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.

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.

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.

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.

Active hexose-correlated compound down-regulates sex-determining region Y-box 2 of pancreatic cancer cells.

BACKGROUND/AIM: Active hexose-correlated compound (AHCC) is an extract of basidiomycete mushroom. It has been used as health food due to its efficacy of enhancing antitumor effects and reducing adverse effects of chemotherapy. Our previous research showed that AHCC down-regulated heat-shock protein (HSP)-27 and exhibited cytotoxic effects against gemcitabine-resistant pancreatic cancer cells. Sex-determining region Y-box 2 (SOX2) is reported to be up-regulated in other kinds of cancer cells and involved in carcinogenesis and malignancy. The aim of this study was to investigate the effects of AHCC on protein expression of SOX2 in the gemcitabine-resistant pancreatic cancer cell line KLM1-R. MATERIALS AND METHODS: AHCC was applied to KLM1-R cells and expression of SOX2 was analyzed by western blotting. RESULTS: AHCC down-regulated SOX2 in KLM1-R cells. Nanog and Oct4, co-workers of SOX2 in maintaining pluripotency, did not exhibit any significant change in protein expression. CONCLUSION: We showed the potential of AHCC to be a candidate for combinatorial therapy in anticancer drug regimens. This result suggests that the target of AHCC in expressing therapeutic efficacy was not the pluripotent cells such as cancer stem cells (CSCs) but SOX2-specific.