Effect of Oxidative Stress on Intracellular Ca2+ and Na+ Concentration in Duchenne Muscular Dystrophy Myotubes

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Duchenne muscular dystrophy (DMD) is an X-linked myopathy leading to progressive muscle weakness that is the most common and devastating type of muscular dystrophy in boys. Oxidative stress has been suggested as a potential upstream contributor to the skeletal muscle damage in DMD. Here we test this hypothesis by comparing the effect of ROS in intracellular concentrations of Ca2+ and Na+. Intracellular calcium concentration ([Ca2+]i) and intracellular Na+ concentration ([Na+]i) were measured using Ca2+ and Na+ selective microelectrodes in Wt and mdx myotubes. Mdx myotubes showed significantly elevated [Ca2+]i and [Na+]i in comparison to Wt cells. In mdx myotubes [Ca2+]i was 333±35 nM (mean±SD, n=35) and [Na+]i was 17.5±1.3 mM (n=15)versus115±10 nM (n=40) and 7.9±0.6 mM, (n=15) in Wt. Ca2+ influx at rest measured by Mn2+ quenching also reveled increased Ca2+ entry in mdx. The effect of ROS on cultured myotubes was tested by exposing myotubes to 10μM H2O2, a concentration that did not alter cell viability. H2O2 caused a significant elevation of [Ca2+]i and [Na+]i in both mdx and Wt. However, the relative increase of both ions was much greater in mdx than Wt. Whereas in mdx [Ca2+]i reached 1167±138 nM (n=14) and [Na+]i 23.6±2 mM (n=11) in Wt cells these concentration were 152±11 nM (n=15) and 10.5±0.5 mM (n=14), respectively. In addition, pre-treatment of mdx and Wt myotubes with nonselective TRPC blockers 5μM BTP2 or 20μM gadolinium prevented the increase in [Ca2+]i and [Na+]i induced by H2O2. These results suggest that mdx myotubes are more susceptible than Wt to elevation of [Ca2+]i and [Na+]I induced by oxidative stress. Moreover, H2O2-induced dysregulation of [Ca2+]i and [Na+]i seems primarily mediated by Ca2+ entry.Supported by Grants P01AR47605 (to PDA) and 5K01AR054818 (to CFP)