Simulated microgravity leads to altered intracellular calcium homeostasis and protein expression in C2C12 cultures.

Abstract

Skeletal muscle has the role to maintain body posture against a constant gravitational load. During aging, in low physical activity or even in spaceflight, the muscle mass is decreasing, resulting in impaired myogenesis and regeneration. The cytoskeleton, mechanosensitive ion channels, and calcium homeostasis play crucial role in maintaining normal myogenic processes. Simulated microgravity (SM) with 3D clinorotation is one of the accepted techniques to model gravitational unloading in vitro. C2C12 mouse myoblast cell line is a verified in vitro model system of myogenesis. Using RPM 2.0, a Random Positioning Machine as a partial g simulator, by randomly rotating the accommodated experiment package around the Earth's gravity vector, we are able to investigate alteration of physiological processes caused by SM. In our experiments, territorial gravity is compared to 0 g, respectively. However, fusion of myoblasts was preserved with SM, the formation of myotubes was different as compared to the control environment. Expression of Myosin Heavy Chain 2 (MYH2) protein, which is an important structural protein and a marker molecule of myotube differentiation program, was significantly reduced in samples kept in RPM. Immunocytochemistry of myogenic cultures revealed decreased size and average nuclei content in differentiated myotubes. The expression level of Piezo1 channels, which are mechanically activated cation channels are also decreased under SM. Altered calcium transients evoked by KCl have been observed in myotubes formed under simulated microgravity. Our results are in coherence with the recently published data. Our future goal is to analyze the architecture of cytoskeletal Septin7 protein and investigate how intracellular calcium concentrations and mechanotransduction is regulated by Piezo1 channels under microgravity condition.