RyR2-targeting therapy prevents left ventricular remodeling and ventricular tachycardia in post-infarction heart failure.

BACKGROUND: Dantrolene binds to the Leu601-Cys620 region of the N-terminal domain of cardiac ryanodine receptor (RyR2), which corresponds to the Leu590-Cys609 region of the skeletal ryanodine receptor, and suppresses diastolic Ca2+ leakage through RyR2. OBJECTIVE: We investigated whether the chronic administration of dantrolene prevented left ventricular (LV) remodeling and ventricular tachycardia (VT) after myocardial infarction (MI) by the same mechanism with the mutation V3599K of RyR2, which indicated that the inhibition of diastolic Ca2+ leakage occurred by enhancing the binding affinity of calmodulin (CaM) to RyR2. METHODS AND RESULTS: A left anterior descending coronary artery ligation MI model was developed in mice. Wild-type (WT) were divided into four groups: sham-operated mice (WT-Sham), sham-operated mice treated with dantrolene (WT-Sham-DAN), MI mice (WT-MI), and MI mice treated with dantrolene (WT-MI-DAN). Homozygous V3599K RyR2 knock-in (KI) mice were divided into two groups: sham-operated mice (KI-Sham) and MI mice (KI-MI). The mice were followed for 12 weeks. Survival was significantly higher in the WT-MI-DAN (73%) and KI-MI groups (70%) than the WT-MI group (40%). Echocardiography, pathological tissue, and epinephrine-induced VT studies showed that LV remodeling and VT were prevented in the WT-MI-DAN and KI-MI groups compared to the WT-MI group. An increase in diastolic Ca2+ spark frequency and a decrease in the binding affinity of CaM to the RyR2 were observed at 12 weeks after MI in the WT-MI group, although significant improvements in these values were observed in the WT-MI-DAN and KI-MI groups. CONCLUSIONS: Pharmacological or genetic stabilization of RyR2 tetrameric structure improves survival after MI by suppressing LV remodeling and proarrhythmia.

Stabilizing cardiac ryanodine receptor with dantrolene treatment prevents left ventricular remodeling in pressure-overloaded heart failure mice.

Dantrolene (DAN) directly binds to cardiac ryanodine receptor 2 (RyR2) through Leu601-Cys620 in the N-terminal domain and subsequently inhibits diastolic Ca2+ leakage through RyR2. We previously reported that therapy using RyR2 V3599K mutation, which inhibits diastolic Ca2+ leakage by enhancing calmodulin (CaM) binding ability to RyR2, prevents left ventricular (LV) remodeling in transverse aortic constriction (TAC) heart failure. Here, we examined whether chronic administration of DAN prevents LV remodeling in TAC heart failure via the same mechanism as genetic therapy. A pressure-overloaded hypertrophy mouse model was developed using TAC. Wild-type (WT) mice were divided into three groups: sham-operated mice (Sham group), TAC mice (TAC group), and TAC mice treated with DAN (TAC-DAN group, 20 mg/kg/day, i.p.). They were then followed up for 8 weeks. The survival rate was higher in the TAC-DAN group (83%) than in the TAC group (49%), and serial echocardiography studies and pathological tissue analysis showed that LV remodeling was significantly prevented in the TAC-DAN group compared to the TAC group. An increase in the diastolic Ca2+ spark frequency and a decrease in the binding affinity of CaM to RyR2 were observed at 8 weeks in the TAC group but not in the TAC-DAN group. Stabilization of RyR2 with DAN prevented LV remodeling and improved survival after TAC by enhancing CaM binding to RyR2 and inhibiting RyR2-mediated diastolic Ca2+ leakage.

Endoplasmic reticulum stress promotes nuclear translocation of calmodulin, which activates phenotypic switching of vascular smooth muscle cells.

