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.

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.

Urinary 8-hydroxy-2'-deoxyguanosine reflects symptomatic status and severity of systolic dysfunction in patients with chronic heart failure.

AIMS: Oxidative stress is known to play a crucial role in the pathogenesis of heart failure (HF). We investigated whether urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG), a product of oxidative DNA damage, is a clinically useful biomarker of the severity of chronic heart failure (CHF) and oxidative stress levels in failing hearts. METHODS AND RESULTS: We measured 8-OHdG in the serum obtained from the coronary sinus (CS) and aortic root (Ao) in small groups of control subjects and CHF patients. We then measured urinary 8-OHdG and other biomarkers (brain natriuretic peptide, 8-isoplastane, high-sensitivity C-reactive protein, interleukin-6, and tumour necrosis factor-α) in 31 control subjects and 194 patients with CHF [left-ventricular ejection fraction (LVEF): 28.3 ± 8.1%]. Serum 8-OHdG was significantly higher in the CS than the Ao in CHF patients only. Urinary 8-OHdG was also significantly higher in CHF patients than in control subjects, and urinary 8-OHdG became higher as New York Heart Association class increased. Moreover, there was a significant correlation between urinary 8-OHdG and LVEF (r = -0.27), pulmonary capillary wedge pressure (r = 0.31), or left-ventricular end-diastolic volume index (r = 0.22). In contrast, there was poor correlation between the severity of CHF and the other neurohumoral biomarkers. CONCLUSION: In HF, urinary 8-OHdG seems to reflect the level of oxidative stress and various parameters related to symptomatic status and functional severity of CHF.

A case of mitochondrial disease with severe left ventricular hypertrophy.

A 35-year-old woman was admitted for progressive dyspnea with lower limb edema. Transthoracic echocardiography showed severe left ventricular hypertrophy (LVH) and heart failure with preserved ejection fraction (HFPEF). Electron microscopy of an endomyocardial biopsy sample revealed a high density of mitochondria of abnormal size and shape. We report a case of mitochondrial disease with severe LVH and HFPEF.

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.

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.

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.

Impact of intraoperative transesophageal echocardiography in cardiac and thoracic aortic surgery: experience in 1011 cases.

BACKGROUND: Although intraoperative transesophageal echocardiography (IOTEE) has been widely used in cardiovascular surgery, the exact incidence of abnormalities detected by IOTEE in each type of surgical procedure is still unclear. The aim of this study was to review our experiences of IOTEE, in patients who underwent different types of cardiovascular surgery and to evaluate the clinical usefulness of IOTEE. METHODS AND RESULTS: Our database of 1011 consecutive patients, who underwent cardiovascular surgery and IOTEE monitoring was reviewed. The incidence of abnormal findings was 115 of 1011 patients (11.4%), and the highest incidence was the appearance of new wall motion abnormalities after cardiopulmonary bypass. These findings influenced surgical decision-making in 59 of the evaluated 1011 patients (5.8%). CONCLUSIONS: IOTEE provides important intraoperative and postoperative information that may influence surgical decision-making in various cardiovascular surgeries.

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.

AT1 receptor antagonist restores cardiac ryanodine receptor function, rendering isoproterenol-induced failing heart less susceptible to Ca2+ -leak induced by oxidative stress.

BACKGROUND: The Ca(2+) regulatory proteins in the sarcoplasmic reticulum (SR) play a key role in the pathogenesis of heart failure. In the present study the effect of chronic beta-receptor-stimulation on cardiac and SR functions was assessed, with or without angiotensin-II receptor antagonist treatment recently reported to have anti-beta-adrenergic activity. METHODS AND RESULTS: Rats were treated with isoproterenol with (+) or without (-) candesartan (CAN) and then SR vesicles were isolated from the left ventricular muscle. Both Ca(2+)-uptake and the amount of SR Ca(2+)-ATPase were significantly lower in the CAN (-) group than in the shams, but those were almost normally restored in the CAN (+). Although the level of the protein kinase A (PKA)-phosphorylation of the SR Ca(2+) release channel, known as the ryanodine receptor (RyR2), was elevated in the CAN (-), no Ca(2+)-leak was detected. However, SIN-1 (O(2) (-) donor) induced Ca(2+)-leak in the CAN (-) at a 10-fold lower dose than in the sham and CAN (+). In cardiomyocytes, SIN-1 decreased cell shortening and the peak Ca(2+) transient and prolonged time from peak to 70% decline in CAN (-), again at 10-fold lower dose than in the sham and CAN (+). CONCLUSION: Chronic beta-receptor-stimulation did not induce any Ca(2+)-leak from the SR, whereas Ca(2+)-leak was easily induced when oxidative stress was applied to the PKA-phosphorylated RyR2. Candesartan not only improved Ca(2+)-uptake, but also prevented PKA-phosphorylation, rendering the SR less susceptible to Ca(2+)-leak.

Defective regulation of interdomain interactions within the ryanodine receptor plays a key role in the pathogenesis of heart failure.

