Objectives To investigate the effect of dantrolene, a drug generally used to treat Malignant Hyperthermia (MH), on the Ca2+ release and cardiomyocyte function in failing hearts. Background The N-terminal (N: 1-600) and Central (C: 2000-2500) domains of the ryanodine receptor (RyR), harbor many mutations associated with MH in skeletal muscle RyR (RyR1) and polymorphic ventricular tachycardia in cardiac RyR (RyR2). There is strong evidence that inter-domain interaction between these regions plays an important role in the mechanism of channel regulation. Methods Sarcoplasmic reticulum (SR) vesicles and cardiomyocytes were isolated from dog LV muscles (normal or rapid ventricular pacing for 4 weeks), for Ca2+ leak, transient, and spark assays. To assess the zipped or unzipped state of the interacting domains, the RyR was fluorescently labeled with methylcoumarin acetate in a site-directed manner. We employed a quartz-crystal microbalance technique to identify the dantrolene binding site within the RyR2. Results Dantrolene specifically bound to domain 601-620 in RyR2. In the SR isolated from pacing-induced dog failing hearts, the defective inter-domain interaction_(domain unzipping) has already occurred, causing spontaneous Ca2+ leak. Dantrolene suppressed both domain unzipping and the Ca2+ leak, showing identical drug concentration-dependence (IC50=0.3 μmol/L). In failing cardiomyocytes, both diastolic Ca2+ sparks and delayed afterdepolarization were frequently observed, but 1 μmol/L dantrolene inhibited both events. Conclusions Dantrolene corrects defective inter-domain interactions within RyR2 in failing hearts, inhibits spontaneous Ca2+ leak, in turn improves cardiomyocyte function in failing hearts. Thus, dantrolene may have a potential to treat heart failure, specifically targeting the RyR2.

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 Key Words: ryanodine receptor Ⅲ calcium Ⅲ ventricular tachycardia Ⅲ sarcoplasmic reticulum T o date, more than 70 cardiac ryanodine receptor (RyR)2 missense mutations have been identified that are linked with 2 inherited forms of sudden cardiac death: catecholaminergic polymorphic ventricular tachycardia (CPVT) 1 and arrhythmogenic right ventricular cardiomyopathy type 2. 1 These mutations cluster in 3 well-defined regions of the RyR2 that correspond to malignant hyperthermia or the central core disease mutable regions, designated as the N-terminal domain (amino acids 1 to 600), central domain (amino acids 2000 to 2500), and the C-terminal transmembrane channel domain of the skeletal muscle-type ryanodine receptor (RyR1). 1 This suggests that the RyR2 shares a common domain-mediated channel regulation mechanism with RyR1. Mutations at different positions in each of these domains result in the nearly identical phenotype of channel dysfunctions such as hyperactivation of the Ca 2ϩ channel and hypersensitization to agonists. To account for these phenomena, Ikemoto et al 2,3 proposed the so-called "domain switch hypothesis" and stated that in the resting or nonactivated state, the N-terminal domain and the central domain make close contact at several subdomains (domain zipping). Then, on physiological or pharmacological stimulation, these critical interdomain contacts are weakened, resulting in the loss of conformational constraints (domain unzipping), thus lowering the energy barrier for Ca 2ϩ channel opening. Consistent with this hypothesis, single particle analysis of the 3D structure of the RyR2 molecule revealed that the N-terminal and central domains (located in domains 5 and 6 of the so-called clamp Original received September 15, 2009; revision received February 9, 2010; accepted February 26, 2010 region, respectively) are in a close apposition ...

Rationale Calmodulin (CaM) associates with cardiac ryanodine receptors (RyR2) as an important regulator. Defective CaM-RyR2 interaction may occur in heart failure (HF), cardiac hypertrophy, and catecholaminergic polymorphic ventricular tachycardia (CPVT). However, the in situ binding properties for CaM-RyR2 are unknown. Objective We sought to measure the in situ binding affinity and kinetics for CaM-RyR2 in normal and HF ventricular myocytes, estimate the percentage of Z-line localized CaM that is RyR2-bound and test cellular function of defective CaM-RyR2 interaction. Methods & Results Using FRET (fluorescence resonance energy transfer) in permeabilized myocytes, we specifically resolved RyR2-bound CaM from other potential binding targets, and measured CaMRyR2 binding affinity in situ (Kd =10-20 nM). Using RyR2ADA/+ knock-in (KI) mice, in which half of the CaM-RyR2 binding is suppressed, we estimated that >90% of Z-line CaM is RyR2-bound. Functional tests indicated a higher propensity for Ca2+ waves production and stress induced ventricular arrhythmia in RyR2ADA/+ mice. In a post myocardial infarction (MI) rat HF model, we detected a decrease in the CaMRyR2 binding affinity (Kd ≈ 51nM, ~3 fold increase) and unaltered FKBP12.6-RyR2 binding affinity (Kd ≈ 0.8nM). Conclusions CaM binds to RyR2 with high affinity in cardiac myocytes. Physiologically, CaM is bound to >70% of RyR2 monomers and inhibits SR Ca2+ release. RyR2 is the major binding site for CaM along the Z-line in cardiomyocytes and dissociating CaM from RyR2 can cause severe ventricular arrhythmia. In HF, RyR2 shows decreased CaM affinity, but unaltered FKBP12.6 affinity.