BACKGROUND AND AIMS: Increased endoplasmic reticulum (ER) stress is strongly associated with the phenotypic switching of vascular smooth muscle cells (VSMCs) in atherosclerosis. Depletion of the ER Ca2+ content is one of the leading causes of increased ER stress in VSMCs. The ryanodine receptor (RyR) is a major Ca2+ release channel in the sarcoplasmic reticulum membrane. Calmodulin (CaM), which binds to RyR (CaM-RyR), stabilizes the closed state of RyR in the resting state in normal cells. Defective CaM-RyR interactions can cause abnormal Ca2+ leakage through RyR, resulting in decreased Ca2+ content, indicating that defective CaM-RyR interactions may be a cause of increased ER stress. Herein, we used a mouse VSMCs to assess whether CaM-RyR plays a pivotal role in VSMCs phenotypic switching, which is caused by ER stress, and whether dantrolene, which enhances the binding affinity of CaM to RyR, affects VSMCs phenotypic switching. METHODS AND RESULTS: Tunicamycin was used to mimic ER stress in vitro. Tunicamycin-induced ER stress caused CaM to dissociate from the RyR and translocate to the nucleus, which stimulated phenotypic switching through the activation of MEF2 and KLF5. Dantrolene suppressed tunicamycin-induced apoptosis, ER stress (restoring ER Ca2+ content), and phenotypic switching of VSMCs. Suramin, which directly unbinds CaM from RyR, promoted nuclear CaM accumulation with parallel VSMCs phenotypic switching, and dantrolene prevented these effects. CONCLUSIONS: We observed that ER stress causes CaM translocation to the nucleus and drives the phenotypic switching of VSMCs. Thus, restoration of the binding affinity of CaM to RyR may be a therapeutic target for atherosclerosis.

Dantrolene reduces platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell proliferation and neointimal formation following vascular injury in mice.

Dantrolene is a ryanodine receptor blocker that is used clinically for treatment of malignant hyperthermia. This study was conducted using murine aortic vascular smooth muscle cells (MOVAS) and a mouse arterial injury model to investigate the inhibitory effect of dantrolene on smooth muscle cell proliferation and migration. We investigated whether dantrolene suppressed platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell proliferation and migration in vitro. The effect of dantrolene on smooth muscle phenotype was evaluated using immunostaining. In addition, smooth muscle cell proliferation and phenotype switching were tested by applying dantrolene around blood vessels using a mouse arterial injury model. Dantrolene inhibited PDGF-induced cell proliferation and migration of MOVAS. Dantrolene also inhibited the switch from contractile to synthetic phenotype both in vitro and in vivo. Dantrolene is effective at inhibiting vascular smooth muscle cell proliferation, migration, and neointimal formation following arterial injury in mice.

Serial changes in the quantitative flow ratio in patients with intermediate residual stenosis after percutaneous coronary intervention.

A beneficial surrogate marker for evaluating the effect of medical therapy is warranted to avoid deferred lesion revascularization. Similar to coronary artery imaging for monitoring the effects of medical therapy by analyzing plaque regression and stabilization, we hypothesized that evaluation of serial changes in the quantitative flow ratio (QFR) would serve as a surrogate marker of the effects of medical therapy against deferred lesion revascularization. Here, we investigated serial changes in QFR over time after percutaneous coronary intervention in patients who underwent medical therapy as a secondary prevention. Patients with intermediate stenosis in an untreated vessel observed at the baseline (BL) coronary angiography and follow-up (FU) coronary angiography performed 6-18 months after BL angiography were screened in 2 centers. A total of 52 patients were able to analyze both BL and FU QFR. The median QFR was 0.83 (IQR, 0.69, 0.89) at BL and 0.80 (IQR, 0.70, 0.86) at FU. The number of positive ΔQFR and negative ΔQFR were 21 and 31, respectively. The median ΔQFR was 0.05 (IQR, 0.03, 0.09) in positive ΔQFR and - 0.05 (IQR, - 0.07, - 0.03) in negative ΔQFR (p < 0.0001). Univariate and multivariate analyses revealed that LDL-C at FU predicted improvement in the QFR (OR 0.95, 95% confidence interval [0.91, 0.98], P = 0.001). Assessment of serial changes in the QFR may serve as a surrogate marker for the effects of medical therapy in patients with residual intermediate coronary stenosis.

Inflammation and NLRP3 Inflammasome Activation Initiated in Response to Pressure Overload by Ca2+/Calmodulin-Dependent Protein Kinase II δ Signaling in Cardiomyocytes Are Essential for Adverse Cardiac Remodeling.

