Abstract 12222: Cardiomyocyte Ryanodine Receptor Oxidation in Situ Causes Defective Interdomain Interaction and Reduces Calmodulin Binding
Oxidation is an important factor for cardiac ryanodine receptor (RyR2) dysfunction. This can enhance diastolic Ca leak contributing to heart failure and arrhythmia, but the mechanism for this RyR2 dysregulation is not clear. Here, directly in cardiac myocytes, we investigate effects of oxidation on RyR2 conformation and the binding of channel regulators calmodulin (CaM) and FKBP12.6. We have shown that fluorescent domain peptide-DP2460-2495 (F-DPc10) binding kinetics are a sensitive measure of local RyR2 conformation (domain-domain interaction state between N-terminal and central domains) which may be linked to altered RyR2 gating.
Methods and results: We use permeabilized ventricular myocytes and fluorescent proteins (F-CaM, F-FKBP12.6 and F-DPc10). H2O2 (50 μM) pretreatment is used to provide cellular oxidative stress. H2O2 pretreatment significantly reduces the F-CaM binding to the RyR2 (by 32%) for [CaM] in the Kd range (20nM), but has no effect on F-FKBP12.6 binding (1 nM, also Kd range). Interestingly, the treatment of dantrolene, which can correct the defective domain interaction in RyR2 (unzipped state), can also restore the CaM-RyR2 binding without any change to cellular oxidation level. Furthermore, H2O2 significantly accelerates F-DPc10 association rate (by 40%), which is diagnostic of domain unzipping between N-terminal and central domains of the RyR2 (allowing better access of free F-DPc10 to its binding site). Dantrolene dramatically reduces F-DPc10 binding Bmax either in normal (by 73%) or H2O2 treated myocytes (by 34%), indicating that dantrolene can stabilize the domain interaction and keep RyR2 in the zipped state both in normal or oxidative conditions.
Conclusions: In permeabilized cardiomyocytes, oxidation of RyR2 by H2O2 markedly reduces the CaM binding (but not FKBP12.6) affinity to the RyR2, and causes defective domain interaction between the N-terminal and central domains. But dantrolene can prevent all of these effects.
- © 2013 by American Heart Association, Inc.