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.

Detection of Active Inflammation Status Around Ventricular Aneurysms in Patients With Cardiac Sarcoidosis.

BACKGROUND: Little is known about the pattern of isotope accumulation in the heart on 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography in patients with cardiac sarcoidosis (CS) complicated by ventricular aneurysm (VA).Methods and Results:We prospectively enrolled 82 consecutive patients with CS; 54 patients with active CS (presence of abnormal 18F-FDG accumulation in the heart) were subdivided into VA (n=17) and non-VA groups (n=37). Strong 18F-FDG accumulation surrounding the VA and its disappearance in the VA center was observed in all patients with VA, probably because of scar formation at the VA. Peak standardized uptake value was higher around the VA than in the VA center (5.1±2.1 vs. 2.2±0.6, P=0.0003) and the VA center had no 18F-FDG accumulation (VA center: 2.2±0.6 vs. control area: 2.1±0.6, P=0.37). On the other hand, in non-VA patients with LV wall thinning (n=28), 18F-FDG accumulation was significantly high, even in the area of LV wall thinning (LV wall thinning area: 3.1±0.8 vs. control area: 2.0±0.6, P=0.00002). CONCLUSIONS: A pattern of strong 18F-FDG accumulation surrounding the VA and its disappearance in the VA center might be characteristic in patients with CS complicated by VA. Careful attention to FDG uptake would further elucidate CS pathophysiology and aid in the early treatment of VA.

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.

Inter-Technique Consistency and Prognostic Value of Intra-Procedural Angiographic and Echocardiographic Assessment of Aortic Regurgitation After Transcatheter Aortic Valve Implantation.

BACKGROUND: We investigated the relationship between intraprocedural angiographic and echocardiographic AR severity after TAVI, and the clinical robustness of angiographic assessment. Methods and Results: In 74 consecutive patients, the echocardiographic circumferential extent (CE) of the paravalvular regurgitant jet was retrospectively measured and graded based on the VARC-2 cut-points; and angiographic post-TAVI AR was retrospectively quantified using contrast videodensitometry (VD) software that calculates the ratio of the contrast time-density integral in the LV outflow tract to that in the ascending aorta (LVOT-AR). Seventy-four echocardiograms immediately after TAVI were analyzable, while 51 aortograms were analyzable for VD. These 51 echocardiograms and VD were evaluated. Median LVOT-AR across the echocardiographic AR grades was as follows: none-trace, 0.07 (IQR, 0.05-0.11); mild, 0.12 (IQR, 0.09-0.15); and moderate, 0.17 (IQR, 0.15-0.22; P<0.05 for none-trace vs. mild, and mild vs. moderate). LVOT-AR strongly correlated with %CE (r=0.72, P<0.0001). At 1 year, the rate of the composite end-point of all-cause death or HF re-hospitalization was significantly higher in >mild AR patients compared with no-mild AR on intra-procedural echocardiography (41.5% vs. 12.4%, P=0.03) as well as in patients with LVOT-AR >0.17 compared with LVOT-AR ≤0.17 (59.5% vs. 16.6%, P=0.03). CONCLUSIONS: VD (LVOT-AR) has good intra-procedural inter-technique consistency and clinical robustness. Greater than mild post-TAVI AR, but not mild post-TAVI AR, is associated with late mortality.

Enhanced oxidative stress and presence of ventricular aneurysm for risk prediction in cardiac sarcoidosis.

OBJECTIVE: Sudden cardiac death (SCD) is the major cause of death in cardiac sarcoidosis (CS). We aimed to identify the prognostic markers for sustained ventricular tachycardia (sVT) and SCD in patients with CS. METHODS: We performed a prospective observational cohort study for patients with CS diagnosed according to the Japanese or Heart Rhythm Society guidelines between June 2008 and March 2020 in our hospital. The primary endpoint was a composite of the first sVT and SCD. The levels of urinary 8-hydroxy-2'-deoxyguanosine (U-8-OHdG), a marker of oxidative DNA damage that reflects the inflammatory activity of CS, other biomarkers, and indices of cardiac function and renal function were measured on admission. RESULTS: Eighty-nine consecutive patients with CS were enrolled; 28 patients with no abnormal 18F-fluorodeoxyglucose (18F-FDG) accumulation in the heart were excluded and 61 patients with abnormal 18F-FDG accumulation were followed up for a median of 46 months (IQR: 20-84). During the follow-up period, 15 of 61 patients showed sVT (n=12) or SCD (n=3). A Cox proportional hazard model showed that U-8-OHdG concentration and presence of ventricular aneurysm (VA) were independent predictors of first sVT/SCD. The cut-off U-8-OHdG concentration for predicting first sVT/SCD was 14.9 ng/mg·Cr. Patients with U-8-OHdG concentration ≥14.9 ng/mg·Cr and VA showed a significantly increased risk of sVT/SCD. CONCLUSIONS: U-8-OHdG and presence of VA were powerful predictors of first sVT/SCD in patients with CS, facilitating the stratification of cardiac events and providing relevant information about the substrates of ventricular tachycardia.

