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.

Dantrolene, a stabilizer of the ryanodine receptor, prevents collagen-induced arthritis.

Dantrolene inhibits Ca2+ leakage from destabilized ryanodine receptors and therefore may serve as a therapeutic agent against endoplasmic reticulum stress-associated diseases. However, its effectiveness in treating autoimmune diseases remains unclear. Here, we investigated the effect of dantrolene on collagen-induced arthritis (CIA) in mice. Oral administration of dantrolene resulted in significantly lower arthritic scores in both male and female CIA mice than in the control mice. Micro-computed tomographic and histological analyses showed that dantrolene suppressed bone and chondral destruction. The serum levels of anti-type II collagen (CII) IgG were positively correlated with the arthritic scores (r = 0.704, p < 0.01). In addition, the serum levels of anti-CII IgG were significantly lower in the dantrolene group than in the control group (p < 0.05). These results demonstrate that oral administration of dantrolene to CIA mice inhibits the production of serum anti-CII IgG and consequently prevents arthritis. Therefore, dantrolene may be a potential anti-rheumatic drug.

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.

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.

Increasing SERCA function promotes initiation of calcium sparks and breakup of calcium waves.

KEY POINTS: Increasing sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump activity enhances sarcoplasmic reticulum calcium (Ca) load, which increases both ryanodine receptor opening and driving force of Ca release flux. Both of these effects promote Ca spark formation and wave propagation. However, increasing SERCA activity also accelerates local cytosolic Ca decay as the wave front travels to the next cluster, which limits wave propagation. As a result, increasing SERCA pump activity has a biphasic effect on the propensity of arrhythmogenic Ca waves, but a monotonic effect to increase Ca spark frequency and amplitude. ABSTRACT: Waves of sarcoplasmic reticulum (SR) calcium (Ca) release can cause arrhythmogenic afterdepolarizations in cardiac myocytes. Ca waves propagate when Ca sparks at one Ca release unit (CRU) recruit new Ca sparks in neighbouring CRUs. Under normal conditions, Ca sparks are too small to recruit neighbouring Ca sparks where Ca sensitivity is also low. However, under pathological conditions such as a Ca overload or ryanodine receptor (RyR) sensitization, Ca sparks can be larger and propagate more readily as macro-sparks or full Ca waves. Increasing SERCA pump activity promotes SR Ca load, which promotes RyR opening and increases driving force of the Ca release flux from SR to cytosol, promoting Ca waves. However, high sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) activity can also decrease local cytosolic [Ca] as it approaches the next CRU, thereby reducing wave appearance and propagation. In this study, we use a physiologically detailed model of subcellular Ca cycling and experiments in phospholamban-knockout mice, to show how Ca waves are initiated and propagate and how different conditions contribute to the generation and propagation of Ca waves. We show that reducing diffusive coupling between Ca sparks by increasing SERCA activity prevents Ca waves by reducing [Ca] at the next CRU, as do Ca buffers, low intra-SR Ca diffusion and distance between CRUs. Increasing SR Ca uptake rate has a biphasic effect on Ca wave propagation; initially it enhances Ca spark probability and amplitude and CRU coupling, thereby promoting arrhythmogenic Ca wave propagation, but at higher levels SR Ca uptake can abort those arrhythmogenic Ca waves.

Stabilizing cardiac ryanodine receptor prevents the development of cardiac dysfunction and lethal arrhythmia in Ca2+/calmodulin-dependent protein kinase IIδc transgenic mice.

