The cardiac ryanodine receptor, but not sarcoplasmic reticulum Ca2+-ATPase, is a major determinant of Ca2+ alternans in intact mouse hearts

Bo Sun,Jinhong Wei,Xiaowei Zhong,Wenting Guo,Jinjing Yao,Ruiwu Wang,Alexander Vallmitjana,Raul Benitez,Leif Hove-Madsen,S R Wayne Chen

doi:10.1074/jbc.ra118.003760

Abstract

Sarcoplasmic reticulum (SR) Ca2+ cycling is governed by the cardiac ryanodine receptor (RyR2) and SR Ca2+-ATPase (SERCA2a). Abnormal SR Ca2+ cycling is thought to be the primary cause of Ca2+ alternans that can elicit ventricular arrhythmias and sudden cardiac arrest. Although alterations in either RyR2 or SERCA2a function are expected to affect SR Ca2+ cycling, whether and to what extent altered RyR2 or SERCA2a function affects Ca2+ alternans is unclear. Here, we employed a gain-of-function RyR2 variant (R4496C) and the phospholamban-knockout (PLB-KO) mouse model to assess the effect of genetically enhanced RyR2 or SERCA2a function on Ca2+ alternans. Confocal Ca2+ imaging revealed that RyR2-R4496C shortened SR Ca2+ release refractoriness and markedly suppressed rapid pacing-induced Ca2+ alternans. Interestingly, despite enhancing RyR2 function, intact RyR2-R4496C hearts exhibited no detectable spontaneous SR Ca2+ release events during pacing. Unlike for RyR2, enhancing SERCA2a function by ablating PLB exerted a relatively minor effect on Ca2+ alternans in intact hearts expressing RyR2 WT or a loss-of-function RyR2 variant, E4872Q, that promotes Ca2+ alternans. Furthermore, partial SERCA2a inhibition with 3 μm 2,5-di-tert-butylhydroquinone (tBHQ) also had little impact on Ca2+ alternans, whereas strong SERCA2a inhibition with 10 μm tBHQ markedly reduced the amplitude of Ca2+ transients and suppressed Ca2+ alternans in intact hearts. Our results demonstrate that enhanced RyR2 function suppresses Ca2+ alternans in the absence of spontaneous Ca2+ release and that RyR2, but not SERCA2a, is a key determinant of Ca2+ alternans in intact working hearts, making RyR2 an important therapeutic target for cardiac alternans.

Highlights

Sarcoplasmic reticulum (SR) Ca2؉ cycling is governed by the cardiac ryanodine receptor (RyR2) and SR Ca2؉-ATPase (SERCA2a)
Abnormal SR Ca2؉ cycling is thought to be the primary cause of Ca2؉ alternans that can elicit ventricular arrhythmias and sudden cardiac arrest
An increasing body of evidence indicates that Ca2ϩ alternans can occur in the absence of other forms of cardiac alternans, supporting the notion that Ca2ϩ alternans plays a primary role in the genesis of cardiac alternans [1,2,3,4,5,6,7,8,9,10,11]

Summary

Results

Enhancing RyR2 function reduces the refractoriness of SR Ca2؉ release in intact hearts. Despite its significant impact on SERCA2a, tBHQ at 3 ␮M did not significantly affect the average alternans ratio or duration in intact RyR2 WT hearts stimulated at a wide range of frequencies (from 5 to 14 Hz) (Fig. 7). TBHQ at 10 ␮M markedly reduced both the average alternans ratio and duration in intact RyR2 WT hearts at stimulation frequencies of 11–14 Hz (Fig. 7) These data indicate that, depending on the extent of SERCA2a inhibition, reducing. TBHQ at 3 ␮M had a minor effect on the average alternans ratio or duration in intact E4872Q hearts (Fig. 8) These data indicate that compared with RyR2, SERCA2a plays a relatively minor role in Ca2ϩ alternans in intact hearts

Discussion

Although genetically engineered mouse models harboring a

Animal studies

Image and signal processing

Statistical analysis