Smooth Muscle Cells of Dystrophic (mdx) Mice Are More Susceptible to Hypoxia; The Protective Effect of Reducing Ca(2+) Influx

Duchenne muscular dystrophy (DMD) is an inherited muscular disorder caused by mutations in the dystrophin gene. DMD patients have hypoxemic events due to sleep-disordered breathing. We reported an anomalous regulation of resting intracellular Ca(2+) ([Ca(2+)](i)) in vascular smooth muscle cells (VSMCs) from a mouse (mdx) model of DMD. We investigated the effect of hypoxia on [Ca(2+)](i) in isolated and quiescent VSMCs from C57BL/10SnJ (WT) and C57BL/10ScSn-Dmd (mdx) male mice. [Ca(2+)](i) was measured using Ca(2+)-selective microelectrodes under normoxic conditions (95% air, 5% CO(2)) and after hypoxia (glucose-free solution aerated with 95% N2-5% CO(2) for 30 min). [Ca(2+)](i) in mdx VSMCs was significantly elevated compared to WT under normoxia. Hypoxia-induced [Ca(2+)](i) overload, which was significantly greater in mdx than in WT VSMCs. A low Ca(2+) solution caused a reduction in [Ca(2+)](i) and prevented [Ca(2+)](i) overload secondary to hypoxia. Nifedipine (10 µM), a Ca(2+) channel blocker, did not modify resting [Ca(2+)](i) in VSMCs but partially prevented the hypoxia-induced elevation of [Ca(2+)](i) in both genotypes. SAR7334 (1 µM), an antagonist of TRPC3 and TRPC6, reduced the basal and [Ca(2+)](i) overload caused by hypoxia. Cell viability, assessed by tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, was significantly reduced in mdx compared to WT VSMCs. Pretreatment with SAR7341 increases cell viability in normoxic mdx (p < 0.001) and during hypoxia in WT and mdx VSMCs. These results provide evidence that the lack of dystrophin makes VSMCs more susceptible to hypoxia-induced [Ca(2+)](i) overload, which appears to be mediated by increased Ca(2+) entry through L-type Ca(2+) and TRPC channels.