AIMS: Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been shown to induce aberrant Ca2+ release from the cardiac ryanodine receptor (RyR2) in various diseased hearts. However, the precise pathogenic mechanism remains to be elucidated. Here, we investigated the effect of dantrolene (DAN): a RyR2 stabilizer on local Ca2+ release, cardiac function, and lethal arrhythmia in CaMKIIδc transgenic (TG) mice. METHODS AND RESULTS: The TG mice showed an increase in left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) with a reduction in LV fractional shortening (LVFS). The phosphorylation levels of Ser2814 in RyR2 and Thr287 in CaMKII increased in TG mice. In TG cardiomyocytes, peak cell shortening (CS) decreased, and the frequency of spontaneous Ca2+ transients (sCaTs) increased. Endogenous RyR2-associated calmodulin (CaM) markedly decreased in TG cardiomyocytes. After chronic DAN treatment for 1 month, LVESD (but not LVEDD) decreased with an increase in LVFS. In the chronic DAN-treated cardiomyocytes, CS increased, sCaTs decreased, and the endogenous CaM binding to RyR2 normally restored. The phosphorylation levels of Ser2814 in RyR2 and Thr287 in CaMKII remained elevated even after DAN treatment. Moreover, in TG mice, chronic DAN treatment prevented sustained ventricular tachycardia induced by epinephrine. CONCLUSIONS: Defective association of CaM with RyR2 is most likely to be involved in the pathogenesis of CaMKII-mediated cardiac dysfunction and lethal arrhythmia.

Dantrolene prevents ventricular tachycardia by stabilizing the ryanodine receptor in pressure- overload induced failing hearts.

Aberrant Ca2+ release from cardiac ryanodine receptors (RyR2) has been shown to be one of the most important causes of lethal arrhythmia in various types of failing hearts. We previously showed that dantrolene, a specific agent for the treatment of malignant hyperthermia, inhibits Ca2+ leakage from the RyR2 by correcting the defective inter-domain interaction between the N-terminal (1-619 amino acids) and central (2000-2500 amino acids) domains of the RyR2 and allosterically enhancing the binding affinity of calmodulin to the RyR2 in diseased hearts. In this study, we examined whether dantrolene inhibits this Ca2+ leakage, thereby preventing the pharmacologically inducible ventricular tachycardia in ventricular pressure-overloaded failing hearts. Ventricular tachycardia (VT) was easily induced after an injection of epinephrine in mice after 8 weeks of transverse aortic constriction-induced pressure-overload. Pretreatment with dantrolene almost completely inhibited the pharmacologically inducible VT. In the presence of dantrolene, the occurrence of both Ca2+ sparks and spontaneous Ca2+ transients was inhibited, which was associated with enhanced calmodulin binding affinity to the RyR2. These results suggest that dantrolene could be a new potent agent in the treatment of lethal arrhythmia in cases of acquired heart failure.

Addition of a ß1-Blocker to Milrinone Treatment Improves Cardiac Function in Patients with Acute Heart Failure and Rapid Atrial Fibrillation.

BACKGROUND: Tachycardia worsens cardiac performance in acute decompensated heart failure (ADHF). We investigated whether heart rate (HR) optimization by landiolol, an ultra-short-acting ß1-selective blocker, in combination with milrinone improved cardiac function in patients with ADHF and rapid atrial fibrillation (AF). METHODS AND RESULTS: We enrolled9 ADHF patients (New York Heart Association classification IV; HR, 138 ± 18 bpm; left ventricular [LV] ejection fraction, 28 ± 8%; cardiac index [CI], 2.1 ± 0.3 L/min-1/m-2; pulmonary capillary wedge pressure [PCWP], 24 ± 3 mm Hg), whose HRs could not be reduced using standard treatments, including diuretics, vasodilators, and milrinone. Landiolol (1.5-6.0 µg/kg-1/min-1, intravenous) was added to milrinone treatment to study its effect on hemodynamics. The addition of landiolol (1.5 µg/kg-1/min-1) significantly reduced HR by 11% without changing systolic blood pressure (BP) and resulted in a significant decrease in PCWP and a significant increase in stroke volume index (SVI), suggesting that HR reduction restores incomplete LV relaxation. Administration of more than 3.0 µg/kg-1/min-1 of landiolol decreased BP, CI, and SVI. CONCLUSION: The addition of landiolol at doses of <3.0 µg/kg/min to milrinone improved cardiac function in decompensated chronic heart failure with rapid atrial fibrillation by selectively reducing HR.

Nuclear translocation of calmodulin in pathological cardiac hypertrophy originates from ryanodine receptor bound calmodulin.

