Insights into RyRs Dysfunctions via Studies of Intracellular Calcium Signals

Duchenne muscular dystrophy (DMD) is a striated muscle disease with severe cardiac manifestations. The mdx mouse, an animal model of DMD, develops dilated cardiomyopathy. Several studies associated changes in Ca2+ homeostasis and oxidative stress with DMD. In particular, posttranslational modifications of Ca2+ release channels (RyRs) increase their sensitivity, leading to augmented Ca2+ responses during mechanical challenges, and to cellular and mitochondrial Na+ overload, dysfunction and cell death. We examined whether changes in RyR function were causal for or a consequence of cardiac failure and which posttranslational modifications of RyRs drive the development of the pathology. Fluorescent indicators and imaging techniques make it possible to study the function of RyR channels in the natural cellular environment on a near-molecular level. Young mdx mice show no changes in cardiac performance, but do so after ∼8 months. However, even myocytes from 1 month old mdx mice produced exaggerated Ca2+ sparks and waves after osmotic shock, and exhibited “hypersensitive” excitation-contraction coupling. Both were nearly abolished by antioxidants and NOX inhibitors and reduced by CaMKII but not by NOS- and PKA-inhibitors. SR Ca2+ load, leak or resting [Ca2+]i were unchanged in young mdx cells. However, by the age of 4-5 months and in senescence, load was reduced, leak and resting [Ca2+]i increased, indicating disease progression. By this age, all agents listed above reduced intracellular Ca2+ responses and prevented changes in ECC, Ca2+ load and leak. Thus 1) increased Ca2+ sensitivity of RyRs precedes and presumably contributes to the development of dystrophic cardiomyopathy and 2) oxidative stress drives its development. RyR oxidation, nitrosylation and phosphorylation, first by CaMKII followed by PKA, lead to even further sensitization. This synergistic sensitization of RyRs by several pathways results in cardiac muscle deterioration and heart failure.