Ca SR Leak is Modulated by CaMKII Phosphorylation on RyR2-S2814

Abstract

CaMKII has been shown to increase cardiac SR Ca leak through RyRs. RyR2-S2814 has been suggested as the phosphorylation site responsible for SR Ca leak triggering cardiac arrhythmias in heart failure. Here we test the requirement of S2814 for these effects, in knock-in mice expressing only RyR2-S2814A or -S2814D. Ca spark frequency (CaSpF) and SR load were studied in intact and permeabilized cardiomyocytes using confocal microscopy. At baseline CaSpF is higher in S2814D vs. WT (8.7±0.4 (n=9) vs 6.44±0.3, n=8, p<0.01) without altered SR Ca load. Activation of endogenous CaMKII (1.2 μM Ca-calmodulin) in WT increases CaSpF as described by Guo et al (Circ. Res, 2006), but only to the level seen in S2814D at baseline. Moreover, CaMKII did not further increase CaSpF in S2814D myocytes. In RyR2-S2814A CaMKII activation produces a very small CaSpF increase vs WT (19 vs 60%), and that response in S2814A is secondary to increased SR Ca load (by 15%, n=10, p<0.01). In intact myocytes (as above), basal CaSpF was highest in S2814D vs. WT and S2814A (which were similar). Baseline Ca transients were not different among the groups. The cAMP activated GTP exchange factor Epac may activate CaMKII to induce SR Ca leak (Pereira et al, J Physiol, 2007). In WT mice the Epac activator 8-CPT enhanced CaSpF and decreased both SR Ca load and Ca transient amplitude (consistent with a primary effect on SR Ca leak). However, 8-CPT had no effect on any of these parameters in either S2814A or S2814D myocytes. These data indicate that RyR2-S2814 is the critical RyR2 site responsible for CaMKII-dependent enhancement of SR Ca leak and potential arrhythmogenesis in heart failure, and confirm that Epac signaling can work through this same pathway.