BACKGROUND: Inflammation is associated with cardiac remodeling and heart failure, but how it is initiated in response to nonischemic interventions in the absence of cell death is not known. We tested the hypothesis that activation of Ca2+/calmodulin-dependent protein kinase II δ (CaMKIIδ) in cardiomyocytes (CMs) in response to pressure overload elicits inflammatory responses leading to adverse remodeling. METHODS: Mice in which CaMKIIδ was selectively deleted from CMs (cardiac-specific knockout [CKO]) and floxed control mice were subjected to transverse aortic constriction (TAC). The effects of CM-specific CaMKIIδ deletion on inflammatory gene expression, inflammasome activation, macrophage accumulation, and fibrosis were assessed by quantitative polymerase chain reaction, histochemistry, and ventricular remodeling by echocardiography. RESULTS: TAC induced increases in cardiac mRNA levels for proinflammatory chemokines and cytokines in ≤3 days, and these responses were significantly blunted when CM CaMKIIδ was deleted. Apoptotic and necrotic cell death were absent at this time. CMs isolated from TAC hearts mirrored these robust increases in gene expression, which were markedly attenuated in CKO. Priming and activation of the NOD-like receptor pyrin domain-containing protein 3 inflammasome, assessed by measuring interleukin-1ß and NOD-like receptor pyrin domain-containing protein 3 mRNA levels, caspase-1 activity, and interleukin-18 cleavage, were increased at day 3 after TAC in control hearts and in CMs isolated from these hearts. These responses were dependent on CaMKIIδ and associated with activation of Nuclear Factor-kappa B and reactive oxygen species. Accumulation of macrophages observed at days 7 to 14 after TAC was diminished in CKO and, by blocking Monocyte Chemotactic Protein-1 signaling, deletion of CM Monocyte Chemotactic Protein-1 or inhibition of inflammasome activation. Fibrosis was also attenuated by these interventions and in the CKO heart. Ventricular dilation and contractile dysfunction observed at day 42 after TAC were diminished in the CKO. Inhibition of CaMKII, Nuclear Factor-kappa B, inflammasome, or Monocyte Chemotactic Protein-1 signaling in the first 1 or 2 weeks after TAC decreased remodeling, but inhibition of CaMKII after 2 weeks did not. CONCLUSIONS: Activation of CaMKIIδ in response to pressure overload triggers inflammatory gene expression and activation of the NOD-like receptor pyrin domain-containing protein 3 inflammasome in CMs. These responses provide signals for macrophage recruitment, fibrosis, and myocardial dysfunction in the heart. Our work suggests the importance of targeting early inflammatory responses induced by CM CaMKIIδ signaling to prevent progression to heart failure.

Inflammation in nonischemic heart disease: initiation by cardiomyocyte CaMKII and NLRP3 inflammasome signaling.

There is substantial evidence that chronic heart failure in humans and in animal models is associated with inflammation. Ischemic interventions such as myocardial infarction lead to necrotic cell death and release of damage associated molecular patterns, factors that signal cell damage and induce expression of proinflammatory chemokines and cytokines. It has recently become evident that nonischemic interventions are also associated with increases in inflammatory genes and immune cell accumulation in the heart and that these contribute to fibrosis and ventricular dysfunction. How proinflammatory responses are elicited in nonischemic heart disease which is not, at least initially, associated with cell death is a critical unanswered question. In this review we provide evidence supporting the hypothesis that cardiomyocytes are an initiating site of inflammatory gene expression in response to nonischemic stress. Furthermore we discuss the role of the multifunctional Ca2+/calmodulin-regulated kinase, CaMKIIδ, as a transducer of stress signals to nuclear factor-κB activation, expression of proinflammatory cytokines and chemokines, and priming and activation of the NOD-like pyrin domain-containing protein 3 (NLRP3) inflammasome in cardiomyocytes. We summarize recent evidence that subsequent macrophage recruitment, fibrosis and contractile dysfunction induced by angiotensin II infusion or transverse aortic constriction are ameliorated by blockade of CaMKII, of monocyte chemoattractant protein-1/C-C chemokine receptor type 2 signaling, or of NLRP3 inflammasome activation.

CaMKIIδ subtypes differentially regulate infarct formation following ex vivo myocardial ischemia/reperfusion through NF-κB and TNF-α.