BACKGROUND: According to our hypothesis, 2 domains within the ryanodine receptor (RyR) of sarcoplasmic reticulum (SR) (N-terminal [0 to 600] and central [2000 to 2500] domains), where many mutations have been found in patients with polymorphic ventricular tachycardia, interact with each other as a regulatory switch for channel gating. Here, we investigated whether the defective FKBP12.6-mediated stabilization of RyR in heart failure is produced by an abnormal interdomain interaction. METHODS AND RESULTS: SR vesicles were isolated from dog left ventricular muscles, and then the RyR moiety of the SR was fluorescently labeled with methylcoumarin acetate (MCA) using DPc10, a synthetic peptide corresponding to Gly2460-Pro2495 of RyR (one of the mutable domains in polymorphic ventricular tachycardia), as a site-directing carrier; the carrier was removed from the RyR after MCA labeling. Addition of DPc10 induced an unzipped state of the interacting N-terminal and central domains, as evidenced by an increase in the accessibility of the RyR-bound MCA fluorescence to a large fluorescence quencher. Domain unzipping resulted in Ca2+ leak through the RyR and facilitated cAMP-dependent hyperphosphorylation of RyR and FKBP12.6 dissociation from RyR. When DPc10 was introduced into the isolated myocytes, the magnitude of intracellular Ca2+ transient decreased, and its decay time was prolonged. In the SR isolated from pacing-induced dog failing hearts, the domain unzipping has already occurred, together with FKBP12.6 dissociation and Ca2+ leak. CONCLUSIONS: The specific domain interaction within the RyR regulates the channel gating property, and the defectiveness in the mode of the interdomain interaction seems to be the initial critical step of the pathogenesis of heart failure.

Valsartan restores sarcoplasmic reticulum function with no appreciable effect on resting cardiac function in pacing-induced heart failure.

BACKGROUND: Although angiotensin II receptor blockade is considered to be useful for the treatment of human heart failure, little beneficial hemodynamic effect has been shown in some experimental failing hearts. In this study, we assessed the effect of an angiotensin II receptor blocker, valsartan, on sarcoplasmic reticulum (SR) function, defectiveness of which is a major pathogenic mechanism in heart failure. METHODS AND RESULTS: SR vesicles were isolated from dog left ventricular muscle (normal or exposed to 4-week rapid ventricular pacing with or without valsartan). In the untreated and valsartan-treated paced dogs, cardiac function showed similar deterioration (compared with before pacing). However, both the density of beta-receptors and the contractile response to dobutamine were greater in the valsartan-treated paced dogs than in the untreated paced dogs. In untreated paced hearts, the ryanodine receptor was protein kinase A-hyperphosphorylated, showed an abnormal Ca2+ leak, and had a decreased amount of ryanodine receptor-bound FKBP12.6. No such phenomena were seen in the valsartan-treated paced hearts. Both the SR Ca2+ uptake function and the amount of Ca2+-ATPase were decreased in the untreated failing SR, but both were restored in the valsartan-treated SR. CONCLUSIONS: During the development of pacing-induced heart failure, valsartan preserved the density of beta-receptors and concurrently restored SR function without improving resting cardiac function.

FKBP12.6-mediated stabilization of calcium-release channel (ryanodine receptor) as a novel therapeutic strategy against heart failure.

BACKGROUND: The development of heart failure is tightly correlated with a decrease in the stoichiometric ratio for FKBP12.6 binding to the ryanodine receptor (RyR) in the sarcoplasmic reticulum (SR). We report that a new drug, the 1,4-benzothiazepine derivative JTV519, reverses this pathogenic process. JTV519 is known to have a protective effect against Ca2+ overload-induced myocardial injury. METHODS AND RESULTS: Heart failure was produced by 4 weeks of rapid right ventricular pacing, with or without JTV519; SR were then isolated from dog left ventricular (LV) muscles. First, in JTV519-treated dogs, no signs of heart failure were observed after 4 weeks of chronic right ventricular pacing, LV systolic and diastolic functions were largely preserved, and LV remodeling was prevented. Second, JTV519 acutely inhibited both the FK506-induced Ca2+ leak from RyR in normal SR and the spontaneous Ca2+ leak in failing SR. Third, there was no abnormal Ca2+ leak in SR vesicles isolated from JTV519-treated hearts. Fourth, in JTV519-treated hearts, both the stoichiometry of FKBP12.6 binding to RyR and the amount of RyR-bound FKBP12.6 were restored toward the values seen in normal SR. Fifth, in JTV519-untreated hearts, RyR was PKA-hyperphosphorylated, whereas it was reversed in JTV519-treated hearts, returning the channel phosphorylation toward the levels seen in normal hearts. CONCLUSIONS: During the development of experimental heart failure, JTV519 prevented the amount of RyR-bound FKBP12.6 from decreasing. This in turn reduced the abnormal Ca2+ leak through the RyR, prevented LV remodeling, and led to less severe heart failure.