Hiroki Tateishi

Dantrolene, a therapeutic agent for malignant hyperthermia, markedly improves the function of failing cardiomyocytes by stabilizing interdomain interactions within the ryanodine receptor.

OBJECTIVES We sought to investigate the effect of dantrolene, a drug generally used to treat malignant hyperthermia, 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 malignant hyperthermia in skeletal muscle RyR (RyR1) and polymorphic ventricular tachycardia in cardiac RyR (RyR2). There is strong evidence that interdomain interaction between these regions plays an important role in the mechanism of channel regulation. METHODS Sarcoplasmic reticulum vesicles and cardiomyocytes were isolated from the left ventricular muscles of dogs (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 labeled fluorescently with methylcoumarin acetate in a site-directed manner. We used 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 sarcoplasmic reticulum isolated from pacing-induced failing dog hearts, the defective interdomain interaction (domain unzipping) had already occurred, causing spontaneous Ca2+ leak. Dantrolene suppressed both domain unzipping and the Ca2+ leak, demonstrating identical drug concentration-dependence (IC50 = 0.3 micromol/l). In failing cardiomyocytes, both diastolic Ca2+ sparks and delayed afterdepolarization were observed frequently, but 1 micromol/l dantrolene inhibited both events. CONCLUSIONS Dantrolene corrects defective interdomain interactions within RyR2 in failing hearts, inhibits spontaneous Ca2+ leak, and in turn improves cardiomyocyte function in failing hearts. Thus, dantrolene may have a potential to treat heart failure, specifically targeting the RyR2.

Correction of defective interdomain interaction within ryanodine receptor by antioxidant is a new therapeutic strategy against heart failure

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.

Catecholaminergic Polymorphic Ventricular Tachycardia Is Caused by Mutation-Linked Defective Conformational Regulation of the Ryanodine Receptor

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 Ca2+ 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 Ca2+ transients. cAMP-induced aberrant Ca2+ release events (Ca2+ sparks/waves) occurred at much lower sarcoplasmic reticulum Ca2+ 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 [Ca2+] for activation, primarily mediated by defective interdomain interaction within the RyR2.

Defective domain–domain interactions within the ryanodine receptor as a critical cause of diastolic Ca2+ leak in failing hearts

AIMS A domain peptide (DP) matching the Gly(2460)-Pro(2495) 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 Ca(2+) cycling in failing hearts. METHODS AND RESULTS Sarcoplasmic reticulum (SR) vesicles and cardiomyocytes were isolated from dog left ventricular muscles for Ca(2+) 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 Ca(2+) leak and Ca(2+) spark frequency (SpF) were markedly increased in failing cardiomyocytes. Upon introduction of DP163-195 or DP4090-4123 into normal SR or cardiomyocytes, both Ca(2+) 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 Ca(2+) leak and SpF) or those in failing cardiomyocytes, but did not suppress the effects induced by DP4090-4123. CONCLUSION Defective inter-domain interaction between N-terminal and central domains induces diastolic Ca(2+) 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 Ca(2+) leak is similarly induced.

Identification of Target Domains of the Cardiac Ryanodine Receptor to Correct Channel Disorder in Failing Hearts

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 Ca2+ leak in failing hearts and that K201 (JTV519) inhibits the Ca2+ 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 fragment1741–2270, K201 was found to specifically bind to its subfragment2114–2149. With the use of the peptide matching this subfragment (DP2114–2149) 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 (Ab2132). Moreover, of several recombinant RyR2 fragments, only fragment2234–2750 was specifically MCA labeled; this suggests that the K201-binding domain2114–2149 binds with domain2234–2750. Addition of DP2114–2149 to the MCA-labeled sarcoplasmic reticulum interfered with the interaction between domain2114–2149 and domain2234–2750, 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 Ca2+ spark was markedly increased compared with normal cardiomyocytes, whereas incorporation of DP2114–2149 markedly decreased the frequency of spontaneous Ca2+ spark. Conclusions— We first identified the K201-binding site as domain2114–2149 of RyR2. Interruption of the interdomain interaction between the domain2114–2149 and central domain2234–2750 seems to mediate stabilization of RyR2 in failing hearts, which may lead to a novel therapeutic strategy against heart failure and perhaps lethal arrhythmia.