In cardiac myocytes Calmodulin (CaM) bound to the ryanodine receptor (RyR2) constitutes a large pool of total myocyte CaM, but the CaM-RyR2 affinity is reduced in pathological conditions. Knock-in mice expressing RyR2 unable to bind CaM also developed hypertrophy and early death. However, it is unknown whether CaM released from this RyR2-bound pool participates in pathological cardiac hypertrophy. We found that angiotensin II (AngII) or phenylephrine (PE) both cause CaM to dissociate from the RyR2 and translocate to the nucleus. To test whether this nuclear CaM accumulation depends on CaM released from RyR2, we enhanced CaM-RyR2 binding affinity (with dantrolene), or caused CaM dissociation from RyR2 (using suramin). Dantrolene dramatically reduced AngII- and PE-induced nuclear CaM accumulation. Conversely, suramin enhanced nuclear CaM accumulation. This is consistent with nuclear CaM accumulation coming largely from the CaM-RyR2 pool. CaM lacks a nuclear localization signal (NLS), but G-protein coupled receptor kinase 5 (GRK5) binds CaM, has a NLS and translocates like CaM in response to AngII or PE. Suramin also promoted GRK5 nuclear import, and caused nuclear export of histone deacetylase 5 (HDAC5). Dantrolene prevented these effects. After 2-8 weeks of pressure overload (TAC) CaM binding to RyR2 was reduced, nuclear CaM and GRK5 were both elevated and there was enhanced nuclear export of HDAC5. Stress (acute AngII or TAC) causes CaM dissociation from RyR2 and translocation to the nucleus with GRK5 with parallel HDAC5 nuclear export. Thus CaM dissociation from RyR2 may be an important step in driving pathological hypertrophic gene transcription.

CaMKII-dependent phosphorylation of RyR2 promotes targetable pathological RyR2 conformational shift.

Diastolic calcium (Ca) leak via cardiac ryanodine receptors (RyR2) can cause arrhythmias and heart failure (HF). Ca/calmodulin (CaM)-dependent kinase II (CaMKII) is upregulated and more active in HF, promoting RyR2-mediated Ca leak by RyR2-Ser2814 phosphorylation. Here, we tested a mechanistic hypothesis that RyR2 phosphorylation by CaMKII increases Ca leak by promoting a pathological RyR2 conformation with reduced CaM affinity. Acute CaMKII activation in wild-type RyR2, and phosphomimetic RyR2-S2814D (vs. non-phosphorylatable RyR2-S2814A) knock-in mouse myocytes increased SR Ca leak, reduced CaM-RyR2 affinity, and caused a pathological shift in RyR2 conformation (detected via increased access of the RyR2 structural peptide DPc10). This same trio of effects was seen in myocytes from rabbits with pressure/volume-overload induced HF. Excess CaM quieted leak and restored control conformation, consistent with negative allosteric coupling between CaM affinity and DPc10 accessible conformation. Dantrolene (DAN) also restored CaM affinity, reduced DPc10 access, and suppressed RyR2-mediated Ca leak and ventricular tachycardia in RyR2-S2814D mice. We propose that a common pathological RyR2 conformational state (low CaM affinity, high DPc10 access, and elevated leak) may be caused by CaMKII-dependent phosphorylation, oxidation, and HF. Moreover, DAN (or excess CaM) can shift this pathological gating state back to the normal physiological conformation, a potentially important therapeutic approach.

S-Nitrosylation Induces Both Autonomous Activation and Inhibition of Calcium/Calmodulin-dependent Protein Kinase II δ.

