A domain peptide (DP) matching the Gly²⁴⁶⁰–Pro²⁴⁹⁵ region of the cardiac type-2 ryanodine receptor (RyR2), DPc10, is known to mimic channel dysfunction associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), owing to its interference in a normal interaction of the N-terminal (1–600) and central (2000–2500) domains (viz. domain unzipping). Using DPc10 and two other DPs harboring different mutation sites, we investigated the underlying mechanism of abnormal Ca²⁺ cycling in failing hearts.

Sarcoplasmic reticulum (SR) vesicles and cardiomyocytes were isolated from dog left ventricular muscles for Ca²⁺ leak and spark assays. The RyR2 moiety of the SR was fluorescently labelled with methylcoumarin acetate (MCA) using DPs corresponding to the 163–195 and 4090–4123 regions of RyR2 (DP163–195 and DP4090–4123, respectively) as site-directed carriers. Both DPs mediated a specific MCA fluorescence labelling of RyR2. Addition of either DP to the MCA-labelled SR induced domain unzipping, as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. Both SR Ca²⁺ leak and Ca²⁺ spark frequency (SpF) were markedly increased in failing cardiomyocytes. Upon introduction of DP163–195 or DP4090–4123 into normal SR or cardiomyocytes, both Ca²⁺ leak and SpF increased to the levels comparable with those of failing myocytes. K201 (JTV519) suppressed all of the effects induced by DP163–195 (domain unzipping and increased Ca²⁺ leak and SpF) or those in failing cardiomyocytes, but did not suppress the effects induced by DP4090–4123.

Defective inter-domain interaction between N-terminal and central domains induces diastolic Ca²⁺ leak, leading to heart failure and lethal arrhythmia. Mutation at the C-terminal region seen in CPVT does not seem to communicate with the aforementioned N-terminal and central inter-domain interaction, although spontaneous Ca²⁺ leak is similarly induced.