Abstract | RATIONALE: 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.
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Authors | Yi Yang, Tao Guo, Tetsuro Oda, Asima Chakraborty, Le Chen, Hitoshi Uchinoumi, Anne A Knowlton, Bradley R Fruen, Razvan L Cornea, Gerhard Meissner, Donald M Bers |
Journal | Circulation research
(Circ Res)
Vol. 114
Issue 2
Pg. 295-306
(Jan 17 2014)
ISSN: 1524-4571 [Electronic] United States |
PMID | 24186966
(Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
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Chemical References |
- Calmodulin
- Ryanodine Receptor Calcium Release Channel
- Tacrolimus Binding Proteins
- tacrolimus binding protein 1B
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Topics |
- Animals
- Arrhythmias, Cardiac
(etiology, genetics, metabolism, physiopathology)
- Calcium Signaling
- Calmodulin
(metabolism)
- Disease Models, Animal
- Fluorescence Resonance Energy Transfer
- Heart Failure
(complications, genetics, metabolism, physiopathology)
- Kinetics
- Mice
- Mice, Transgenic
- Myocytes, Cardiac
(metabolism)
- Protein Binding
- Protein Interaction Mapping
- Rats
- Rats, Sprague-Dawley
- Ryanodine Receptor Calcium Release Channel
(genetics, metabolism)
- Sarcoplasmic Reticulum
(metabolism)
- Tacrolimus Binding Proteins
(metabolism)
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