Evaluation Of Calcium Homeostasis Within The Mdx Skeletal Muscle

Abstract

Background: Duchenne muscular dystrophy is a terminal X-linked muscle wasting disease occurring in 1 out of every 3,500 live male births. Currently, no cure exists. However, current data implicate a relationship between reactive oxygen species (ROS), Ca2+, and membrane permeability possibly affecting the muscle degeneration associated with DMD. Design: I assayed myoplasmic ROS during field-stimulated contractions using a green fluorescent protein targeted to the mitochondria. Its fluorescence changed depending on the oxidizing or reducing conditions. My findings suggested the mitochondria are not the primary source of intracellular ROS generation during field-stimulated contractions. Instead, NADPH oxidase may provide an important source for ROS generation during unloaded contraction. I then evaluated the sensitivity of Excitation Coupled Calcium Entry (ECCE) to ROS, using manganese (Mn2+) quench (a surrogate to Ca2+ influx), after treating the muscle fibers with oxidizing or reducing solutions. Results: Exogenous H2O2 treatment of myofibers did not alter either the basal or ECCE Ca2+ permeability in the WT fibers when compared to the non-treated controls. Oxidation with H2O2 did not significantly affect the basal Ca2+ permeability in the mdx fibers, but did greatly increase ECCE permeability by almost 8 fold greater than WT basal rate with 100 μM [H2O2]. In opposition to oxidation, scavenging ROS with n-acetylcysteine decreased Mn2+ permeability during ECCE in mdx muscle. The non-specific TRP channel inhibitor BTP2, reduced the Mn2+ permeability during basal conditions and Mn2+ permeability during ECCE in mdx muscle fibers by 87% and 96%, p <.05, respectively. In WT fibers, BTP2 reduced Mn2+ permeability ECCE by 67%, p<.05, but did not inhibit the basal permeability. Conclusions: We demonstrate that the ECCE Ca2+ entry pathway is exuberant in mdx muscle. Furthermore, we determine that ECCE is ROS sensitive. We conclude that ECCE contributes to the altered Ca2+ homeostasis in mdx skeletal muscle. The increased ROS generation in mdx skeletal muscle may directly or indirectly mediate TRPC activity to generate the Ca2+ dyshomeostasis associated with dystrophin deficiency