The oxidation-resistant CaMKII-MM281/282VV mutation does not prevent arrhythmias in CPVT1

Abstract

Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited arrhythmogenic disorder caused by missense mutations in the cardiac ryanodine receptors (RyR2), that result in increased β-adrenoceptor stimulation-induced diastolic Ca²⁺ leak. We have previously shown that exercise training prevents arrhythmias in CPVT1, potentially by reducing the oxidation of Ca²⁺ /calmodulin-dependent protein kinase type II (CaMKII). Therefore, we tested whether an oxidation-resistant form of CaMKII protects mice carrying the CPVT1-causative mutation RyR2-R2474S (RyR2-RS) against arrhythmias. Antioxidant treatment (N-acetyl-L-cysteine) reduced the frequency of β-adrenoceptor stimulation-induced arrhythmogenic Ca²⁺ waves in isolated cardiomyocytes from RyR2-RS mice. To test whether the prevention of CaMKII oxidation exerts an antiarrhythmic effect, mice expressing the oxidation-resistant CaMKII-MM281/282VV variant (MMVV) were crossed with RyR2-RS mice to create a double transgenic model (RyR2-RS/MMVV). Wild-type mice served as controls. Telemetric ECG surveillance revealed an increased incidence of ventricular tachycardia and an increased arrhythmia score in both RyR2-RS and RyR2-RS/MMVV compared to wild-type mice, both following a β-adrenoceptor challenge (isoprenaline i.p.), and following treadmill exercise combined with a β-adrenoceptor challenge. There were no differences in the incidence of arrhythmias between RyR2-RS and RyR2-RS/MMVV mice. Furthermore, no differences were observed in β-adrenoceptor stimulation-induced Ca²⁺ waves in RyR2-RS/MMVV compared to RyR2-RS. In conclusion, antioxidant treatment reduces β-adrenoceptor stimulation-induced Ca²⁺ waves in RyR2-RS cardiomyocytes. However, oxidation-resistant CaMKII-MM281/282VV does not protect RyR2-RS mice from β-adrenoceptor stimulation-induced Ca²⁺ waves or arrhythmias. Hence, alternative oxidation-sensitive targets need to be considered to explain the beneficial effect of antioxidant treatment on Ca²⁺ waves in cardiomyocytes from RyR2-RS mice.

Keywords: CPVT; CaMKII; RyR2; arrhythmias; oxidation.

FIGURE 1

Antioxidant treatment reduces Ca²⁺ waves: (a) Illustrative tracings from a vehicle‐treated RyR2‐RS cardiomyocyte (left) paced at 1 Hz during β‐adrenoceptor stimulation with 100 nM ISO showing Ca²⁺ waves (indicated by arrows) in the post‐pacing rest period. Illustrative tracing from a NAC‐treated RyR2‐RS cardiomyocyte (right) during the same experimental conditions. (b) The frequency of Ca²⁺ waves was measured in a 10 s post‐pacing rest period in cardiomyocytes paced at 1 and 4 Hz under baseline conditions and during β‐adrenoceptor stimulation (ISO). (c) The time from the beginning of the last paced Ca²⁺ transient to the beginning of the first Ca²⁺ wave was analyzed, that is, Ca²⁺ wave latency. Nested ANOVA; *p = 0.024 compared to the vehicle; 11 hearts, 20–23 cells per group at baseline and 62–65 cells per group during ISO stimulation

FIGURE 2

Genetic inhibition of CaMKII MM281/282 oxidation does not affect arrhythmias in CPVT1: (a) Illustrative ECG tracings from a WT, a RyR2‐RS, and a RyR2‐RS/MMVV mouse following i.p. administration of 20 mg/kg ISO. The WT tracing shows sinus rhythm, while the tracings from RyR2‐RS and RyR2‐RS/MMVV show a few sinus rhythms preceding bidirectional VT. (b) Arrhythmias were studied for 5 min following isoprenaline (ISO) administration. An arrhythmia score was used based on the severity of arrhythmias. (c) The percentage of animals that developed the most severe arrhythmia, that is, VT. (d) The numbers of the subtypes of arrhythmias counted in 5 min following ISO administration. The experiment was repeated with treadmill running preceding the ISO administration with (e) arrhythmia score, (f) percentage of animals with VT and (g) subtypes of arrhythmias shown. Kruskal–Wallis test for arrhythmia score and arrhythmia events; Fisher's exact test for incidence of VT; *p < 0.05 compared to WT; 9 WT, 12 RyR2‐RS, and 12 RyR2‐RS/MMVV animals for isoprenaline only; 5 WT, 12 RyR2‐RS, and 11 RyR2‐RS/MMVV animals for running + ISO

