Effect of Chronic Muscle Contraction on Endurance Training Associated Protein Expression in Mouse Primary Cultured Myotubes

Abstract

Introduction: Endurance training induces a fiber type shift from fast to slow, mitochondria biogenesis, and increased oxidative capacity, which together are known as the skeletal muscle adaptation. To understand how training induces the skeletal muscle adaptation, the study has generally been done an in vivo model because of the lack of an appropriate cell culture model representing these phenomena. Yet the underlying mechanism remains unknown. Therefore, an in vitro training model with skeletal muscle cells is required to elucidate the molecular mechanism of muscle adaptation induced by training. The purpose of this study is to establish a chronic muscle contraction model of cultured myotubes that mimic the in vivo endurance-training-induced adaptation.

Methods: Mouse primary myotubes derived from skeletal muscle satellite cells were used for this study. Mouse extensor digitorum longus was digested in DMEM supplemented with collagenase, and satellite cells were cultured in growth medium including 30% FBS. Satellite-cell-derived myoblasts were differentiated to myotubes by switching the differentiation medium (5% horse serum in DMEM). Three days after differentiation, the myotubes were stimulated with electric pulses at 10 Hz (twitch condition) for different periods (consecutively for 24 h, 72 h, or intermittently for 8 days) or 100 Hz (tetanic condition) for 48 h or 96 h. The myotubes were harvested after stimulation, and the protein expressions of myosin heavy chain (MyHC), MyHC II, hexokinase II, glucose transporter 4, myoglobin, and COX IV were quantified by immunoblotting.

Results: The contraction of cultured primary myotubes persisted for 8 days under the twitch condition without any visible changes. Contrary to expectations, the protein expressions were not changed by twitch contraction for 24 h, 72 h or 8 days. Under tetanic contraction for 48 h and 96 h, the protein expressions were unchanged.

Discussions: We successfully made primary myotubes that endured the twitch condition for 8 days and the tetanic condition for 96 h. However, the protein expressions induced by endurance training were not accompanied by either chronic twitch or tetanic contraction. We suggest two possible reasons that the protein expressions were unchanged after continuous contraction. First, the stimulus conditions adopted in this study were not sufficient to induce a change of protein expression. Second, the protein expression accompanied by endurance training is not only induced by physical contraction but also by other factors such as neurotransmitters and cytokines derived from immune cells.