Ero1α-Dependent ERp44 Dissociation From RyR2 Contributes to Cardiac Arrhythmia

Abstract

Background: Oxidative stress in cardiac disease promotes proarrhythmic disturbances in Ca²⁺ homeostasis, impairing luminal Ca²⁺ regulation of the sarcoplasmic reticulum (SR) Ca²⁺ 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 Ca²⁺ 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 Ero1α (endoplasmic reticulum oxidoreductase 1 alpha). Pharmacological (EN460) or genetic Ero1α inhibition normalized SR redox state, increased Ca²⁺ transient amplitude and SR Ca²⁺ content, and reduced proarrhythmic spontaneous Ca²⁺ waves in TAB VMs under β-adrenergic stimulation (isoproterenol). Ero1α overexpression in Sham VMs had opposite effects. Ero1α inhibition attenuated Ca²⁺-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 Ca²⁺-channel activity involves dissociation of intraluminal protein ERp44 (endoplasmic reticulum protein 44) 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 Ca²⁺ release and Ca²⁺-dependent tachyarrhythmias in hypertrophic hearts, without causing hypo-oxidative stress in the SR.

Keywords: cardiovascular diseases; constriction; heart failure; homeostasis; oxidoreductases.

Figure 1.

Upregulation of Ero1α (endoplasmic reticulum oxidoreductase 1 alpha) increases intrasarcoplasmic reticulum (SR) reactive oxygen species (ROS) in cardiac disease. A, Relative mRNA expression data from Sham and thoracic aortic banding (TAB) ventricular myocytes (VMs). Data were normalized to housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and are presented as the ratio of the expression values obtained with TAB RNA vs Sham RNA, which was normalized to 1.0. Relative expression levels were obtained after ΔΔCt calculation. P values were calculated Mann-Whitney test. B, Representative Western blot of Sham and TAB rat VMs and (C) mean±SEM Ero1α signal, normalized to GAPDH. N=8 Sham, N=8 TAB animals. P value obtained using 2-sample Student t test. D, Cultured VM expressing intra-SR redox probe ERroGFP. E, Representative ERroGFP fluorescence traces. VMs were paced (2 Hz, 5 min) and treated with isoproterenol (ISO; 50 nmol/L) or ISO and Ero1α (endoplasmic reticulum oxidoreductase 1 alpha) inhibitor EN460 (20 μmol/L). Signal normalized to minimum (dithiothreitol [DTT], 5 mmol/L) and maximum (2,2′-dithiodipyridine [DTDP], 200 µmol/L) fluorescence. F, Mean±SEM fluorescence (%). N=6 Sham, N=6 TAB animals, n=9 Sham, n=11 TAB, n=5 TAB+EN460 VMs. P values obtained using 2-level random intercept model with Tukey posthoc.

Figure 2.

Inhibition of Ero1α (endoplasmic reticulum oxidoreductase 1 alpha) oxidoreductase reduces arrhythmogenic potential in hypertrophic rat hearts. A, ECG recordings of ventricular fibrillation (VF) induction in thoracic aortic banding (TAB) hearts under isoproterenol (ISO; 50 nmol/L). After EN460 (10 µmol/L, 30 min), transient ventricular tachycardia (VTs) were observed rather than long-lasting VFs. B, Propagation maps of Sham heart (left), VF in TAB heart (center), and transient VT in the presence of EN460 (right). Arrows represent reentry. C, Rapid pacing followed by pause-induced spontaneous Ca²⁺ release that triggered delayed after depolarizations. EN460 suppressed spontaneous Ca²⁺ release. D, Number of ex vivo Sham, TAB, and TAB+EN460 hearts exhibiting VF. N=7 Sham+ISO, N=7 TAB+ISO, N=8 TAB+ISO+EN460 hearts. *P=2.1×10⁻⁴, obtained using Freeman-Halton extension of the Fisher exact test.

Figure 3.