FIGURE 3

Oxidation‐resistant CaMKII does not affect Ca²⁺ waves in CPVT1: (a) Illustrative tracings from WT, MMVV, RyR2‐RS, and RyR2‐RS/MMVV cardiomyocytes with paced Ca²⁺ transients at 1 Hz during ISO stimulation (100 nM ISO) and post‐pacing rest period with Ca²⁺ waves indicated by arrows. (b) Frequency of Ca²⁺ waves at baseline and during ISO stimulation. (c) Latency of Ca²⁺ waves at baseline and during ISO stimulation. Nested ANOVA; all genotypes were compared to WT, RyR2‐RS/MMVV was also compared to RyR2‐RS; *p < 0.05 compared to WT; 15–16 animals in each group; 37–53 cells per group at baseline; 84–95 cells per group during ISO stimulation

FIGURE 4

The effect of oxidation‐resistant CaMKII on Ca²⁺ handling in CPVT1: (a) Illustrative tracings from 1 Hz‐paced Ca²⁺ transients (left) and caffeine‐induced transients (right), both during β‐adrenoceptor stimulation with 100 nM ISO. (b) Amplitude of the paced Ca²⁺ transients at 1 and 4 Hz, under baseline conditions and during β‐adrenoceptor stimulation (ISO). (c) SR Ca²⁺ content following 1 Hz pacing measured as the amplitude of the caffeine‐induced transient (10 mM caffeine), at baseline and during ISO stimulation. (d) Exponential fitting to the decay of the Ca²⁺ transient was used to assess the Ca²⁺ removal rate. (e) SERCA‐dependent Ca²⁺ reuptake was calculated by subtracting the decay rate constant of the caffeine‐induced transient (following 1 Hz pacing) from the decay rate constant of the paced Ca²⁺ transient at 1 Hz from the same cardiomyocyte. Nested ANOVA; all genotypes were compared to WT, RyR2‐RS/MMVV was also compared to RyR2‐RS; *p < 0.05 compared to WT; ^# p < 0.05 compared to RyR2‐RS; 15–16 animals in each group; 38–53 cells for Ca²⁺ transients and 21–28 cells for caffeine transients at baseline, 79–97 cells for Ca²⁺ transients and 27–71 for caffeine transients during ISO stimulation

FIGURE 5

The effect of oxidation‐resistant CaMKII on Ca²⁺ handling phosphoproteins: Immunoblots were performed on left ventricular homogenates from untreated mice (baseline) and mice three min after the administration of 20 mg/kg isoprenaline i.p. (isoprenaline). (a) Illustrative immunoblots of phosphorylated CaMKII on T286, total CaMKII and vinculin as loading control with averaged data for T286 phosphorylated CaMKII normalized to total CaMKII. (b) Illustrative immunoblots of phosphorylated RyR2 on the CaMKII phosphorylation site S2814, total RyR2, and GAPDH as loading control with averaged data for S2814‐RyR2 normalized to total RyR2. (c) Illustrative immunoblots of phosphorylated PLB on T17, total PLB, and GAPDH or vinculin as loading controls with averaged data for T17‐PLB normalized to total PLB. (d) Illustrative immunoblots of RyR2 phosphorylation on the protein kinase A phosphorylation site S2808, total RyR2, and GAPDH as loading control with averaged data for S2808 normalized to total RyR2. (e) Illustrative immunoblots of phosphorylated PLB on S16, total PLB, and GAPDH or vinculin as loading control with averaged data for S16‐PLB normalized to total PLB are shown. T‐test; all genotypes were compared to WT, RyR2‐RS/MMVV was also compared to RyR2‐RS; *p < 0.05; 5 WT, 8 MMVV, 8 RyR2‐RS, and 8 RyR2‐RS/MMVV mice