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

Figures

• 

  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…

• 

  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…