Abstract 19322: Cardiac Function and Beta-Adrenergic Response in Mice with Genetic Ablation of Two Major Phosphorylation Sites in the Cardiac Ryanodine Receptor

Abstract

Despite intensive research, the functional consequences of cardiac ryanodine receptor (RyR2) phosphorylation remain highly controversial. Two major phosphorylation sites, S2808 and S2814, are respective targets for PKA and CaMKII and form a RyR2 “phosphorylation hot spot”. We previously showed that genetic ablation of S2808 has no structural or functional consequences for cardiac function. Here we generated a novel double knock-in mouse with genetic ablation of S2808 and S2814 (S2808A/S2814A) and tested the hypothesis that preventing the “hot spot” phosphorylation has discernible impact in cardiac function. Echocardiography and intraventricular pressure measurements revealed that, under basal conditions, S2808A/S2814A mice have higher heart rate (553±33 vs. 460±28 bpm, p <0.05, n= 8/group) and decreased ejection fraction (55±3 vs. 65±2%, p <0.05, n=12/group) compared to WT. In addition, ventricular myocytes isolated from the S2808A/S2814A hearts displayed a significant increase in Ca 2+ transient amplitude (357±27 vs. 257±31 nM, p <0.05, n=13 cells/group), a trend towards higher Ca 2+ spark frequency (0.51±0.14 vs. 0.30±0.17 sparks.100 μm -1 .sec -1 , n=7 cells/group) while the sarcoplasmic reticulum (SR) Ca 2+ load was comparable to that of WT cells (107±7 vs. 97±4 μmol/L cytosol). Surprisingly, β-adrenergic stimulation eliminated the differences between the two groups: Langerdorff-perfused hearts displayed a robust increase in the amplitude of contraction in response to 200 nM isoprenaline (51±4 vs. 62±14% above control in double knock-in and WT mice, respectively; n=5/group) and in isolated ventricular myocytes, 100 nM isoproterenol significantly increased the Ca 2+ transient amplitude (1.99±0.18 vs. 1.47±0.15 μM, n= 7 cells/group) and SR Ca 2+ load (175±12 vs. 183±14 μmol/L cytosol) in double knock-in and WT mice, respectively. Thus, ablation of the “phosphorylation hot spot” has discernible effects on cardiac function at baseline, but under β-adrenergic stimulation other RyR2 phosphorylation sites appear to normalize Ca 2+ transient and cardiac contraction. This is compatible with a multi-phosphorylation site model of RyR2 where the β-adrenergic stimulation response is dictated by a complex interplay of phosphorylations.