Smooth Muscle Cells of Dystrophic (mdx) Mice Are More Susceptible to Hypoxia; The Protective Effect of Reducing Ca2+ Influx

Abstract

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 Ca2+ ([Ca2+]i) in vascular smooth muscle cells (VSMCs) from a mouse (mdx) model of DMD. We investigated the effect of hypoxia on [Ca2+]i in isolated and quiescent VSMCs from C57BL/10SnJ (WT) and C57BL/10ScSn-Dmd (mdx) male mice. [Ca2+]i was measured using Ca2+-selective microelectrodes under normoxic conditions (95% air, 5% CO2) and after hypoxia (glucose-free solution aerated with 95% N2-5% CO2 for 30 min). [Ca2+]i in mdx VSMCs was significantly elevated compared to WT under normoxia. Hypoxia-induced [Ca2+]i overload, which was significantly greater in mdx than in WT VSMCs. A low Ca2+ solution caused a reduction in [Ca2+]i and prevented [Ca2+]i overload secondary to hypoxia. Nifedipine (10 µM), a Ca2+ channel blocker, did not modify resting [Ca2+]i in VSMCs but partially prevented the hypoxia-induced elevation of [Ca2+]i in both genotypes. SAR7334 (1 µM), an antagonist of TRPC3 and TRPC6, reduced the basal and [Ca2+]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 [Ca2+]i overload, which appears to be mediated by increased Ca2+ entry through L-type Ca2+ and TRPC channels.

Re-examination of therapeutic management of muscular dystrophies using a vascular smooth muscle-centered approach

“…Dystrophic pathogenesis, including that of VSMCs, results in an increase in [Ca 2+ ]. Failure to maintain low [Ca 2+ ] levels in VSMCs activates enzymes such as proteases, nucleases, and lipases that impair energy production, compromise muscle function, and cause cell death ( 26 ). Additionally, myocytes are more vulnerable to mechanical stress due to structural defects in DMD.…”

“…Smooth muscle dystrophin is involved in the dystrophin-glycoprotein complex (DGC) and its association with the anchorage proteins of the complex, such as NO-synthase (nNOS), aquaporin-4 (AQP4), and acetylcholine receptors, thus making DMD a systemic disease that requires systemic intervention rather than skeletal muscle disease-targeted intervention ( 29 ). The “functional ischemia” perspective of this disease, rather than the muscular fatigue that contributes to the disease phenotype, indicates the importance of addressing ischemia for effective therapy (both current and future) ( 26 ). In addition to the above alterations in smooth muscle cells and the endothelium of blood vessels, muscle stem cells, also known as satellite cells, from DMD mice have also been shown to have a reduced capacity to promote angiogenesis.…”