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

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.

Keywords: Ca2+ alternans; Ca2+ release refractoriness; calcium; calcium ATPase; calcium imaging; calcium intracellular release; cardiac dysfunction; confocal Ca2+ imaging; endoplasmic reticulum (ER); intact heart imaging; phospholamban; ryanodine receptor; sarcoplasmic reticulum (SR); ventricular tachyarrhythmia.