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 4

Functional characterization of cardiac RyR2 channels from WT and RyR2-S2808D+/+ mice.

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Functional characterization of cardiac RyR2 channels from WT and RyR2-S2...
(A) Representative single channel current traces of cardiac RyR2 channels isolated from WT and RyR2-S2808D+/+ mice at 1.5 months of age. (B) Bar graph summarizing average Po in WT (n = 3) and RyR2-S2808D+/+ (n = 6) channels from 1.5-month-old mice (P = NS). The number of channels recorded from each sample is indicated by the parenthetical numbers over each bar. (C) Representative single channel current traces of cardiac RyR2 channels isolated from 10-month-old WT and RyR2-S2808D+/+ mice. (D) Bar graph summarizing average Po in WT (n = 6) and S2808D+/+ (n = 9) channels from 10-month-old mice (*P < 0.05). The number of channels recorded from each sample is indicated by the parenthetical numbers over each bar. Channel openings are shown as upward deflections; the open and closed (c) states of the channel are indicated by horizontal bars in the beginning of each trace. Examples of the channel activity are shown at 2 different time scales (5 s for upper trace and 500 ms for lower trace depicted by the thick gray bar) as indicated by dimension bars. The respective Po, To (average open time), and Tc (average closed time) are shown above each 5 second trace and correspond to that particular experiment. (E) Amplitude histogram of a representative WT cardiac RyR2 channel (at 10 months old), showing 2 distinct peaks corresponding to fully open (~4 pA) and closed (0 pA) states of the channel. (F) Samples of amplitude histograms from 3 different experiments, using RyR2-S2808D+/+ channels (10-month-old mice), showing subconductance states.