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 8

S107 but not metoprolol or carvedilol improves cardiac function in RyR-S2808D+/+ mice after MI.

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S107 but not metoprolol or carvedilol improves cardiac function in RyR-S...
(A and B) Echocardiographic measurements at baseline, 1, 3, and 5 weeks after MI. Treatment started 1 week after MI (arrows). (A) In the WT group, S107, metoprolol (Met), and carvedilol (Carv) showed a beneficial effect at the end of study (week 4 of treatment). (B) In the RyR2-S2808D+/+ group, only S107 inhibited HF progression. *P < 0.05 versus vehicle group. (C and D) Hemodynamic data were obtained by cardiac catheterization at the end of study. (C) In the WT group, S107, metoprolol, and carvedilol improved LV systolic function (dP/dtmax). (D) In the RyR2-S2808D+/+ group, only S107 improved LV systolic function. The number of mice in each treatment group is indicated by the parenthetical numbers over each bar. Black represents vehicle; light blue represents low-dose metoprolol (30 mg/kg/d); dark blue represents high-dose metoprolol (300 mg/kg/d); green represents carvedilol (10 mg/kg/d); and red represents S107 (30 mg/kg/d). *P < 0.05 versus vehicle group. (E) Representative immunoblots from in vivo studies. Equivalent amounts of RyR2 were immunoprecipitated from cardiac lysates using an anti-RyR2 antibody. In the WT group, MI increased RyR2-S2808 PKA phosphorylation and calstabin2 depletion from the RyR2 channel complex and metoprolol decreased RyR2-S2808 PKA phosphorylation and reduced depletion of calstabin2 from the cardiac RyR2 channel complex. In the RyR2-S2808D+/+ group, calstabin2 was depleted from RyR2 channel complex in both vehicle- and metoprolol-treated groups, whereas S107 reduced depletion of calstabin2 from the cardiac RyR2 channel complex. (F) Pooled data from 3 separate immunoblots. *P < 0.05 versus sham; #P < 0.05 versus metoprolol treated.