Defective calmodulin binding to the cardiac ryanodine receptor plays a key role in CPVT-associated channel dysfunction.

Calmodulin (CaM), one of the accessory proteins of the cardiac ryanodine receptor (RyR2), is known to play a significant role in the channel regulation of the RyR2. However, the possible involvement of calmodulin in the pathogenic process of catecholaminergic polymorphic ventricular tachycardia (CPVT) has not been investigated. In this study, we investigated the state of RyR2-bound CaM and channel dysfunctions using a knock-in (KI) mouse model with CPVT-linked RyR2 mutation (R2474S). Without added effectors, the affinity of CaM binding to the RyR2 was indistinguishable between KI and WT hearts. In response to cAMP (1 micromol/L), the RyR2 phosphorylation at Ser2808 increased in both WT and KI hearts to the same extent. However, cAMP caused a significant decrease of the CaM-binding affinity in KI hearts, but the affinity was unchanged in WT. Dantrolene restored a normal level of CaM-binding affinity in the cAMP-treated KI hearts, suggesting that defective inter-domain interaction between the N-terminal domain and the central domain of the RyR2 (the target of therapeutic effect of dantrolene) is involved in the cAMP-induced reduction of the CaM-binding affinity. In saponin-permeabilized cardiomyocytes, the addition of cAMP increased the frequency of spontaneous Ca(2+) sparks to a significantly larger extent in KI cardiomyocytes than in WT cardiomyocytes, whereas the addition of a high concentration of CaM attenuated the aberrant increase of Ca(2+) sparks. In conclusion, CPVT mutation causes defective inter-domain interaction, significant reduction in the ability of CaM binding to the RyR2, spontaneous Ca(2+) leak, and then lethal arrhythmia.

Dissociation of calmodulin from cardiac ryanodine receptor causes aberrant Ca2+ release in heart failure

AIMS Calmodulin (CaM) is well known to modulate the channel function of the cardiac ryanodine receptor (RyR2). However, the possible role of CaM on the aberrant Ca(2+) release in diseased hearts remains unclear. In this study, we investigated the state of RyR2-bound CaM and channel dysfunctions in pacing-induced failing hearts. METHODS AND RESULTS The characteristics of CaM binding to RyR2 and the role of CaM on the aberrant Ca(2+) release were assessed in normal and failing canine hearts. The affinity of CaM binding to RyR2 was lower in failing sarcoplasmic reticulum (SR) than in normal SR. Addition of FK506, which dissociates FKBP12.6 from RyR2, to normal SR reduced the CaM-binding affinity. Dantrolene restored a normal level of the CaM-binding affinity in either FK506-treated (normal) SR or failing SR, suggesting that the defective inter-domain interaction between the N-terminal domain and the central domain of RyR2 (the therapeutic target of dantrolene) is involved in the reduction of the CaM-binding affinity in failing hearts. In saponin-permeabilized cardiomyocytes, the frequency of spontaneous Ca(2+) sparks was much more increased in failing cardiomyocytes than in normal cardiomyocytes, whereas the addition of a high concentration of CaM attenuated the aberrant increase of Ca(2+) sparks. CONCLUSION 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.

3D optical coherence tomography: new insights into the process of optimal rewiring of side branches during bifurcational stenting.

AIMS We describe three-dimensional optical coherence tomography (3D-OCT) guided bifurcation stenting and the clinical utility of 3D-OCT. METHODS AND RESULTS Twenty-two consecutive patients who underwent OCT examination to confirm the recrossing position after stent implantation in a bifurcation lesion were enrolled. Frequency domain OCT images were obtained to check the recrossing position and 3D reconstructions were performed off-line. The recrossing position was clearly visualised in 18/22 (81.8%) cases. In 13 cases, serial 3D-OCT could be assessed both before and after final kissing balloon post-dilation (FKBD). We divided these cases into two groups according to the presence of the link between hoops at the carina: free carina type (n=7) and connecting to carina type (n=6). All free carina types complied with the distal rewiring. The percentage of incomplete stent apposition (%ISA) of free carina type at the bifurcation segment after FKBD was significantly smaller than that of the connecting to carina type (0.7±0.9% vs. 12.2±6.5%, p=0.0074). CONCLUSIONS 3D-OCT confirmation of the recrossing into the jailed side branch is feasible during PCI and may help to achieve distal rewiring and favourable stent positioning against the side branch ostium, leading to reduction in ISA and potentially better clinical outcomes.

