Cardiac Myocyte Z-line Calmodulin is Mainly RyR2-Bound and Reduction is Arrhythmogenic and Occurs in Heart Failure
Rationale: Calmodulin (CaM) associates with cardiac ryanodine receptors (RyR2) as an important regulator. Defective CaM-RyR2 interaction may occur in heart failure (HF), cardiac hypertrophy, and catecholaminergic polymorphic ventricular tachycardia (CPVT). 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 HF 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 FRET (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 (Kd =10-20 nM). Using RyR2ADA/+ knock-in (KI) 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 Ca2+ waves production and stress induced ventricular arrhythmia in RyR2ADA/+ mice. In a post myocardial infarction (MI) rat HF model, we detected a decrease in the CaM-RyR2 binding affinity (Kd ≈ 51nM, ~3 fold increase) and unaltered FKBP12.6-RyR2 binding affinity (Kd ≈ 0.8nM).
Conclusions: CaM binds to RyR2 with high affinity in cardiac myocytes. Physiologically, CaM is bound to >70% of RyR2 monomers and inhibits SR Ca2+ 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 HF, RyR2 shows decreased CaM affinity, but unaltered FKBP12.6 affinity.
- Binding properties
- fluorescent imaging
- ryanodine receptor
- heart failure
- arrhythmia (mechanisms)
- Received October 14, 2013.
- Revision received October 30, 2013.
- Accepted October 31, 2013.