Deletion of Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) has been shown to protect against in vivo ischemia/reperfusion (I/R) injury. It remains unclear which CaMKIIδ isoforms and downstream mechanisms are responsible for the salutary effects of CaMKIIδ gene deletion. In this study we sought to compare the roles of the CaMKIIδB and CaMKIIδC subtypes and the mechanisms by which they contribute to ex vivo I/R damage. WT, CaMKIIδKO, and mice expressing only CaMKIIδB or δC were subjected to ex vivo global ischemia for 25min followed by reperfusion. Infarct formation was assessed at 60min reperfusion by triphenyl tetrazolium chloride (TTC) staining. Deletion of CaMKIIδ conferred significant protection from ex vivo I/R. Re-expression of CaMKIIδC in the CaMKIIδKO background reversed this effect and exacerbated myocardial damage and dysfunction following I/R, while re-expression of CaMKIIδB was protective. Selective activation of CaMKIIδC in response to I/R was evident in a subcellular fraction enriched for cytosolic/membrane proteins. Further studies demonstrated differential regulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling and tumor necrosis factor alpha (TNF-α) expression by CaMKIIδB and CaMKIIδC. Selective activation of CaMKIIδC was also observed and associated with NF-κB activation in neonatal rat ventricular myocytes (NRVMs) subjected to oxidative stress. Pharmacological inhibition of NF-κB or TNF-α significantly ameliorated infarct formation in WT mice and those that re-express CaMKIIδC, demonstrating distinct roles for CaMKIIδ subtypes in I/R and implicating acute activation of CaMKIIδC and NF-κB in the pathogenesis of reperfusion injury.

Differences of side branch jailing between left main-left anterior descending artery stenting and left main-left circumflex artery stenting with Nobori biolimus-eluting stent.

The aim of this study is to indicate differences of side branch jailing between the left main (LM)-left anterior descending artery (LAD) stenting and the LM-left circumflex artery (LCx) stenting. Thirty-one patients who underwent single-stenting using a two-link ten-crowns biolimus-eluting stent (Japanese design of BES, J-BES) and subsequent kissing balloon dilation (KBD) on an LM bifurcation with optical coherence tomography (OCT) were divided into two groups according to the stented vessel. Bifurcation angles were measured by three-dimensional (3D) quantitative coronary analysis. The jailing pattern on a side branch ostium was evaluated by stent-enhanced 3D-OCT. Incomplete stent apposition (ISA) after KBD was compared between the stented vessels. The to-be-stented angle of the LM-LCx stenting (n = 11) was significantly steeper than that of the LM-LAD stenting (n = 20) (132.6° ± 16.9° vs. 150.7° ± 10.6°, p < 0.01). The incidence of the free carina type, which has no stent links bridging from a carina, in the LM-LCx stenting was significantly higher than that in the LM-LAD stenting (90.9 vs. 45.0 %, p = 0.02). The percentage of ISA at the bifurcation segment in the LM-LCx stenting was significantly smaller than that in the LM-LAD stenting (4.4 ± 8.2 vs. 12.7 ± 9.2 %, p = 0.0003). This study showed, by higher incidence of the favorable configuration, that the LM-LCx stenting achieved a smaller percentage of ISA than the LM-LAD stenting. These insights may help guide LM bifurcation stenting with J-BES.

Flow Grade-Based Success Rates, Complication Rates, and Balloon Pulmonary Angioplasty Patency for Total Occlusions.

BACKGROUND: The number of successfully recanalized total occlusions affects hemodynamic improvement after balloon pulmonary angioplasty (BPA) in patients with chronic thromboembolic pulmonary hypertension (CTEPH). We aimed to clarify the current efficacy, patency, and success rate of BPA for total occlusions. METHODS: Between April 2016 and August 2021, 178 BPAs were performed in 100 patients with CTEPH and total occlusions. The primary success and subsequent patency rates immediately before the second BPA procedure (follow-up) were compared between the segmental and subsegmental groups, based on the flow grade, which was defined as follows: 0, no reperfusion; 1, minimal reperfusion; 2, partial reperfusion; and 3, complete reperfusion. RESULTS: Total occlusions were mainly located in the right lung (70%) and lower lobes (48%). The primary success rate was 88%, with significant improvements in oxygenation, hemodynamic parameters, and 6-minute walk test. The primary flow grade did not differ between groups. However, the proportion of lesions with a flow grade of 2 or 3 at follow-up was significantly higher in the subsegmental group than in the segmental group (84% vs 45%, respectively; P < 0.01). In multivariate analysis, flow grade in the acute phase (odds ratio [OR], 46.9; 95% confidence interval [CI], 12.54-176.78; P < 0.01) and subsegmental lesions (OR, 13.8; 95% CI, 3.24-58.94; P < 0.01) were independently associated with better patency (flow grade of 2 or 3) at follow-up. CONCLUSIONS: Total occlusions can be safely and effectively treated with BPA. BPA for total occlusions may be preferable for subsegmental over segmental lesions.

