Oxidative stress may contribute to cardiac ryanodine receptor (RyR2) dysfunction in heart failure (HF) and arrhythmias. Altered RyR2 domain-domain interaction (domain unzipping) and calmodulin (CaM) binding affinity are allosterically coupled indices of RyR2 conformation. In HF RyR2 exhibits reduced CaM binding, increased domain unzipping and greater SR Ca leak, and dantrolene can reverse these changes. However, effects of oxidative stress on RyR2 conformation and leak in myocytes are poorly understood. We used fluorescent CaM, FKBP12.6, and domain-peptide biosensor (F-DPc10) to measure, directly in cardiac myocytes, (1) RyR2 activation by hydrogen peroxide (H<inf>2</inf>O<inf>2</inf>)-induced oxidation, (2) RyR2 conformation change caused by oxidation, (3) CaM-RyR2 and FK506-binding protein (FKBP12.6)-RyR2 interaction upon oxidation, and (4) whether dantrolene affects 1-3. H<inf>2</inf>O<inf>2</inf> was used to mimic oxidative stress. H<inf>2</inf>O<inf>2</inf> significantly increased the frequency of Ca<sup>2+</sup> sparks and spontaneous Ca<sup>2+</sup> waves, and dantrolene almost completely blocked these effects. H<inf>2</inf>O<inf>2</inf> pretreatment significantly reduced CaM-RyR2 binding, but had no effect on FKBP12.6-RyR2 binding. Dantrolene restored CaM-RyR2 binding but had no effect on intracellular and RyR2 oxidation levels. H<inf>2</inf>O<inf>2</inf> also accelerated F-DPc10-RyR2 association while dantrolene slowed it. Thus, H<inf>2</inf>O<inf>2</inf> causes conformational changes (sensed by CaM and DPc10 binding) associated with Ca leak, and dantrolene reverses these RyR2 effects. In conclusion, in cardiomyocytes, H<inf>2</inf>O<inf>2</inf> treatment markedly reduces the CaM-RyR2 affinity, has no effect on FKBP12.6-RyR2 affinity, and causes domain unzipping. Dantrolene can correct domain unzipping, restore CaM-RyR2 affinity, and quiet pathological RyR2 channel gating. F-DPc10 and CaM are useful biosensors of a pathophysiological RyR2 state.
- Reactive oxygen species
- Ryanodine receptor
ASJC Scopus subject areas
- Molecular Biology
- Cardiology and Cardiovascular Medicine