Cardiac Myocyte Z-Line Calmodulin Is Mainly RyR2-Bound, and Reduction Is Arrhythmogenic and Occurs in Heart Failure

Abstract

Rationale: Calmodulin (CaM) associates with cardiac ryanodine receptor type-2 (RyR2) as an important regulator. Defective CaM–RyR2 interaction may occur in heart failure, cardiac hypertrophy, and catecholaminergic polymorphic ventricular tachycardia. However, the in situ binding properties for CaM–RyR2 are unknown. Objective: We sought to measure the in situ binding affinity and kinetics for CaM–RyR2 in normal and heart failure ventricular myocytes, estimate the percentage of Z-line–localized CaM that is RyR2-bound, and test cellular function of defective CaM–RyR2 interaction. Methods and Results: Using fluorescence resonance energy transfer in permeabilized myocytes, we specifically resolved RyR2-bound CaM from other potential binding targets and measured CaM–RyR2 binding affinity in situ (K d =10–20 nmol/L). Using RyR2 ADA/+ knock-in mice, in which half of the CaM–RyR2 binding is suppressed, we estimated that >90% of Z-line CaM is RyR2-bound. Functional tests indicated a higher propensity for Ca 2+ wave production and stress-induced ventricular arrhythmia in RyR2 ADA/+ mice. In a post–myocardial infarction rat heart failure model, we detected a decrease in the CaM–RyR2 binding affinity (K d ≈51 nmol/L; ≈3-fold increase) and unaltered RyR2 affinity for the FK506-binding protein FKBP12.6 (Kd~0.8 nmol/L). Conclusions: CaM binds to RyR2 with high affinity in cardiac myocytes. Physiologically, CaM is bound to >70% of RyR2 monomers and inhibits sarcoplasmic reticulum Ca 2+ release. RyR2 is the major binding site for CaM along the Z-line in cardiomyocytes, and dissociating CaM from RyR2 can cause severe ventricular arrhythmia. In heart failure, RyR2 shows decreased CaM affinity, but unaltered FKBP 12.6 affinity.