The Effect Of Mechanical Loading On Mitophaging In Aged Myoblasts

During aging, skeletal muscle mass constantly diminishes and myogenic potential declines. At the cellular level, a decline in mitochondrial function is a hallmark of the aging process and the deficiency of mitochondrial network contributes to a progressive reduction of muscle mass. Autophagic clearance of mitochondria through mitophagy is required to remove impaired or damaged mitochondria, while mitophagy is a key regulator of muscle maintenance. Dysfunctional degradation of mitochondria is associated with aging (mitophaging), while mechanical stimuli have been shown to ameliorate the aging-induced impaired muscle mass, however, less is known about the potential effects of mechanical loading on mitophaging. PURPOSE: This study investigated the effect of mechanical stretching on mitophagy in aged myoblasts, in vitro. METHODS: Cell senescence was replicated using a multiple cell division model of C2C12 myoblasts. Control and aged cells were cultured on elastic membranes and underwent passive stretching using a mechanical loading protocol of 15% elongation for 12 hrs at a frequency of 1 Hz. Cell signaling and gene expression responses of mitophagy-associated factors were assessed by immunoblotting of cell lysates derived from stretched and non-stretched control and aged myoblasts. RESULTS: Mitophagy factor AMP-activated protein kinase (AMPK) and mitochondrial biogenesis stimulator PGC1a were downregulated in control stretched myoblasts compared to non-stretched cells [AMPK, Fold change (FC): 0.244; PGC1a, FC: 0.890; p < 0.05], while the specific loading protocol used also reduced the phosphorylation of unc-51 like autophagy activating kinase 1 (pULK1) (FC: 0.527; p < 0.05). Interestingly, this mechanical loading resulted in increased PGC1a expression (FC:1.125; p < 0.05) inducing the previously undetected AMPK expression and pULK1activation in the aged myoblasts. CONCLUSIONS: These findings suggest the beneficial effects of mechanical loading on the impaired mitophagy in aged myoblasts, as indicated by the mitophagy initiation and the promotion of mitochondrial biogenesis in these cells. The mechanical loading-induced downregulation of mitophagy in the control cells might indicate loading-specific differential responses of control myoblasts compared to the aged cells.