Altered expression levels of Ero1α (endoplasmic reticulum oxidoreductase 1 alpha) modulate intrasarcoplasmic reticulum (SR) redox state and RyR2 (ryanodine receptor type 2)-mediated Ca²⁺ release. A, Representative Western blot demonstrating adenoviral overexpression of Ero1α, and shRNA-mediated Ero1α knockdown in cultured Sham and thoracic aortic banding (TAB) ventricular myocytes (VMs), respectively. B, Mean±SEM Ero1α signal, normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase). N=4 Sham, N=4 TAB animals. P values calculated using Kruskal-Wallis with Dunn posthoc. C, Representative SR redox probe ERroGFP fluorescence traces. VMs were treated with isoproterenol (ISO; 50 nmol/L) and paced at 2 Hz (5 min). Dithiothreitol (DTT; 5 mmol/L) and 2,2′-dithiodipyridine (DTDP; 200 µmol/L) were used to obtain minimum and maximum fluorescence. D, Mean±SEM ERroGFP fluorescence (%). N=6 Sham, N=5 TAB animals, n=11 Sham, n=12 Sham+Ero1α, n=9 TAB, n=13 TAB+Ero1α-shRNA VMs. P values obtained using 2-level random intercept model with Tukey posthoc. E, Fluo-3 fluorescence (F/F₀) profiles of ISO-treated VMs undergoing 2 Hz pace-pause protocol. F, Mean±SEM Ca²⁺ transient amplitude at 2 Hz (ΔF/F₀) and spontaneous Ca²⁺ wave (SCW) latency (s). N=9 Sham, N=9 TAB animals, n=25 Sham, n=18 Sham+Ero1α, n=29 TAB VMs, n=14 TAB+Ero1α-shRNA VMs. P values obtained using 2-level random intercept model with Tukey posthoc. G, Representative traces of caffeine-induced Ca²⁺ transients (10 mmol/L). H, Mean±SEM caffeine-sensitive Ca²⁺ store load. n=15 Sham, n=14 Sham+Ero1α, n=15 TAB, n=12 TAB+Ero1α-shRNA VMs; from the same animals used in E and F. P values obtained using 2-level random intercept model with Tukey posthoc.

Figure 4.

ERp44 (endoplasmic reticulum protein 44) is a binding partner of RyR2 (ryanodine receptor type 2) and this interaction is reduced in cardiac disease. A, Representative images of Sham and thoracic aortic banding (TAB) ventricular myocytes (VMs) probed with anti-RyR2 and anti-ERp44 antibodies. VMs were treated with EN460 (20 µmol/L, 30 min) or dithiothreitol (DTT; 5 mmol/L, 10 min). B, Mean±SEM for Manders coefficients M1 and M2. N=4 Sham, N=6 TAB animals, n=36 Sham, n=81 TAB, n=26 TAB+DTT, N=14 TAB+EN460 VMs. P values obtained using 2-level random intercept model with Tukey posthoc. C, Representative Western blot demonstrating expression of RyR2 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) in Sham and TAB VMs, and mean±SEM RyR2 signal, normalized to GAPDH. N=7 Sham animals, N=7 TAB animals. P values obtained using 1-way ANOVA with Bonferroni posthoc. D, Representative Western blot of Sham and TAB VMs probed for RyR2 and ERp44 expression, and mean±SEM normalized ERp44/RyR2 signal (%). N=6 Sham, N=6 TAB animals. P value obtained by Mann-Whitney test. E, Representative BN-PAGE images of RyR2 from fresh Sham and TAB VMs, immunoblotted for RyR2 and ERp44. VMs were treated with EN460 (20 µmol/L, 30 min) or DTT (5 mmol/L, 10 min). Arrow indicates native RyR2 protein complexes. F, Mean±SEM normalized ERp44/RyR2 signal (%). N=5 Sham, N=5 TAB animals. P values obtained using Kruskal-Wallis with Dunn posthoc.

Figure 5.

