Author(s): CHATZINIKITA, E., TOLIS, E., GIANNOPOULOS, A., MARIDAKI, M., PHILIPPOU, A., Institution: NATIONAL AND KAPODISTRIAN UNIVERCITY OF ATHENS, Country: GREECE, Abstract-ID: 2099

INTRODUCTION:
Mechanical loading potently stimulates skeletal muscle adaptation, however excessive mechanical strain can lead to muscle damage, activating inflammatory, regenerative and remodeling processes. Even though mechanical loading has been widely studied in the context of skeletal muscle adaptation, data directly quantifying mechanically induced cellular damage in differentiated myotubes remain sparse.
The purpose of this study was to investigate the effects of a prolonged mechanical stretching protocol on the plasma membrane integrity of differentiated C2C12 myotubes, as an indication of mechanical stress-induced cell injury, necrosis or apoptosis, and hence to establish an in vitro model of mechanically induced muscle cell damage.
METHODS:
C2C12 myotubes (at day 6 of differentiation) underwent a passive cyclic stretching protocol using the Flexcell FX-5000 system. The protocol consisted of sustained 6% elongation with additional cyclic stretching up to 12% elongation at a frequency of 2 Hz for 3 hours. Non-stretched myotubes were used as controls. Potential cell membrane damage was assessed via LDH release into the culture medium at baseline, 1 hour, 24 hours and 72 hours post mechanical loading.
RESULTS:
Mechanically loaded myotubes presented increased LDH release at 1-hour post-loading (71.42±9.68%; p<0.05), which further increased at 24 (146.40±2.01%; p<0.001) and 72 hours (171.31±16.66%; p=0.01) compared to baseline (12.70±1.09%). Control (non-stretched) cells exhibited lower levels of LDH release at 24 (104.07±0.08; p<0.001) and 72 hours (128.96±11.69; p=0.01) compared to baseline, possibly due to its spontaneous release during the myotubes’ differentiation. These LDH release levels remained significantly lower in comparison to the loaded cells (24 hours: 104.07±0.08 vs 146.40±2.01; p<0.01; 72 hours: 128.96±11.69 vs 171.31±16.66; p=0.08).
CONCLUSION:
Sustained mechanical loading induces increased and time-dependent cytotoxicity in differentiated C2C12 myotubes, confirming the effectiveness of the protocol utilized in eliciting mechanically induced muscle cell damage. This in vitro model might be utilized as a useful source for future investigation of various cellular and molecular responses following mechanical loading-associated damage of muscle cells.