Rationale : Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by a single point mutation in a well-defined region of the cardiac type 2 ryanodine receptor (RyR)2. However, the underlying mechanism by which a single mutation in such a large molecule produces drastic effects on channel function remains unresolved. Objective : Using a knock-in (KI) mouse model with a human CPVT-associated RyR2 mutation (R2474S), we investigated the molecular mechanism by which CPVT is induced by a single point mutation within the RyR2. Methods and Results : The R2474S/+ KI mice showed no apparent structural or histological abnormalities in the heart, but they showed clear indications of other abnormalities. Bidirectional or polymorphic ventricular tachycardia was induced after exercise on a treadmill. The interaction between the N-terminal (amino acids 1 to 600) and central (amino acids 2000 to 2500) domains of the RyR2 (an intrinsic mechanism to close Ca ²⁺ channels) was weakened (domain unzipping). On protein kinase A-mediated phosphorylation of the RyR2, this domain unzipping further increased, resulting in a significant increase in the frequency of spontaneous Ca ²⁺ transients. cAMP-induced aberrant Ca ²⁺ release events (Ca ²⁺ sparks/waves) occurred at much lower sarcoplasmic reticulum Ca ²⁺ content as compared to the wild type. Addition of a domain-unzipping peptide, DPc10 (amino acids 2460 to 2495), to the wild type reproduced the aforementioned abnormalities that are characteristic of the R2474S/+ KI mice. Addition of DPc10 to the (cAMP-treated) KI cardiomyocytes produced no further effect. Conclusions : A single point mutation within the RyR2 sensitizes the channel to agonists and reduces the threshold of luminal [Ca ²⁺ ] for activation, primarily mediated by defective interdomain interaction within the RyR2.

Background—Defective interdomain interaction within the ryanodine receptor (RyR2) seems to play a key role in the pathogenesis of heart failure, as shown in recent studies. In the present study we investigated the effect of oxidative stress on the interdomain interaction, its outcome in the cardiac function in heart failure, and the possibility of preventing the problem with antioxidants.Methods and Results—Sarcoplasmic reticulum (SR) vesicles were isolated from dog left ventricular (LV) muscle (normal or rapid ventricular pacing for 4 weeks with or without the antioxidant edaravone). In the edaravone-treated paced dogs (EV+), but not in the untreated paced dogs (EV−), normal cardiac function was restored almost completely. In the SR vesicles isolated from the EV−, oxidative stress of the RyR2 (reduction in the number of free thiols) was severe, but it was negligible in EV+. The oxidative stress of the RyR2 destabilized interdomain interactions within the RyR2 (EV−), but its effect was reversed in EV+. Abnormal Ca²⁺leak through the RyR2 was found in EV− but not in EV+. The amount of the RyR2-bound FKBP12.6 was less in EV− than in normal dogs, whereas it was restored almost to a normal amount in EV+. The NO donor 3-morpholinosydnonimine (SIN-1) reproduced, in normal SR, several abnormal features seen in failing SR, such as defective interdomain interaction and abnormal Ca²⁺leak. Both cell shortening and Ca²⁺transients were impaired by SIN-1 in isolated normal myocytes, mimicking the pathophysiological conditions in failing myocytes. Incubation of failing myocytes with edaravone restored the normal properties.Conclusions—During the development of heart failure, edaravone ameliorated the defective interdomain interaction of the RyR2. This prevented Ca²⁺leak and LV remodeling, leading to an improvement of cardiac function and an attenuation of LV remodeling.

Background— We previously demonstrated that defective interdomain interaction between N-terminal (0 to 600) and central regions (2000 to 2500) of ryanodine receptor 2 (RyR2) induces Ca ²⁺ leak in failing hearts and that K201 (JTV519) inhibits the Ca ²⁺ leak by correcting the defective interdomain interaction. In the present report, we identified the K201-binding domain and characterized the role of this novel domain in the regulation of the RyR2 channel. Methods and Results— An assay using a quartz-crystal microbalance technique (a very sensitive mass-measuring technique) revealed that K201 specifically bound to recombinant RyR2 fragments 1741 to 2270 and 1981 to 2520 but not to other RyR2 fragments from the 1 to 2750 region (1 to 610, 494 to 1000, 741 to 1260, 985 to 1503, 1245 to 1768, 2234 to 2750). By further analysis of the fragment ^1741–2270 , K201 was found to specifically bind to its subfragment ^2114–2149 . With the use of the peptide matching this subfragment (DP ^2114–2149 ) as a carrier, the RyR2 was fluorescently labeled with methylcoumarin acetate (MCA) in a site-directed manner. After tryptic digestion, the major MCA-labeled fragment of RyR2 (155 kDa) was detected by an antibody raised against the central region (Ab ²¹³² ). Moreover, of several recombinant RyR2 fragments, only fragment ^2234–2750 was specifically MCA labeled; this suggests that the K201-binding domain ^2114–2149 binds with domain ^2234–2750 . Addition of DP ^2114–2149 to the MCA-labeled sarcoplasmic reticulum interfered with the interaction between domain ^2114–2149 and domain ^2234–2750 , causing domain unzipping, as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. In failing cardiomyocytes, the frequency of spontaneous Ca ²⁺ spark was markedly increased compared with normal cardiomyocytes, whereas incorporation of DP ^2114–2149 markedly decreased the frequency of spontaneous Ca ²⁺ spark. Conclusions— We first identified the K201-binding site as domain ^2114–2149 of RyR2. Interruption of the interdomain interaction between the domain ^2114–2149 and central domain ^2234–2750 seems to mediate stabilization of RyR2 in failing hearts, which may lead to a novel therapeutic strategy against heart failure and perhaps lethal arrhythmia.

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.