Changes of EC-coupling and RyR Calcium Sensitivity in Dystrophic mdx Mouse Cardiomyocytes

Abstract

Duchenne muscular dystrophy represents a severe inherited disease of striated muscle, caused by a mutation in the dystrophin gene and characterized by progressive loss of skeletal muscle function. Most patients also develop dystrophic cardiomyopathy resulting in dilated hypertrophy and heart failure. On the cellular level, absence of dystrophin affects mechanical membrane stability and intracellular Ca signaling in cardiomyocytes. Cellular mechanisms leading to deterioration of cardiac function remain elusive. We tested whether defective excitation-contraction (EC) coupling contributes to impaired cardiac performance. EC-coupling gain, a measure for the effectiveness to amplify the Ca signal by Ca release from the sarcoplasmic reticulum (SR), was determined from control and dystrophin-deficient mdx hearts. Ca currents were measured with the whole-cell patch-clamp technique, while Ca transients were simultaneously recorded with confocal imaging of fluo-3. Initial findings indicated subtle problems of EC-coupling in mdx cells despite matched SR Ca loading. However, lowering extracellular Ca, a maneuver used to unmask latent EC-coupling problems, was surprisingly much better tolerated by mdx myocytes. Normalized to control conditions, the EC-coupling gain in mdx cells reached 112% compared with 31% in control cells, suggesting hypersensitive EC-coupling. Further investigation of this apparent increase in Ca sensitivity by inducing slow elevations of intracellular Ca resulted in Ca oscillations after a much shorter delay in mdx cells, consistent with enhanced Ca sensitivity of SR Ca release channels (ryanodine receptors, RyRs). Elevated cellular reactive oxygen species (ROS) generation in dystrophy suggests redox-modifications on the RyR, enhancing its Ca sensitivity. Preincubation of mdx cells with a ROS scavenger normalized the EC-coupling hypersensitivity back to control cardiomyocytes. Our data suggest that in dystrophin-deficient cardiomyocytes, EC-coupling mechanisms are altered, partly due to potentially arrhythmogenic changes in Ca sensitivity of redox-modified RyRs. Supported by SNF, MDA, SSEM.