Role of chronic ryanodine receptor phosphorylation in heart failure and β-adrenergic receptor blockade in mice
Jian Shan, … , Bi-Xing Chen, Andrew R. Marks
Jian Shan, … , Bi-Xing Chen, Andrew R. Marks
Published November 22, 2010
Citation Information: J Clin Invest. 2010;120(12):4375-4387. https://doi.org/10.1172/JCI37649.
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Research Article

Role of chronic ryanodine receptor phosphorylation in heart failure and β-adrenergic receptor blockade in mice

Abstract

Increased sarcoplasmic reticulum (SR) Ca2+ leak via the cardiac ryanodine receptor/calcium release channel (RyR2) is thought to play a role in heart failure (HF) progression. Inhibition of this leak is an emerging therapeutic strategy. To explore the role of chronic PKA phosphorylation of RyR2 in HF pathogenesis and treatment, we generated a knockin mouse with aspartic acid replacing serine 2808 (mice are referred to herein as RyR2-S2808D+/+ mice). This mutation mimics constitutive PKA hyperphosphorylation of RyR2, which causes depletion of the stabilizing subunit FKBP12.6 (also known as calstabin2), resulting in leaky RyR2. RyR2-S2808D+/+ mice developed age-dependent cardiomyopathy, elevated RyR2 oxidation and nitrosylation, reduced SR Ca2+ store content, and increased diastolic SR Ca2+ leak. After myocardial infarction, RyR2-S2808D+/+ mice exhibited increased mortality compared with WT littermates. Treatment with S107, a 1,4-benzothiazepine derivative that stabilizes RyR2-calstabin2 interactions, inhibited the RyR2-mediated diastolic SR Ca2+ leak and reduced HF progression in WT and RyR2-S2808D+/+ mice. In contrast, β-adrenergic receptor blockers improved cardiac function in WT but not in RyR2-S2808D+/+ mice.Thus, chronic PKA hyperphosphorylation of RyR2 results in a diastolic leak that causes cardiac dysfunction. Reversing PKA hyperphosphorylation of RyR2 is an important mechanism underlying the therapeutic action of β-blocker therapy in HF.

Authors

Jian Shan, Matthew J. Betzenhauser, Alexander Kushnir, Steven Reiken, Albano C. Meli, Anetta Wronska, Miroslav Dura, Bi-Xing Chen, Andrew R. Marks

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Figure 5

Reduced store content and increased diastolic Ca2+ leak in RyR2-S2808D+/+ mice.

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Reduced store content and increased diastolic Ca2+ leak in RyR2-S2808D+/...
(A) Representative line scans obtained from ventricular myocytes isolated from WT and RyR2-S2808D+/+ mice, showing an increased spark frequency in RyR2-S2808D myocytes. (B) Pooled data showing mean ± SEM spark frequency from the number of myocytes indicated parenthetically. *P < 0.05. (C) Pooled data showing mean ± SEM amplitude of caffeine-evoked signals from the number of cells indicated parenthetically. *P < 0.05. (D) Representative trace depicting the protocol for determining the level of SR Ca2+ leak in ventricular myocytes. After termination of 3-Hz pacing, cells were superfused with Na+- and Ca2+-free solution. Application of tetracaine (1 mM) reduced the baseline fluorescence (leak). Caffeine (Caff; 10 mM) was applied at the end of the protocol to assess the SR Ca2+ load. The box made of dashed lines indicates the region expanded in E. (E) Representative signals during tetracaine application from WT, RyR2-S2808D+/+, and S107-treated RyR2-S2808D+/+ myocytes. (F) Pooled data from the number of cells indicated parenthetically, showing the mean ± SEM leak/load relationship of WT and RyR2-S2808D+/+ myocytes in the presence and absence of S107 (1 μM). Values represent the magnitude of reduction due to tetracaine expressed as a percentage of the increase in signal in response to caffeine (*P < 0.05). Myocytes were prepared from 5 or 6 mice in each experimental group.