Relation between Oscillometric Measurement of Central Hemodynamics and Left Ventricular Hypertrophy in Hypertensive Patients.

INTRODUCTION: The augmentation index (AIx) or central systolic blood pressure (SBP), measured by radial applanation tonometry, has been reported to be independently associated with left ventricular hypertrophy (LVH) in Japanese hypertensive patients. Cuff-based oscillometric measurement of the AIx using Mobil-O-Graph® showed a low or moderate agreement with the AIx measurement with other devices. METHODS: The AIx measured using the Mobil-O-Graph was validated against the tonometric measurements of the radial AIx measured using HEM-9000AI in 110 normotensive healthy individuals (age, 21-76 years; 50 men). We investigated the relationship between the central hemodynamics assessed using the Mobil-O-Graph and LVH in 100 hypertensive patients (age, 54-75 years; 48 men), presenting a wall thickness of ≥11 mm and ≥10 mm in men and women, respectively. RESULTS: Although the Mobil-O-Graph-measured central AIx showed no negative values, it correlated moderately with the HEM-9000AI-measured radial AIx (r = 0.602, p < 0.001) in the normotensive individuals. The hypertensive patients did not show a significant difference in the central SBP between the sexes, but the central AIx was lower in men than in women. The independent determinants influencing left ventricle (LV) mass index (LVMI) (R2 = 0.362; adjusted R2 = 0.329, p < 0.001) were heart rate (ß = -0.568 ± 0.149, p < 0.001), central SBP (ß = 0.290 ± 0.100, p = 0.005), and aortic root diameter (ß = 1.355 ± 0.344, p = 0.001). Age (ß = -0.025 ± 0.124, p = 0.841) and the central AIx (ß = 0.120 ± 0.131, p = 0.361) were not independently associated with the LVMI. The area under the receiver operator characteristic curve to evaluate the diagnostic performance of the central AIx for the presence of LVH (LVMI >118 g/m2 in men or >108 g/m2 in women) was statistically significant in men (0.875, p < 0.001) but not in women (0.622, p = 0.132). In men, a central AIx of 28.06% had a sensitivity of 83.3% and specificity of 80.0% for detecting LVH. CONCLUSIONS: AIx measurement in men provided useful prognostic information for the presence of LVH. Pulse-wave analysis assessed using the Mobil-O-Graph may be a valuable tool for detecting LVH in hypertensive patients.

G790del mutation in DSC2 alone is insufficient to develop the pathogenesis of ARVC in a mouse model.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart disease that causes heart failure and/or sudden cardiac death. Several desmosomal genes (DSC2, PKG, PKP2, DSP, and RyR2) are thought to be the causative gene involved in ARVC. Out of them, DSC2 mutations account for 2% of ARVC genetic abnormalities. This study aimed to clarify the effect of G790del mutation in DSC2 on the arrhythmogenic mechanism and cardiac function in a mouse model. RESULT: Neither the heterozygous +/G790del nor homozygous G790del/G790del mice showed structural and functional defects in the right ventricle (RV) or lethal arrhythmia. The homozygous G790del/G790del 6-month-old mice slightly showed left ventricular (LV) dysfunction. Cell shortening decreased with prolongation of intracellular Ca2+ transient in cardiomyocytes isolated from the homozygous G790del/G790del mice, and spontaneous Ca2+ transients were frequently observed in response to isoproterenol. CONCLUSIONS: G790del mutation in DSC2 was not relevant to the pathogenesis of ARVC, but showed a slight contractile dysfunction and Ca2+ dysregulation in the LV.

Mutation-linked, excessively tight interaction between the calmodulin binding domain and the C-terminal domain of the cardiac ryanodine receptor as a novel cause of catecholaminergic polymorphic ventricular tachycardia.

