Ero1α-Dependent ERp44 Dissociation From RyR2 Contributes to Cardiac Arrhythmia

Abstract

Background: Oxidative stress in cardiac disease promotes proarrhythmic disturbances in Ca 2+ homeostasis, impairing luminal Ca 2+ regulation of the sarcoplasmic reticulum (SR) Ca 2+ release channel, the RyR2 (ryanodine receptor), and increasing channel activity. However, 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 within the SR are involved in this process. Methods: A rat model of hypertrophy induced by thoracic aortic banding (TAB) was used for ex vivo whole heart optical mapping and for Ca 2+ and reactive oxygen species imaging in isolated ventricular myocytes (VMs). Results: The SR-targeted reactive oxygen species biosensor ERroGFP showed increased intra-SR oxidation in TAB VMs that was associated with increased expression of oxidoreductase Ero1α. Pharmacological (EN460) or genetic Ero1α inhibition normalized SR redox state, increased Ca 2+ transient amplitude and SR Ca 2+ content, and reduced proarrhythmic spontaneous Ca 2+ waves in TAB VMs under β-adrenergic stimulation (isoproterenol). Ero1α overexpression in Sham VMs had opposite effects. Ero1α inhibition attenuated Ca 2+ -dependent ventricular tachyarrhythmias in TAB hearts challenged with isoproterenol. Experiments in TAB VMs and human embryonic kidney 293 cells expressing human RyR2 revealed that an Ero1α-mediated increase in SR Ca 2+ -channel activity involves dissociation of intraluminal protein ERp44 from the RyR2 complex. Site-directed mutagenesis and molecular dynamics simulations demonstrated a novel redox-sensitive association of ERp44 with RyR2 mediated by intraluminal cysteine 4806. ERp44-RyR2 association in TAB VMs was restored by Ero1α inhibition, but not by reducing agent dithiothreitol, as hypo-oxidation precludes formation of covalent bond between RyR2 and ERp44. Conclusions: A novel axis of intraluminal interaction between RyR2, ERp44, and Ero1α has been identified. Ero1α inhibition exhibits promising therapeutic potential by stabilizing RyR2-ERp44 complex, thereby reducing spontaneous Ca 2+ release and Ca 2+ -dependent tachyarrhythmias in hypertrophic hearts, without causing hypo-oxidative stress in the SR.

ER stress and calcium-dependent arrhythmias

“…The PDI-like protein ERp44 was previously reported to inhibit Ca 2+ release channel inositol triphosphate receptor type 1 (IP 3 R1) by direct protein-protein interaction, and this association was redox-dependent ( Anelli et al, 2002 ; Higo et al, 2005 ; Wang et al, 2014 ). Of note, ERp44 interacts with an IP3R1 region that has sequence homology to the RyR2 second intraluminal loop, and using a multi-pronged approach, we revealed ERp44 directly interacts with RyR2 in this region via disulfide bond formation ( Hamilton et al, 2022 ). This protein-protein interaction is redox-dependent and exerts a stabilizing influence on channel function.…”

“…It is well established that UPR is activated in various cardiac pathologies associated with increased arrhythmic risk. For example, in failing human and animal hearts, increased levels of AT4, CHOP and GRP78 are reported ( Hamilton et al, 2022 ). We have recently demonstrated upregulation of CHOP and downstream oxidoreductase ERO1α in hypertrophic rat myocytes ( Hamilton et al, 2022 ).…”

“…For example, in failing human and animal hearts, increased levels of AT4, CHOP and GRP78 are reported ( Hamilton et al, 2022 ). We have recently demonstrated upregulation of CHOP and downstream oxidoreductase ERO1α in hypertrophic rat myocytes ( Hamilton et al, 2022 ). Diabetic cardiomyopathy is also associated with ER stress and activated UPR ( Liu et al, 2014 ; Liu et al, 2021 ), as is cancer chemotherapy-induced cardiotoxicity ( Kerkela et al, 2006 ).…”

“…We recently demonstrated a novel regulatory axis for RyR2-ROS modulation involving ERO1α and luminal binding of RyR2 by protein ERp44 ( Hamilton et al, 2022 ). The PDI-like protein ERp44 was previously reported to inhibit Ca 2+ release channel inositol triphosphate receptor type 1 (IP 3 R1) by direct protein-protein interaction, and this association was redox-dependent ( Anelli et al, 2002 ; Higo et al, 2005 ; Wang et al, 2014 ).…”

“…As H 2 O 2 is a byproduct of oxidative protein folding, the SR is increasingly recognized as a significant source of ROS within the cardiomyocyte, in addition to well-established sources such as NADPH oxidase 2 or mitochondria ( Bertero and Maack, 2018 ; Hamilton et al, 2022 ). While stress of the SR/ER invokes initially adaptive pathways to restore protein-folding capacity and upregulate antioxidant machinery, chronic ER stress invokes maladaptive pathways of the UPR, including excessive ROS production and degradation of protein mRNA.…”