Phosphorylation of RyR2 at S2814 is a necessary step for the protective effect of β-adrenergic stimulation against Ca2+ alternans

AGÜERO, RODRIGO; RACIOPPI, MARÍA FLORENCIA; RIZZARDI, FRANCISCO; CELY-ORTIZ, ALEJANDRA

Berlin

Congreso; 2022 XXIV ISHR WORLD CONGRESS; 2022

IntroductionWhile β-adrenergic stimulation can prevent Ca2+ alternans, pharmacological inhibition of CaMKII precludes this. However, if this is due to phosphorylation of RyR2 or Phospholamban is not known.ObjectiveTo determine the relevance of RyR2 phosphorylation at S2814 for the propensity to Ca2+ alternans with and without β-adrenergic stimulation.Materials & methodsVentricular cardiomyocytes were obtained from 2 to 3 months gender mixed wild-type (C57) and S2814A mice after collagenase-based heart digestion in Langendorff system. Cytosolic Ca2+ was monitored loading the cells with Fluo-4 and these were paced at 1,3,4,5,6 and 8 Hz. The alternansratio (AR) was calculated by measuring the difference in amplitude between consecutive transients normalized by the amplitude of the largest. The alternans threshold was determined as the pacing frequency at which the AR was ≥0.1, if not reached, the threshold was considered as 9 Hz. Unpaired t-test was applied and mean ± SEM is presented.ResultsThe alternans threshold in wt and S2814A cells was not significantly different (5.82 ± 0.38 and 5.92 ± 0.39) and AR was not significantly different at any frequency studied (n = 28 cells per group). However, the incidence of spontaneous Ca2+ waves after the pacing protocol was significantly higher in wt than in S2814A cells. A short incubation (10 min) with isoproterenol 100 nM increased the alternans threshold and reduced the AR at 5 Hz in WT cardiomyocytes (threshold wt = 5.82 ± 0.38 and wt + iso = 7.15 ± 0.31. n = 28 and 13 cells respectively), but there were not significant changes in S2814A treated cells. Interestingly, a longer incubation (60 min), which has been shown to increase RyR2 phosphorylation at a same degree than short treatment, promoted further increase in alternans threshold and reduction in AR only in the WT treated group. The potential mechanism for this additional effect will be discussed.