BACKGROUND: Ryanodine receptor (RyR2) is known to be a causal gene of catecholaminergic polymorphic ventricular tachycardia (CPVT), an important inherited disease. Some of the human CPVT-associated mutations have been found in a domain (4026-4172) that has EF hand motifs, the so-called calmodulin (CaM)-like domain (CaMLD). OBJECTIVE: The purpose of this study was to investigate the underlying mechanism by which CPVT is induced by a mutation at CaMLD. METHODS: A new N4103K/+ knock-in (KI) mice model was generated. RESULTS: Sustained ventricular tachycardia was frequently observed after infusion of caffeine plus epinephrine in KI mice. Endogenous CaM bound to RyR2 decreased even at baseline in isolated KI cardiomyocytes. Ca2+ spark frequency (CaSpF) was much higher in KI cells than in wild-type cells. Addition of GSH-CaM (higher affinity CaM to RyR2) significantly decreased CaSpF. In response to isoproterenol, spontaneous Ca2+ transient (SCaT) was frequently observed in intact KI cells. Incorporation of GSH-CaM into intact KI cells using a protein delivery kit decreased SCaT significantly. An assay using a quartz crystal microbalance technique revealed that mutated CaMLD peptide showed higher binding affinity to CaM binding domain (CaMBD) peptide. CONCLUSION: In the N4103K mutant, CaM binding affinity to RyR2 was significantly reduced regardless of beta-adrenergic stimulation. We found that this was caused by an abnormally tight interaction between CaMBD and mutated CaM-like domain (N4103K-CaMBD). Thus, CaMBD-CaMLD interaction may be a novel therapeutic target for treatment of lethal arrhythmia.

Urinary 8-Hydroxy-2'-Deoxyguanosine as a Myocardial Oxidative Stress Marker Is Associated With Ventricular Tachycardia in Patients With Active Cardiac Sarcoidosis.

BACKGROUND: Recently, we reported that urinary 8-hydroxy-2'-deoxyguanosine (U-8-OHdG), an oxidative stress marker, reflected inflammatory activity in cardiac sarcoidosis (CS). Here, we investigated whether U-8-OHdG levels were associated with ventricular tachycardia (VT) in patients with CS. METHODS AND RESULTS: This prospective cohort study enrolled 62 consecutive patients with CS, of whom 36 were diagnosed as having active CS based on abnormal 18F-flurodeoxyglucose accumulation in the heart on positron-emission tomography/computed tomography. The 36 patients with active CS were subdivided as having CS with sustained VT (CS-VT group; n=18) or CS without sustained VT (CS-nVT group; n=18). Twenty-seven patients diagnosed with idiopathic dilated cardiomyopathy served as heart failure controls. U-8-OHdG, brain natriuretic peptide, cardiac function indices, and immunohistological data from subendomyocardial biopsy samples were compared across groups. Immunohistochemical examination of ventricle biopsy samples revealed that the anti-8-OHdG antibody-positive area of cardiac tissue was significantly greater in CS-VT than in CS-nVT or dilated cardiomyopathy and significantly correlated with U-8-OHdG levels (n=58; R=0.61; P<0.00001), which were significantly higher in CS-VT than in CS-nVT (24.6±7.1 versus 15.2±3.8 ng/mg·Cr; P<0.0001). Other baseline characteristics did not differ between the groups. Multivariate analysis indicated that U-8-OHdG was an independent determinant factor for VT. Receiver operating characteristic curve analysis to identify patients with VT resulted in a U-8-OHdG cutoff value of 17.5 ng/mg·Cr (sensitivity, 89%; specificity, 83%; area under the curve, 0.90). CONCLUSIONS: U-8-OHdG levels are associated with VT in patients with active CS diagnosed by 18F-flurodeoxyglucose positron-emission tomography, providing additive and relevant information about the arrhythmia substrate.

Correction of impaired calmodulin binding to RyR2 as a novel therapy for lethal arrhythmia in the pressure-overloaded heart failure.

BACKGROUND: Calmodulin (CaM) is a key modulator of the channel gating function of the ryanodine receptor (RyR). OBJECTIVE: The purpose of this study was to investigate the pathogenic role of RyR-bound CaM in diastolic Ca2+ leakage from the sarcoplasmic reticulum and arrhythmogenesis in pressure-overloaded heart failure. METHODS: Pressure overload was induced in 12-week-old mice by transverse aortic constriction (TAC) using a 27-gauge needle. RESULTS: TAC operation for 8 weeks produced a significant increase in left ventricular end-diastolic diameter and frequent occurrence of lethal arrhythmias after infusion of epinephrine and caffeine in TAC mice. The amount of RyR-bound CaM decreased significantly in TAC mice compared with sham mice. The apparent affinity of CaM binding to RyR decreased in pressure-overloaded cells compared with sham cells and untreated cells. High-affinity calmodulin (HA-CaM; ie, CaM whose binding affinity to RyR was significantly increased) restored a normal level of CaM-RyR binding properties in pressure-overloaded cells. HA-CaM corrected abnormally increased Ca2+ spark frequency in the pressure-overloaded cells to the level seen in the sham cells. The frequency of spontaneous Ca2+ transients in TAC cells during and after 1-5 Hz of field stimulation was 44%, whereas it was significantly attenuated by HA-CaM but not with CaM. CONCLUSION: Several disorders in the RyR channel function characteristic of pressure-overloaded cells (increased spontaneous Ca2+ leakage, delayed afterdepolarization, triggered activity, Ca2+ spark frequency, spontaneous Ca2+ transients) are caused by deteriorated CaM binding to RyR2. These disorders could be rectified by restoring normal CaM binding to RyR2.

