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Circ Res. 2014 Jan 17;114(2):295-306. doi: 10.1161/CIRCRESAHA.114.302857. Epub 2013 Nov 1.

Cardiac myocyte Z-line calmodulin is mainly RyR2-bound, and reduction is arrhythmogenic and occurs in heart failure.

Author information

1
From the Department of Pharmacology (Y.Y., T.G., T.O., H.U., A.A.K., D.M.B.), and Molecular and Cellular Cardiology Division, Department of Medicine (L.C., A.A.K.), University of California, Davis, CA; Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC (A.C., G.M.); and Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN (B.R.F., R.L.C.).

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 (Kd=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 Ca2+ 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 (Kd≈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 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 heart failure, RyR2 shows decreased CaM affinity, but unaltered FKBP 12.6 affinity.

KEYWORDS:

arrhythmias, cardiac; fluorescence resonance energy transfer; heart failure; ryanodine receptor calcium release channel

PMID:
24186966
PMCID:
PMC4004530
DOI:
10.1161/CIRCRESAHA.114.302857
[Indexed for MEDLINE]
Free PMC Article

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