Nox2-Dependent Redox Regulation of Calcium Influx in Dystrophic Skeletal Muscle

Abstract

Duchenne Muscular Dystrophy (DMD) is an X-linked, muscle-wasting disease caused by deletions in the gene that encodes dystrophin, an integral muscle membrane protein that links the contractile proteins to the muscle membrane. Recent studies have suggested that increased Ca2+ influx into the muscle and increased production of free radicals (or reactive oxygen species, ROS) are essential for increased susceptibility of mdx muscle to damage. However, the source of the ROS, the Ca2+ channels affected, and the mechanism(s) of how mechanical stress results in altered regulation of these signaling pathways have yet to be determined. We hypothesis that NADPH oxidase (Nox2) drives excessive ROS production, increased Ca2+ influx, and muscle damage. Manganes (Mn2+) quench was used to assess the role of Nox2 dependent ROS on sarcolemmal Ca2+ influx in response to a physiological stretch and depolarization. Our results show that stretch-activated Ca2+ entry in mdx is significantly increased 4.2-fold (p<0.001) compared to WT, while K+-induced depolarization results in 20-fold (p<0.001) increase in Mn2+ quench in mdx skeletal muscle. Administration of either gp91ds-TAT, an inhibitor of Nox2, or reduced glutathione ethyl ester, a glutathione analogue, in mdx muscle reduces the Mn2+ quench rates back to the WT levels for both passive stretch and K+depolarization. Addition of BTP2, a calcium release activated calcium channel inhibitor, also significantly attenuates Mn2+ quench rates in mdx compared to WT. Our data supports a model in which Nox2 dependent redox modifications of stretch and depolarization activated Ca2+ channels leads to exuberant Ca2+ influx. Our results identify Nox2 as a potential therapeutic approach to minimize or delay the progressive DMD disease.