The RyR2 central domain peptide DPc10 lowers the threshold for spontaneous Ca2+ release in permeabilized cardiomyocytes

Objective: In vitro experiments have shown that the ryanodine receptor-2 (RyR2) central domain peptide DPc10 (Gly(2460)-Pro(2495)) Mimics channel dysfunction associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) by acting competitively to reduce stabilizing interactions between the N-terminal and central domains. In the present study, DPc10 was used as a tool to establish an adult cell model of the disease and to analyse the underlying mechanisms. Methods: Rat ventricular myocytes were permeabilized with saponin and perfused with solutions approximating the intracellular milieu containing fluo-3. Sarcoplasmic reticulum (SR) Ca2+ release was detected using confocal microscopy. DPc10 (10 or 50 mu M) was compared with 0.2mM caffeine, which is known to activate RyR2 and to facilitate Ca2+-induced Ca2+ release (CICR). Results: Introduction of DPc10 induced a transient increase in spark frequency and a sustained rise in resting [Ca2+]. Under conditions causing initial Ca2+ overload of the SR, DPc10 reduced the frequency and amplitude of spontaneous, propagated Ca2+ release (SPCR). Following equilibration with 10 mu M DPc10, the cytosolic [Ca2+] threshold for SPCR was markedly reduced and the proportion of spontaneously active cells increased. Caffeine induced a similar, transient increase in spark frequency and a reduction in the [Ca2+] threshold for SPCR. However, unlike DPc10, caffeine increased SPCR frequency and had no sustained effect on resting [Ca2+]. These results suggest that the net effect of DPc10 (and CPVT mutations) on RyR2 function in situ is not only to increase the sensitivity to CICR as caffeine does, but also to potentiate Ca2+ leakage from the SR. As SPCR can trigger delayed after-depolarisations, the decrease in [Ca2+] threshold may contribute to arrhythmias in CPVT patients during exercise or stress. (c) 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.