Dantrolene improves left ventricular diastolic property in mineralcorticoid-salt-induced hypertensive rats.

Left ventricular (LV) diastolic dysfunction is increasingly common in heart failure with preserved ejection fraction (HFpEF), and new drug therapy is desired. We recently reported that dantrolene (DAN) attenuates pressure-overload induced hypertrophic signaling through stabilization of tetrameric structure of cardiac ryanodine receptor (RyR2). Because cardiac hypertrophy substantially affects LV diastolic properties, we investigated the effect of DAN on LV diastolic properties in mineralocorticoid-salt-induced hypertensive rat model exhibiting the HFpEF phenotype. Male Sprague-Dawley (SD) rats (8 weeks old) received an uninephrectomy (UNX), subcutaneous implantation of a 200 mg pellet of deoxycorticosterone acetate (DOCA), and 0.9% NaCl water (UNX + DOCA-salt). UNX, a control pellet, and water without NaCl served as controls (UNX control). The effect of oral administration of 100 mg/kg/d DAN was examined in UNX control and UNX + DOCA-salt groups (UNX + DAN and UNX + DOCA-salt + DAN). UNX + DOCA-salt treatment resulted in mild hypertension. Chronic administration of DAN to UNX + DOCA-salt rats (UNX + DOCA-salt + DAN) did not affect blood pressure. DAN treatment increased the mitral annular early relaxation velocity in the UNX + DOCA-salt group. The size of cardiomyocytes increased in the UNX + DOCA-salt group, whereas the increase was suppressed by DAN treatment. LV fibrotic area was significantly smaller in the UNX + DOCA-salt + DAN group than in the UNX + DOCA-salt group (2.0 ± 0.2% vs 4.0 ± 0.4%). The LV chamber stiffness significantly increased in the UNX + DOCA-salt group, whereas the increase was suppressed by DAN treatment. DAN treatment normalized the CaM-RyR2 interaction and inhibited aberrant Ca2+ release. DAN improved left ventricular diastolic properties with respect to both myocardial relaxation and chamber stiffness. DAN may be a new treatment option for HFpEF.

Factors Influencing Acceptance of the Chairperson Position at Annual Scientific Meetings of the Japanese Circulation Society　- A Questionnaire Survey in Chugoku District.

Background: The Japanese Circulation Society survey revealed that Japanese female cardiologists exhibited a trend to refuse the chairperson position; however, the causal factors remain uncertain. Methods and Results: We distributed a questionnaire survey among chairpersons of the Chugoku regional meeting in November 2022. The rate of chair acceptance at the annual meeting tended to increase as the chairperson's experience grew (first time chairing a meeting, 25.0%; 2-3 times, 33.3%; 4-5 times, 53.8%; ≥6 times, 70.0%; P=0.021). Conclusions: Providing inexperienced members with the chance to perform the role of chairperson will lead to them accepting to chair annual meetings.

Mutation-linked defective interdomain interactions within ryanodine receptor cause aberrant Ca²âºrelease 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 Ca²âº 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 Ca²âº channel in a diastolic state reflecting on the increased Ca²âº spark frequency, which then leads to lethal arrhythmia.

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 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.

Balloon pulmonary angioplasty for chronic thromboembolic pulmonary hypertension concomitant with Klippel-Trenaunay-Weber syndrome.

Klippel-Trenaunay-Weber syndrome (KTWS) is a rare congenital disorder characterized by cutaneous capillary malformations, bone hypertrophy, and multiple venous or lymphatic malformations. KTWS is associated with chronic thromboembolic pulmonary hypertension (CTEPH), presumably due to thromboembolism from multiple vascular malformations. Here, we report the first case series of patients with KTWS-CTEPH who underwent balloon pulmonary angioplasty (BPA). Both patients are alive 20 years and 1 year after the initial diagnosis of CTEPH, respectively, and are stable with improved hemodynamics. BPA may be an effective treatment option for patients with KTWS-CTEPH.

