The Sarcoplasmic Reticulum Oxidoreductase System Modulates Luminal Ca2+ Regulation of the Ryanodine Receptor in Cardiac Disease

Abstract

The sarcoplasmic reticulum (SR) Ca2+ release ryanodine receptor channel (RyR2) is susceptible to posttranslational modifications by reactive oxygen species (ROS), impairing luminal Ca2+ regulation and increasing channel activity. Despite significant research efforts, exact mechanisms underlying redox-mediated increase of RyR2 function in cardiac disease remain elusive. We tested whether the oxidoreductase family of proteins that dynamically regulate the oxidative environment in the SR are involved in this process. In ventricular myocytes (VMs) from rats with hypertrophy induced by thoracic aortic banding (TAB), expression of SR oxidoreductase Ero1α was significantly increased compared to Sham VMs. Adenoviral expression of intra-SR ROS probe ERroGFP in cultured TAB rat VMs revealed enhanced oxidative stress in the vicinity of RyR2, in parallel with increased RyR2 oxidation, measured with DNP antibody. Ero1α overexpression in control VMs recapitulated a similar phenotype. Conversely, pharmacological inhibition of Ero1α with EN460 (20 μM) reduced arrhythmogenic potential in ex vivo optically mapped TAB rat hearts by reducing SR Ca2+ leak, and suppressed Ca2+ spark frequency in TAB VMs, indicating stabilization of RyR2 function. Utilizing a heterologous HEK293 cell system and intra-ER Ca2+ sensor R-CEPIAer to probe Ca2+ release dynamics of human RyR2, Ero1α overexpression significantly increased Ca2+ wave frequency, mimicking effects of strong oxidative agent DTDP. Knockdown of endogenous Ero1α had opposite effects. Furthermore, mutation of two intraluminal cysteines of RyR2 in the second intraluminal loop, potentially sensitive to modification by ROS, resulted in more frequent Ca2+ waves and reduced basal intra-ER [Ca2+]. These findings suggest upregulation of SR oxidoreductase Ero1α in cardiac disease is detrimental to luminal Ca2+ homeostasis by modulating RyR2 activity via ROS. Additionally, we have identified two potential intraluminal residues of the channel that may be sensitive to redox modification.