequential activation and coordination of mitocytosis, mitophagy, and apoptosis inresponse to gradient mitochondrial damage, and to explore their impact on myogenesis.

We established a gradient mitochondrial damage model in myoblasts using different concentrations of CCCP. Through fluorescence imaging, western blotting, genetic interventions, and small-molecule inhibitors, we investigated the activation sequence and crosstalk among different clearance pathways, and explored their effects on myotube formation and function.

Escalating mitochondrial damage triggered a sequential activation of clearance mechanisms: KIF5B-mediated mitocytosis was first induced, followed by PINK1-dependent mitophagy, and ultimately Caspase 3-mediated apoptosis. When mitocytosis was inhibited, mitophagy dominated mitochondrial clearance, whereas enhanced mitocytosis suppressed both mitophagy and apoptosis. When mitophagy was impaired, cellular homeostasis could be maintained by upregulating mitocytosis under mild mitochondrial damage, but this led to premature apoptosis under severe mitochondrial damage. Myogenesis was significantly suppressed when either mitocytosis or mitophagy was impaired, whether through small-molecule inhibitors or the genetic knockdown of KIF5B or PINK1. Notably, low-dose CCCP treatment promoted myotube formation and mitochondrial function, and also attenuated the myogenic deficits resulting from KIF5B or PINK1 deficiency. Furthermore, KIF5B overexpression enhanced glycolytic metabolism and accelerated myoblast proliferation, highlighting its role beyond mitochondrial clearance.

These findings provide new insights into the coordinated regulatory network among mitochondrial clearance mechanisms and their roles in myogenic differentiation. These insights advance the understanding of muscle biology and offer potential strategies for enhancing muscle regeneration in biomedical and cellular agriculture applications.