Preventing RyR2-S2808 and RyR2-S2814 Phosphorylation does not Alter the β-Adrenergic Response of Mouse Hearts

Abstract

We previously showed that ablation of the cardiac Ca2+ release channel/ryanodine receptor (RyR2) phosphorylation at Ser2808 (RyR2-S2808A) has no functional impact on cardiac performance and/or heart failure progression. In this study, we investigated whether this lack of effect may be due to compensatory mechanisms involving altered phosphorylation of the nearby Ser2814 CaMKII phospho-site. We generated a new mouse model where Ser2808 and Ser2814 were genetically replaced by Alanine (RyR2-S2808A/S2814A). Anesthetized RyR2-S2808A/S2814A mice have normal ejection fraction at baseline compared to wild type (WT) mice (55.1±2.6% vs. 55.9±2.4%, respectively, n=7/group) but exhibit ∼20% increase in heart rate (553±33 vs. 460±28 bpm, p<0.05, n=8/group). Langendorff-perfused hearts from both, RyR2-S2808A/S2814A and WT mice displayed an equally robust increase in the amplitude of isoprenaline (200nM)-stimulated LV contractions (60% and 63% above control in mutant and WT hearts, respectively, n=10/group, p=0.7). In agreement with the whole-heart results, isoproterenol (100nM)-stimulated RyR2-S2808A/S2814A myocytes displayed a comparable increase in Ca2+ transient amplitude and sarcoplasmic reticulum (SR) Ca2+ content than WT cardiomyocytes. Nevertheless, ablation of the S2808/S2814 phospho-sites significantly decreased RyR2 refractoriness measured by a 2-pulse protocol (142±7 vs. 194±6 ms in the S2808A/S2814A and WT, respectively, n=7, p<0.01). This effect was associated with a ∼27% increase in SR Ca2+ leak (3.22±0.2 vs. 2.53±0.2 ΔF/F0.100µm−1.s−1 in the RyR2-S2808A/S2814A and WT myocytes, respectively, n=17/group, p<0.05) causing earlier, faster and more frequent spontaneous Ca2+ waves. Interestingly, 60% of RyR2-S2808A/S2814A and 40% of the WT Langendorff-perfused hearts (n=10 each) exhibited arrhythmias upon isoprenaline stimulation. In conclusion, our data show that inhibition of S2808 and S2814 phosphorylation leaves intact the β-adrenergic response of the heart but accelerates RyR2 refractoriness, increasing SR Ca2+ leak and promoting arrhythmic events.