Autophagy modulation in primary culture of porcine satellite cells

Abstract

Autophagy is a lysosomal degradation pathway with a role in the turnover of cell components via self-digestion. Over the past decade, it has been recognised as an essential process to maintain cellular and energy homeostasis. Nevertheless, little attention has been paid to this process in farm animals. In pigs, the role of autophagy in skeletal muscle homeostasis and more specifically on the formation of multinucleated muscle fibres needs to be determined. Primary culture of satellite cells, the resident muscle stem cells, is an appropriate model to investigate macroautophagy (hereafter autophagy), the main autophagy process. The objective of the current study was to evaluate tools to monitor autophagy in this cell model and to specify the role of autophagy on cell differentiation. Samples of longissimus muscle were collected from 3- to 4-day-old piglets. After isolation, satellite cells were plated in growth medium, allowed to proliferate up to 80% confluence and then placed in an appropriate culture medium to differentiate into myotubes. Cells were explored from day 0 to day 3 of differentiation. Autophagy-related proteins and Adenosine Mono Phosphate-activated protein kinase (AMPK), a major sensor for cell energy, were detected by Western blotting. Expression of genes related to autophagy were also quantified by qPCR. The Microtubule-associated protein 1 light-chain 3β forms ratio increased during cell differentiation whereas phosphatidylinositol 3-kinase and sequestosome 1 proteins decreased significantly. Mitochondrial protein expression also decreased significantly with satellite cell differentiation. Then, cell treatment with an inhibitor of autophagy flux, Bafilomycin A1, confirmed that autophagy was activated during the conversion of myoblasts into myotubes along with AMPK activation in our satellite cell culture model. In conclusion, we provided tools for porcine autophagy investigation in tissues or cells and demonstrated that basal autophagy and energy metabolism are concomitantly modulated during porcine myogenesis in vitro.