Exercise training prevents ventricular tachycardia in CPVT1 due to reduced CaMKII-dependent arrhythmogenic Ca2+ release

Abstract

Aims: Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is caused by mutations in the cardiac ryanodine receptor (RyR2) that lead to disrupted Ca(2+) handling in cardiomyocytes and ventricular tachycardia. The aim of this study was to test whether exercise training could reduce the propensity for arrhythmias in mice with the CPVT1-causative missense mutation Ryr2-R2474S by restoring normal Ca(2+) handling.

Methods and results: Ryr2-R2474S mice (RyR-RS) performed a 2 week interval treadmill exercise training protocol. Each exercise session comprised five 8 min intervals at 80-90% of the running speed at maximal oxygen uptake (VO2max) and 2 min active rest periods at 60%. VO2max increased by 10 ± 2% in exercise trained RyR-RS (ET), while no changes were found in sedentary controls (SED). RyR-RS ET showed fewer episodes of ventricular tachycardia compared with RyR-RS SED, coinciding with fewer Ca(2+) sparks and waves, less diastolic Ca(2+) leak from the sarcoplasmic reticulum, and lower phosphorylation levels at RyR2 sites associated with Ca(2) (+)-calmodulin-dependent kinase type II (CaMKII) compared with RyR-RS SED. The CaMKII inhibitor autocamtide-2-related inhibitory peptide and also the antioxidant N-acetyl-l-cysteine reduced Ca(2+) wave frequency in RyR-RS equally to exercise training. Protein analysis as well as functional data indicated a mechanism depending on reduced levels of oxidized CaMKII after exercise training. Two weeks of detraining reversed the beneficial effects of the interval treadmill exercise training protocol in RyR-RS ET.

Conclusion: Long-term effects of interval treadmill exercise training reduce ventricular tachycardia episodes in mice with a CPVT1-causative Ryr2 mutation through lower CaMKII-dependent phosphorylation of RyR2.

Keywords: Arrhythmias; CPVT1; Ca2+ homeostasis; CaMKII; Exercise training.

Figure 1

Increased VO_2max and reduced arrhythmias in RyR-RS after exercise training. (A) Illustration of the exercise training protocol. (B) Average VO_2max from RyR-RS ET and RyR-RS SED at the end of Weeks 0 and 2 of the exercise protocol. *P < 0.05 RyR-RS SED vs. RyR-RS ET at Week 2, calculated with nested ANOVA. **P < 0.05 RyR-RS ET Week 0 vs. Week 2, calculated with Student's t-test. Number of RyR-RS mice in each group (SED/ET): 59/71. (C) In vivo ECG recording from a RyR-RS SED mouse towards the end of a VO_2max test close to exhaustion. Left box: ventricular premature beat. Centre box: bidirectional VT. Right box: sinus beats. (D) Average number of VT episodes in a 20 min period immediately after the end of VO_2max test and intraperitoneal ISO injection. Number of RyR-RS mice (SED/ET): 6/5.

Figure 2

Reduced Ca²⁺ wave frequency in RyR-RS mice after exercise training. Example tracing of whole-cell calcium imaging in field-stimulated cardiomyocytes from (A) RyR-RS SED and (B) RyR-RS ET. Average Ca²⁺ wave frequency after (C) 1 and 4 Hz stimulation in the absence of ISO. Average Ca²⁺ wave frequency in the presence of ISO after (D) 1 and 4 Hz stimulation. *P< 0.05. Number of RyR-RS mice (SED/ET): 15/13, number of cells (SED/ET): 40/38, ISO: 46/50.

Figure 3

Reduced SR Ca²⁺ leak in RyR-RS mice after exercise training. SR Ca²⁺ leak in resting cardiomyocytes after 1 Hz stimulation. (A) Illustration of protocol in the presence of ISO. SR Ca²⁺ leak in (B) the absence and presence of ISO. The Y axis shows the SR Ca²⁺ leak levels normalized to SR Ca²⁺ content levels (F/F₀). *P < 0.05, number of RyR-RS mice in protocol in the absence and presence of ISO (SED/ET): 12/10, 5/7, number of cells (SED/ET): 12/9, ISO: 13/19. The cells used in the experiments in the presence and absence of ISO were from different animals.

Figure 4

Reduced Ca²⁺ spark frequency in RyR-RS mice after exercise training. Confocal imaging of cardiomyocytes from (A) RyR-RS SED and (B) RyR-RS ET after 1 Hz stimulation in the presence of ISO. Average Ca²⁺ spark frequency in a 6 s pause after (C) 1 and 4 Hz stimulation in the absence of ISO. Average Ca²⁺ spark frequency in a 6 s pause in the presence of ISO after (D) 1 and 4 Hz stimulation. Density blots illustrate Poisson analysis used for comparison of Ca²⁺ spark frequency in RyR-RS ET and RyR-RS SED groups. *P < 0.05. Number of RyR-RS mice (SED/ET): 6/6, number of cells (SED/ET): 30/31, ISO: 21/25.

Figure 5

Reduced CaMKII-dependent phosphorylation of RyR in RyR-RS mice after exercise training. Left ventricle lysates from RyR-RS SED and RyR-RS ET were immunoblotted with antibodies against (A) pSer2814-RyR2, (B) pSer2808-RyR2, (C) total CaMKII, (D) pThr286-CaMKII, (E) Ox-CaMKII, and (F) MDA. pSer2814-RyR2 and pSer2808-RyR2 were normalized to total RyR2 levels; pThr286-CaMKII and Ox-CaMKII levels were normalized to total CaMKII levels. The values are presented in percentage and are normalized to RyR-RS SED. *P < 0.05, **P < 0.05 with one-sided Student's t-test. Number of hearts used from RyR-RS mice (SED/ET): 9/9.

Figure 6

Reduced Ca²⁺ wave frequency in RyR-RS after CaMKII inhibition and antioxidant treatment. (A) Example tracing from whole-cell calcium imaging in field-stimulated cardiomyocytes isolated from RyR-RS SED with AIP added to the experimental solution in the presence and absence of ISO. Separate experiments were performed with the same protocol with NAC replacing AIP. Average Ca²⁺ wave frequency in pause in the absence of ISO after (B) 1 and 4 Hz stimulation. Average Ca²⁺ wave frequency in pause in the presence of ISO after (C) 1 and 4 Hz stimulation. *P < 0.05. Number of RyR-RS mice experiments with AIP and NAC (SED): 6, number of cells (SED): 22, ISO: 32, SED cells with AIP: 10, ISO: 28, SED cells with NAC: 6, ISO: 35.

Figure 7

Detraining reverses VO_2max, VT episodes, and Ca²⁺ waves in RyR-RS. (A) VO_2max levels for RyR-RS SED. (B) VO_2max levels for RyR-RS ET. (C) Average number of VT episodes during VO_2max test after detraining in Week 4. Data from Week 2 for RyR-RS ET in (C) is equal to the RyR-RS ET data in Figure 1D. Number of RyR-RS mice (SED/ET): 6/5. (D and E) Ca²⁺ wave frequency after (D) 1 and (E) 4 Hz stimulation in the absence of ISO. (F and G) Ca²⁺ wave frequency in the presence of ISO after (F) 1 and (G) 4 Hz stimulation. Data from Week 2 for RyR-RS SED and RyR-RS ET in D–G are equal to data in Figure 2C–D. *P < 0.05. Number of RyR-RS mice exposed to detraining: 6 SED/11 ET, number of cells: absence of ISO 11 SED/38 ET, presence of ISO 25 SED/42 ET.