Oxidative stress may contribute to cardiac ryanodine receptor (RyR2) dysfunction in heart failure (HF) and arrhythmias. Altered RyR2 domain-domain interaction (domain unzipping) and calmodulin (CaM) binding affinity are allosterically coupled indices of RyR2 conformation. In HF RyR2 exhibits reduced CaM binding, increased domain unzipping and greater SR Ca leak, and dantrolene can reverse these changes. However, effects of oxidative stress on RyR2 conformation and leak in myocytes are poorly understood. We used fluorescent CaM, FKBP12.6, and domain-peptide biosensor (F-DPc10) to measure, directly in cardiac myocytes, (1) RyR2 activation by hydrogen peroxide (H2O2)-induced oxidation, (2) RyR2 conformation change caused by oxidation, (3) CaM-RyR2 and FK506-binding protein (FKBP12.6)-RyR2 interaction upon oxidation, and (4) whether dantrolene affects 1–3. H2O2 was used to mimic oxidative stress. H2O2 significantly increased the frequency of Ca2+ sparks and spontaneous Ca2+ waves, and dantrolene almost completely blocked these effects. H2O2 pretreatment significantly reduced CaM-RyR2 binding, but had no effect on FKBP12.6-RyR2 binding. Dantrolene restored CaM-RyR2 binding but had no effect on intracellular and RyR2 oxidation levels. H2O2 also accelerated F-DPc10-RyR2 association while dantrolene slowed it. Thus, H2O2 causes conformational changes (sensed by CaM and DPc10 binding) associated with Ca leak, and dantrolene reverses these RyR2 effects. In conclusion, in cardiomyocytes, H2O2 treatment markedly reduces the CaM-RyR2 affinity, has no effect on FKBP12.6-RyR2 affinity, and causes domain unzipping. Dantrolene can correct domain unzipping, restore CaM-RyR2 affinity, and quiet pathological RyR2 channel gating. F-DPc10 and CaM are useful biosensors of a pathophysiological RyR2 state.

Circulation Journal Official Journal of the Japanese Circulation Society http://www. j-circ.or.jp he N-terminal (1-619 amino acid) and central (2,000-2,500 amino acid) domains of the ryanodine receptors (skeletal: RyR1, cardiac: RyR2) harbor many mutations associated with malignant hyperthermia (MH), catecholaminergic polymorphic ventricular tachycardia (CPVT), and arrhythmogenic right ventricular cardiomyopathy type 2. 1 There is strong evidence to suggest that the interdomain interaction between these regions plays an important role in the mechanism of channel regulation. 2- 16 We reported that dantrolene, a specific agent for the treatment of MH, prevented abnormal Ca 2+ leak by correction of the defective inter-domain interaction between the N-terminal and central domains within MH RyR1 (i.e., aberrant formation of a channel-activating unzipped configuration of the N-terminal/central domain pair in an otherwise resting state). 9 We further showed that, in failing hearts, dantrolene corrected the defective inter-domain interaction within the RyR2, thereby inhibiting Ca 2+ leak through RyR2. 11 More recently, by using the knock-in (KI) mouse model with a human CPVT-associated RyR2 mutation (R2474S), we clarified that a single amino acid mutation within the RyR2 sensitizes the RyR2 channel to activation by luminal [Ca 2+ ] (i.e., a decreased threshold of luminal [Ca 2+ ] for channel activation), and in turn induces spontaneous Ca 2+ sparks and DAD, leading to CPVT, and that danrrolene stabilized the leaky RyR2 by correcting the defective inter-domain interaction. 13 Here, we investigated the in vivo anti-arrhythmic effect of dantrolene in the KI mice model. Background: Dantrolene, a specific agent for the treatment of malignant hyperthermia, was found to inhibit Ca 2+ leak through not only the skeletal ryanodine receptor (RyR1), but also the cardiac ryanodine receptor (RyR2) by correcting the defective inter-domain interaction between N-terminal (1-619 amino acid) and central (2,000-2,500 amino acid) domains of RyRs. Here, the in vivo anti-arrhythmic effect of dantrolene in a human catecholaminergic polymorphic ventricular tachycardia (CPVT)-associated RyR2 R2474S/+ knock-in (KI) mouse model was investigated.

The defective inter-domain interaction between N-terminal and central domains within RyR2 reduces the binding affinity of CaM to RyR2, thereby causing the spontaneous Ca(2+) release events in failing hearts. Correction of the defective CaM binding may be a new strategy to protect against the aberrant Ca(2+) release in heart failure.

Background: CaMKII␦ and NO can modulate cardiac signaling/pathology. Results: NO treatment after calcium/calmodulin binding prolongs CaMKII␦ activation, whereas NO pretreatment inhibits CaMKII␦ activation, effects mediated by Cys-290 and Cys-273, respectively. Conclusion: S-nitrosylation has a dual role in modulating CaMKII␦ in the heart. Significance: Dual regulation by NO is a new pathway by which CaMKII can modulate cardiac function.

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.