Maduramicin inactivation of Akt impairs autophagic flux leading to accumulated autophagosomes-dependent apoptosis in skeletal myoblast cells.

Abstract

It has been clinically documented that maduramicin (Mad), a polyether ionophore antibiotic widely used in the control of coccidiosis in poultry worldwide, can elicit skeletal muscle degeneration, heart failure, and even death in animals and humans, if improperly used. Here, we show that Mad induced apoptosis dose-dependently, which was associated with impaired autophagic flux in skeletal myoblast (C2C12 and L6) cells. This is supported by the findings that Mad treatment resulted in increase of autophagosomes with a concomitant elevation of LC3-II and p62 in the cells. Also, Mad increased co-localization of mCherry and GFP tandem-tagged LC3 puncta in the cells, suggesting a blockage of autophagic flux. Furthermore, addition of chloroquine (CQ) strengthened the basic and Mad-enhanced LC3-II and p62 levels, autophagosome formation and cell apoptosis, whereas pretreatment with rapamycin alleviated the effects in the cells exposed to Mad. Moreover, we noticed that Mad treatment inactivated Akt dose-dependently. Inhibition of Akt with inhibitor X potentiated Mad-induced decrease in phosphorylated Akt, and increases in LC3-II and p62 levels, autophagosome formation and cell apoptosis, whereas ectopic expression of constitutively active Akt rendered resistance to these events. Collectively, these results indicate that Mad inactivation of Akt impairs autophagic flux leading to accumulated autophagosomes-dependent apoptosis in skeletal myoblast cells. Our findings suggest that manipulation of Akt activity to improve autophagic flux is a promising strategy against Mad-induced myotoxicity.