An oxidative stress biomarker, urinary 8-hydroxy-2'-deoxyguanosine, predicts cardiovascular-related death after steroid therapy for patients with active cardiac sarcoidosis.

BACKGROUND: We investigated whether urinary 8-hydroxy-2'-deoxyguanosine (U-8-OHdG), a marker of oxidative DNA damage, is a prognosticator of cardiovascular-related death in patients with cardiac sarcoidosis (CS). METHODS AND RESULTS: In this prospective study, 30 consecutive patients were divided into the active CS (n=20) and non-active CS (n=10) groups, based on abnormal isotope accumulation in the heart on (18)F-fluorodeoxyglucose positron-emission tomography/computed tomography ((18)F-FDG PET/CT) imaging. Nineteen patients in the active CS group underwent corticosteroid therapy. Before corticosteroid therapy initiation, U-8-OHdG, brain natriuretic peptide (BNP), other biomarkers, and indices of cardiac function were measured. Patients were followed-up for a median of 48months. The primary endpoint was the incidence of cardiovascular-related death. During the follow-up period, in the corticosteroid-treated active CS group, 7 of 19 patients experienced cardiovascular-related death. By contrast, in the non-active CS group, 1 of 10 patients died from cardiovascular-related causes. Univariate and multivariate analyses showed that U-8-OHdG and BNP were independent predictors for cardiovascular-related death. The cut-off values for predicting cardiovascular death in corticosteroid-treated patients with active CS were 19.1ng/mg·Cr and 209pg/mL for U-8-OHdG and BNP, respectively. Patients with a U-8-OHdG concentration ≥19.1ng/mg·Cr or a BNP concentration ≥209pg/mL had a significantly higher cardiovascular-related death risk, but U-8-OHdG had better predictive value compared with BNP. CONCLUSION: These findings suggested that U-8-OHdG was a powerful predictor of cardiovascular-related death in patients with CS, suggesting that active CS patients with elevated U-8-OHdG levels might be resistant to corticosteroid therapy.

Recombinant Atrial Natriuretic Peptide Prevents Aberrant Ca2+ Leakage through the Ryanodine Receptor by Suppressing Mitochondrial Reactive Oxygen Species Production Induced by Isoproterenol in Failing Cardiomyocytes.

Catecholamines induce intracellular reactive oxygen species (ROS), thus enhancing diastolic Ca2+ leakage through the ryanodine receptor during heart failure (HF). However, little is known regarding the effect of atrial natriuretic peptide (ANP) on ROS generation and Ca2+ handling in failing cardiomyocytes. The aim of the present study was to clarify the mechanism by which an exogenous ANP exerts cardioprotective effects during HF. Cardiomyocytes were isolated from the left ventricles of a canine tachycardia-induced HF model and sham-operated vehicle controls. The degree of mitochondrial oxidized DNA was evaluated by double immunohistochemical (IHC) staining using an anti-VDAC antibody for the mitochondria and an anti-8-hydroxy-2'-deoxyguanosine antibody for oxidized DNA. The effect of ANP on ROS was investigated using 2,7-dichlorofluorescin diacetate, diastolic Ca2+ sparks assessed by confocal microscopy using Fluo 4-AM, and the survival rate of myocytes after 48 h. The double IHC study revealed that isoproterenol (ISO) markedly increased oxidized DNA in the mitochondria in HF and that the ISO-induced DNA damage was markedly inhibited by the co-presence of ANP. ROS production and Ca2+ spark frequency (CaSF) were increased in HF compared to normal controls, and were further increased in the presence of ISO. Notably, ANP significantly suppressed both ISO-induced ROS and CaSF without changing sarcoplasmic reticulum Ca2+ content in HF (p<0.01, respectively). The survival rate after 48 h in HF was significantly decreased in the presence of ISO compared with baseline (p<0.01), whereas it was significantly improved by the co-presence of ANP (p<0.01). Together, our results suggest that ANP strongly suppresses ISO-induced mitochondrial ROS generation, which might correct aberrant diastolic Ca2+ sparks, eventually contributing to the improvement of cardiomyocyte survival in HF.