Hierarchical accumulation of RyR post-translational modifications drives disease progression in dystrophic cardiomyopathy

Aims Duchenne muscular dystrophy (DMD) is a muscle disease with serious cardiac complications. Changes in Ca2+ homeostasis and oxidative stress were recently associated with cardiac deterioration, but the cellular pathophysiological mechanisms remain elusive. We investigated whether the activity of ryanodine receptor (RyR) Ca2+ release channels is affected, whether changes in function are cause or consequence and which post-translational modifications drive disease progression. Methods and results Electrophysiological, imaging, and biochemical techniques were used to study RyRs in cardiomyocytes from mdx mice, an animal model of DMD. Young mdx mice show no changes in cardiac performance, but do so after ∼8 months. Nevertheless, myocytes from mdx pups exhibited exaggerated Ca2+ responses to mechanical stress and ‘hypersensitive' excitation-contraction coupling, hallmarks of increased RyR Ca2+ sensitivity. Both were normalized by antioxidants, inhibitors of NAD(P)H oxidase and CaMKII, but not by NO synthases and PKA antagonists. Sarcoplasmic reticulum Ca2+ load and leak were unchanged in young mdx mice. However, by the age of 4-5 months and in senescence, leak was increased and load was reduced, indicating disease progression. By this age, all pharmacological interventions listed above normalized Ca2+ signals and corrected changes in ECC, Ca2+ load, and leak. Conclusion Our findings suggest that increased RyR Ca2+ sensitivity precedes and presumably drives the progression of dystrophic cardiomyopathy, with oxidative stress initiating its development. RyR oxidation followed by phosphorylation, first by CaMKII and later by PKA, synergistically contributes to cardiac deterioratio