Mutation-Linked Defective Interdomain Interactions Within Ryanodine Receptor Cause Aberrant Ca2+ Release Leading to Catecholaminergic Polymorphic Ventricular Tachycardia

Background— The molecular mechanism by which catecholaminergic polymorphic ventricular tachycardia is induced by single amino acid mutations within the cardiac ryanodine receptor (RyR2) remains elusive. In the present study, we investigated mutation-induced conformational defects of RyR2 using a knockin mouse model expressing the human catecholaminergic polymorphic ventricular tachycardia–associated RyR2 mutant (S2246L; serine to leucine mutation at the residue 2246). Methods and Results— All knockin mice we examined produced ventricular tachycardia after exercise on a treadmill. cAMP-dependent increase in the frequency of Ca2+ sparks was more pronounced in saponin-permeabilized knockin cardiomyocytes than in wild-type cardiomyocytes. Site-directed fluorescent labeling and quartz microbalance assays of the specific binding of DP2246 (a peptide corresponding to the 2232 to 2266 region: the 2246 domain) showed that DP2246 binds with the K201-binding sequence of RyR2 (1741 to 2270). Introduction of S2246L mutation into the DP2246 increased the affinity of peptide binding. Fluorescence quench assays of interdomain interactions within RyR2 showed that tight interaction of the 2246 domain/K201-binding domain is coupled with domain unzipping of the N-terminal (1 to 600)/central (2000 to 2500) domain pair in an allosteric manner. Dantrolene corrected the mutation-caused domain unzipping of the domain switch and stopped the exercise-induced ventricular tachycardia. Conclusions— The catecholaminergic polymorphic ventricular tachycardia–linked mutation of RyR2, S2246L, causes an abnormally tight local subdomain-subdomain interaction within the central domain involving the mutation site, which induces defective interaction between the N-terminal and central domains. This results in an erroneous activation of Ca2+ channel in a diastolic state reflecting on the increased Ca2+ spark frequency, which then leads to lethal arrhythmia.

A Low-Dose β1-Blocker Effectively and Safely Slows the Heart Rate in Patients with Acute Decompensated Heart Failure and Rapid Atrial Fibrillation

Objective: Recently, we reported that low-dose landiolol (1.5 µg·kg-1·min-1), an ultra-short-acting β-blocker, safely decreased the heart rate (HR) in patients with acute decompensated heart failure (ADHF) and sinus tachycardia, thereby improving cardiac function. We investigated whether low-dose landiolol effectively decreased the HR in ADHF patients with rapid atrial fibrillation (AF). Methods: We enrolled 23 ADHF patients with rapid AF (HR ≥120 beats·min-1 and New York Heart Association class III-IV) and systolic heart failure (SHF: n = 12) or diastolic heart failure (DHF: n = 11) who received conventional therapy with diuretics, vasodilators, and/or low-dose inotropes. They were administered continuous intravenous infusion of low-dose landiolol (1.0-2.0 µg·kg-1·min-1), and their electrocardiograms and blood pressures were monitored for 24 h thereafter. Results: Two hours after starting landiolol, the HR was reduced significantly (22%), without a reduction in blood pressure, and remained constant thereafter. The HR reduction 2 h after landiolol administration was significantly greater in the DHF group than in the SHF group. No incidence of hypotension was recorded. Conclusions: Digitalis or amiodarone is currently recommended for HR control in ADHF patients with rapid AF. Our results showed that continuous infusion of low-dose landiolol may also be useful as first-line therapy in these patients.