Stabilization of RyR2 maintains right ventricular function, reduces the development of ventricular arrhythmias, and improves prognosis in pulmonary hypertension.

BACKGROUND: Right ventricular (RV) dysfunction and its associated arrhythmias are recognized as important determinants of the prognosis of pulmonary arterial hypertension (PAH). OBJECTIVE: Here, we aimed to investigate whether direct pharmacological intervention in the RV muscle with dantrolene (DAN), a stabilizer of the cardiac ryanodine receptor (RyR2), has a protective effect against RV dysfunction and arrhythmia in a monocrotaline (MCT)-induced PAH rat model. METHODS: Male 8-week-old Sprague-Dawley rats were injected with MCT for the induction of PAH. Induction of ventricular tachycardia (VT) by catecholamines was also evaluated in association with RyR2-mediated Ca2+ release properties in isolated cardiomyocytes. A pulmonary artery-banding model has also been established to assess the independent effects of chronic pressure overload on RV morphology and function. RESULTS: In the MCT-induced PAH rat model, RV hypertrophy, dilation, and functional decline were observed, with a survival rate of 0% 2 months after MCT induction. In contrast, chronic DAN treatment improved all these RV parameters and increased survival by 80%. Chronic DAN treatment also prevented the dissociation of calmodulin from RyR2, thereby inhibiting Ca2+ sparks and spontaneous Ca2+ transients in MCT-induced hypertrophied RV cardiomyocytes. Epinephrine induced VT in more than 50% of rats with MCT-induced PAH, but complete suppression of VT was achieved by chronic DAN treatment. CONCLUSION: Stabilization of RyR2 by DAN has potential as a new therapeutic agent against the development of RV dysfunction and fatal arrhythmia associated with PAH.

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.

Dantrolene, a therapeutic agent for malignant hyperthermia, inhibits catecholaminergic polymorphic ventricular tachycardia in a RyR2(R2474S/+) knock-in mouse 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. METHODS AND RESULTS: ECG was monitored in KI mice (n=6) and wild-type (WT) mice (n=6), before and after an injection of epinephrine (1.0mg/kg) or on exercise using a treadmill. In all KI (but not WT) mice, bi-directional ventricular tachycardia (VT) was induced after an injection of epinephrine or on exercise. Pre-treatment with dantrolene (for 7-10 days) significantly inhibited the inducible VT (P<0.01). In KI cardiomyocytes, Ca(2+) spark frequency (SpF; s(-1)·100µm(-1): 5.8±0.3, P<0.01) was much more increased after the addition of isoproterenol than in WT cardiomyocytes (SpF: 3.6±0.2). The increase in SpF seen in KI cardiomyocytes was attenuated by 1.0µmol/L dantrolene (SpF: 3.6±0.5, P<0.01). CONCLUSIONS: Dantrolene prevents CPVT, presumably by inhibiting Ca(2+) leak through the RyR2.

CaMKIIδ-mediated inflammatory gene expression and inflammasome activation in cardiomyocytes initiate inflammation and induce fibrosis.

Inflammation accompanies heart failure and is a mediator of cardiac fibrosis. CaMKIIδ plays an essential role in adverse remodeling and decompensation to heart failure. We postulated that inflammation is the mechanism by which CaMKIIδ contributes to adverse remodeling in response to nonischemic interventions. We demonstrate that deletion of CaMKIIδ in the cardiomyocyte (CKO) significantly attenuates activation of NF-κB, expression of inflammatory chemokines and cytokines, and macrophage accumulation induced by angiotensin II (Ang II) infusion. The inflammasome was activated by Ang II, and this response was also diminished in CKO mice. These events occurred prior to any evidence of Ang II-induced cell death. In addition, CaMKII-dependent inflammatory gene expression and inflammasome priming were observed as early as the third hour of infusion, a time point at which macrophage recruitment was not evident. Inhibition of either the inflammasome or monocyte chemoattractant protein 1 (MCP1) signaling attenuated macrophage accumulation, and these interventions, like cardiomyocyte CaMKIIδ deletion, diminished the fibrotic response to Ang II. Thus, activation of CaMKIIδ in the cardiomyocyte represents what we believe to be a novel mechanism for initiating inflammasome activation and an inflammatory gene program that leads to macrophage recruitment and ultimately to development of fibrosis.