NO is known to modulate calcium handling and cellular signaling in the myocardium, but key targets for NO in the heart remain unidentified. Recent reports have implied that NO can activate calcium/calmodulin (Ca(2+)/CaM)-dependent protein kinase II (CaMKII) in neurons and the heart. Here we use our novel sensor of CaMKII activation, Camui, to monitor changes in the conformation and activation of cardiac CaMKII (CaMKIIδ) activity after treatment with the NO donor S-nitrosoglutathione (GSNO). We demonstrate that exposure to NO after Ca(2+)/CaM binding to CaMKIIδ results in autonomous kinase activation, which is abolished by mutation of the Cys-290 site. However, exposure of CaMKIIδ to GSNO prior to Ca(2+)/CaM exposure strongly suppresses kinase activation and conformational change by Ca(2+)/CaM. This NO-induced inhibition was ablated by mutation of the Cys-273 site. We found parallel effects of GSNO on CaM/CaMKIIδ binding and CaMKIIδ-dependent ryanodine receptor activation in adult cardiac myocytes. We conclude that NO can play a dual role in regulating cardiac CaMKIIδ activity.

Cardiac myocyte Z-line calmodulin is mainly RyR2-bound, and reduction is arrhythmogenic and occurs in heart failure.

RATIONALE: Calmodulin (CaM) associates with cardiac ryanodine receptor type-2 (RyR2) as an important regulator. Defective CaM-RyR2 interaction may occur in heart failure, cardiac hypertrophy, and catecholaminergic polymorphic ventricular tachycardia. 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 heart failure ventricular myocytes, estimate the percentage of Z-line-localized CaM that is RyR2-bound, and test cellular function of defective CaM-RyR2 interaction. METHODS AND RESULTS: Using fluorescence resonance energy transfer in permeabilized myocytes, we specifically resolved RyR2-bound CaM from other potential binding targets and measured CaM-RyR2 binding affinity in situ (Kd=10-20 nmol/L). Using RyR2(ADA/+) knock-in 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+ wave production and stress-induced ventricular arrhythmia in RyR2(ADA/+) mice. In a post-myocardial infarction rat heart failure model, we detected a decrease in the CaM-RyR2 binding affinity (Kd≈51 nmol/L; ≈3-fold increase) and unaltered RyR2 affinity for the FK506-binding protein FKBP12.6 (Kd~0.8 nmol/L). CONCLUSIONS: CaM binds to RyR2 with high affinity in cardiac myocytes. Physiologically, CaM is bound to >70% of RyR2 monomers and inhibits sarcoplasmic reticulum 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 heart failure, RyR2 shows decreased CaM affinity, but unaltered FKBP 12.6 affinity.

Oxidation of ryanodine receptor (RyR) and calmodulin enhance Ca release and pathologically alter, RyR structure and calmodulin 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 Ca(2+) sparks and spontaneous Ca(2+) 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.

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.

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.

Feasibility, reproducibility and characteristics of coronary bifurcation type assessment by three-dimensional optical coherence tomography.

AIM: To investigate the characteristics of coronary artery bifurcation type (parallel or perpendicular type) using three-dimensional (3D) optical coherence tomography (OCT), and determine the feasibility, reproducibility, assessment time and correlation with bifurcation angles measured by 3D quantitative coronary angiography (QCA). METHODS AND RESULTS: We evaluated 60 lesions at the coronary bifurcation that were treated by main vessel (MV) stenting with kissing balloon inflation (KBI) under OCT/optical frequency domain imaging (OFDI) guidance. Inter- and intra-observer agreement regarding the assessment of 3D bifurcation types were 0.88 and 0.94, respectively. The assessment times of 3D-OCT bifurcation type with OCT and OFDI were within about 30 seconds. 3D-OCT bifurcation types showed the greatest correlation with the distal bifurcation angle assessed by 3D-QCA among the three bifurcation angles (distal bifurcation angle, proximal bifurcation angle and main vessel angle), and the optimal cut-off distal bifurcation angle to predict a perpendicular type bifurcation, as determined by ROC analysis, was 51.0° (AUC 0.773, sensitivity 0.80, specificity 0.67). Based on this cut-off value for the distal bifurcation angle (51°), the diagnostic accuracy for perpendicular type bifurcation in cases with a BA ≥ 51° (n = 34) was 70.6% (24/34) and that of the parallel type bifurcation in cases of BA < 51° (n = 26) was 76.9% (20/26). CONCLUSION: Performing 3D-OCT for assessment of coronary artery bifurcation type is feasible and simple, and can be done in a short time with high reproducibility.

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.