Role of chronic ryanodine receptor phosphorylation in heart failure and β-adrenergic receptor blockade in mice
Jian Shan, Matthew J. Betzenhauser, Alexander Kushnir, Steven Reiken, Albano C. Meli, Anetta Wronska, Miroslav Dura, Bi-Xing Chen, Andrew R. Marks
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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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 3

Combined effects of PKA phosphorylation and oxidation on calstabin2 binding to RyR2.

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Combined effects of PKA phosphorylation and oxidation on calstabin2 bind...
(A) CSR preparations were treated with 1 mM H2O2, and RyR2 was immunoprecipitated, size fractionated, and immunoblotted for oxidation (DNP) and calstabin2 in the RyR2 complex. (B) Levels of oxidation and calstabin2 in the RyR2 complex were normalized to the total amount of RyR2 (AU). *P < 0.05 untreated versus H2O2-treated samples. (C) GSH/GSSG ratios (n = 2) were compared between WT and RyR2-S2808D+/+ mice. *P < 0.05. (D) 35S-calstabin binding was measured in samples treated with PKA, H2O2, or a combination of the 2, in the presence or absence or S107. Radioactivity counts were normalized to the untreated control samples. Data are presented as mean ± SEM (n = 4). The numbers of replicates for each condition are indicated by the parenthetical numbers over each bar. *P < 0.05 compared with control; #P < 0.05 compared with PKA/H2O2 treatment without S107. (E) CSR was treated with 1 mM H2O2, and RyR2 was immunoprecipitated and immunoblotted for oxidation (DNP) and PDE4D3 in the RyR2 complex. (F) RyR2 was expressed in CHO cells using an inducible vector (tetracycline), transient transfection, or stable transfection and immunoblotted with anti-DNP antibody to determine oxidation of the channel.