Knockdown of ERp44 (endoplasmic reticulum protein 44) and overexpression of Ero1α (endoplasmic reticulum oxidoreductase 1 alpha) increases human RyR2 (ryanodine receptor type 2) activity in a heterologous cell system. A, Images of HEK293 IP3R (inositol triphosphate receptor)-3KO cell co-expressing EGFP-hRyR2-WT (wild type) and R-CEPIAer. B, Western blot demonstrating ERp44 knockdown. C, Western blot demonstrating Ero1α overexpression. D, Representative R-CEPIAer fluorescence traces in cells co-expressing hRyR2±ERp44-shRNA or Ero1α. Signal was normalized to minimum (2 mmol/L EGTA and 20 μmol/L ionomycin) and maximum (20 mmol/L Ca²⁺ and 20 μmol/L ionomycin) fluorescence. E, Mean±SEM Ca²⁺ wave frequency (min⁻¹). Cells were assessed from four transfections. n=17 hRyR2-WT, n=10 hRyR2-WT+ERp44-shRNA, n=7 hRyR2-WT+Ero1α cells. P values obtained using 1-way ANOVA with Bonferroni posthoc.

Figure 6.

Mutation of intraluminal cysteine Cys4806 increases human RyR2 (ryanodine receptor type 2) activity. A, Schematic of RyR2 monomer with proposed ERp44 (endoplasmic reticulum protein 44)-interacting Cys4806. B, DNA chromatogram of recombinant hRyR2 (human RyR2) plasmid indicating Cys4806Ser substitution, to prevent interaction with ERp44. Image created with Biorender.com. C, HEK293 IP3R-3KO cell transfected with hRyR2-WT (wildtype) or hRyR2-MUT (hRyR2-Cys4806Ser) and R-CEPIAer. D, Representative R-CEPIAer fluorescence traces in cells co-transfected with hRyR2-WT or hRyR2-MUT. Caffeine (20 mmol/L) was applied to deplete the store. Signal normalized to minimum (2 mmol/L EGTA and 20 μmol/L ionomycin) and maximum (20 mmol/L Ca²⁺ and 20 μmol/L ionomycin) fluorescence. E, Mean±SEM frequency of Ca²⁺ waves. Cells were assessed from 6 transfections. n=17 hRyR2-WT, n=22 hRyR2-MUT cells. P value obtained using 2-sample Student t test. F, Representative R-CEPIAer fluorescence traces in cells co-expressing hRyR2-MUT±ERp44-shRNA or Ero1α (endoplasmic reticulum oxidoreductase 1 alpha). G, Mean±SEM for Ca²⁺ wave frequency (min⁻¹). Cells were assessed from 6 transfections. n=20 hRyR2-MUT, n=5 hRyR2-MUT+ERp44-shRNA, n=6 hRyR2-MUT+Ero1α cells. P values obtained using Kruskal-Wallis with Dunn posthoc.

Figure 7.

Molecular dynamics simulations suggest RyR2 (ryanodine receptor type 2)-ERp44 (endoplasmic reticulum protein 44) interaction is redox dependent. A, Structural alignment of RyR2 and IP3R1 (inositol triphosphate receptor type 1) intraluminal regions to peroxiredoxin-4 (PRDX4) region involved in formation of a disulfide bridge with ERp44. RyR2 is in purple (PDB ID: 6JH6), IP3R1 is in green (PDB ID: 3JAV), reduced PRXD4 is in blue (PDB ID: 3TJF). B, General view of docked RyR2-ERp44 complex. The intraluminal region of RyR2 with Cys4806 is exposed to the sarcoplasmic reticulum (SR) lumen, making it possible for ERp44 to bind. C, Zoomed disulfide bridge between Cys4806 of RyR2 and Cys29 of ERp44. RyR2 is highlighted in purple (PDB ID: 6JH6), ERp44 is highlighted in blue (PDB ID: 5XWM), and the POPC membrane is translucent. D, Graphs showing distance between β-carbon atom of RyR2’s Cys4806 and sulfur atom of ERp44’s Cys29 for reduced, oxidized, and mutated RyR2-ERp44 complexes as a function of time for 3 full-length simulations (MD1, MD2, and MD3). The reduced form shows increasing distance and completely dissociates during the early period of simulations. Both oxidized and mutated forms show complex retention.

Figure 8.

Scheme depicting the putative multimolecular RyR2 (ryanodine receptor type 2) intraluminal redox sensor and sarcoplasmic reticulum (SR) oxidoreductase-dependent mechanisms of cardiac Ca²⁺-dependent arrhythmia. Figure created with Biorender.com.