Abstract 9785: The Contribution of CaMKII and PKA to the Development of an Increased RyR-dependent SR Ca Leak in Human Cardiac Pathology

Abstract

The mechanisms of a progressive ryanodine receptor type 2 (RyR2) dysregulation in the transition from human cardiac hypertrophy (Hy) to heart failure (HF) are still unclear. Inhibition of Ca 2+ /calmodulin-dependent kinase II (CaMKII) reduces the sarcoplasmic reticulum (SR) Ca 2+- leak and improve contractility in human HF. However, the relative contribution of CaMKII and protein kinase A are still not clear. Western blot experiments from tissue samples (n=12) of patients with afterload-induced Hy and preserved ejection fraction (EF>50%) revealed an increase in RyR2 expression by 82±22% ( P =0.05) compared to healthy controls (n=5). The phosphorylation status of RyR2 at S2809 and S2815 was not altered. In human end-stage HF (n=8), a pronounced hyperphosphorylation of RyR2 could be detected at the CaMKII-dependent site S2815 (by 311±72%, P <0.05) whereas the PKA-dependent site S2809 lacked differential regulation. The comparison of HF patients with and without β-blocker medication (n=7 vs. 4) did not reveal any differences as to RyR2 phosphorylation status. When isolated myocytes were paced at 1 Hz and scanned for diastolic Ca 2+ sparks (confocal microscopy, Fluo 3) a higher Ca 2+ -spark frequency (by 82±26% P <0.05) was detected in HF (n=93) compared to Hy (n=94). Interestingly, PKA inhibition with H89 (5 μM) only accomplished a decrease of SR Ca 2+ -leak in Hy (by 59±13%, P =0.05, n=85 vs. 115) but not in HF (n=85 vs. 74). Stimulated Ca 2+ transients (0.5 Hz, n=31 vs. 23), SR Ca 2+ content (caffeine, n=23 vs. 22) and fractional SR Ca 2+ release (n=21 vs. 15) were not changed by H89 treatment in HF (epifluorescence microscopy, Fura 2). CaMKII inhibition by AIP (1 μM), in contrast, could significantly reduce SR Ca 2+ leak in Hy (by 63±14%, P <0.05, n=93 vs. 103) as well as in HF. Thus, in human Hy with preserved EF both kinases seem to functionally regulate RyR2 gating. In end-stage human HF, however, the diastolic SR Ca 2+ leak seems to be induced by CaMKII rendering an inhibition of CaMKII a promising pharmacologic toehold.