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.

Downregulation of MiR-199b-5p Inducing Differentiation of Bone-Marrow Mesenchymal Stem Cells (BMSCs) Toward Cardiomyocyte-Like Cells via HSF1/HSP70 Pathway.

BACKGROUND Bone-marrow mesenchymal stem cells (BMSCs) are pluripotent stem cells with potent self-renewal and differentiation ability that are widely used in transplantation of cell therapy. But the mechanism on microRNA (miRNA) regulating stem cell differentiation is complicated and unclear. The aim of this study was to investigate whether miR-199b-5p is involved in differentiation of cardiomyocyte-like cells and identify potential signal pathways in BMSCs. MATERIAL AND METHODS Mouse BMSCs were treated with 5-azacytidine and transfected by miR-199b-5p mimic and inhibitor, respectively. qRT-PCR was used to detect the expression of miR-199b-5p in BMSCs, 5-azacytidine treated BMSCs, and neonatal murine cardiomyocytes. The expression of cardiac specific genes and the HSF1/HSP70 signal pathway were examined by qRT-PCR or western blotting. The proliferation and migration of BMSCs were evaluated by CCK-8 assay and wound-healing assay. RESULTS The expression of miR-199b-5p decreased gradually in the process of differentiation of BMSCs toward cardiomyocyte-like cells. The expression of cardiac specific genes and HSF1/HSP70 were increased in the miR-199b-5p inhibitor group; however, the miR-199b-5p mimic group presented an opposite result. Both the miR-199b-5p inhibitor group and the miR-199b-5p mimic group had no influence on BMSCs proliferation and migration. Using lentivirus vectors bearing HSF1 shRNA to silence HSF1 and HSP70, the anticipated elevated expression effect of cardiac specific genes induced by miR-199b-5p inhibitor was suppressed. CONCLUSIONS Downregulation of miR-199b-5p induced differentiation of BMSCs toward cardiomyocyte-like cells partly via the HSF1/HSP70 signaling pathway, and had no influence on BMSCs proliferation and migration.

Role of heat shock transcription factor 1(HSF1)-upregulated macrophage in ameliorating pressure overload-induced heart failure in mice.

In order to explore the role of macrophages in HSF1-mediated alleviation of heart failure, mice model of pressure overload-induced heart failure was established using transverse aortic constriction (TAC). Changes in cardiac function and morphology were studied in TAC and SHAM groups using ultrasonic device, tissue staining, electron microscopy, real-time quantitative polymerase chain reaction (RT-QPCR), and Western blotting. We found that mice in the TAC group showed evidence of impaired cardiac function and aggravation of fibrosis on ultrasonic and histopathological examination when compared to those in the SHAM group. The expressions of HSF1, LC3II/LC3I, Becline-1 and HIF-1, as well as autophagosome formation in TAC group were greater than that in SHAM group. On sub-group analyses in the TAC group, improved cardiac function and alleviation of fibrosis was observed in the HSF1 TG subgroup as compared to that in the wild type subgroup. Expressions of LC3II/LC3I, Becline-1 and HIF-1, too showed an obvious increase; and increased autophagosome formation was observed on electron microscopy. Opposite results were observed in the HSF1 KO subgroup. These results collectively suggest that in the pressure overload heart failure model, HSF1 promoted formation of macrophages by inducing upregulation of HIF-1 expression, through which heart failure was ameliorated.

Role of miR-199b-5p in regulating angiogenesis in mouse myocardial microvascular endothelial cells through HSF1/VEGF pathway.

Our study explored effects of miR-199b-5p on angiogenesis in mouse myocardial microvascular endothelial cells (MMVECs) and the involved working mechanisms. We applied explant culture to incubate C57/BL6 mouse MMVECs. Lipofection was used to transfect miR-199b-5p mimic, miR-199b-5p inhibitor and miR-199b-5p scramble respectively. MMVECs were divided into miR-199b-5p up-regulation, miR-199b-5p down-regulation and control groups based on above sequence. Expressions of miR-199b-5p, heat shock factor protein 1 (HSF1) mRNA were assessed by real-time quantitative polymerase chain reaction (RT-QPCR). Expressions of HSF1 and vascular endothelial growth factor (VEGF) were assessed by Western Blotting. Cell proliferation was assessed by CCK8. Tubule formation assay was conducted to assess formation of blood vessels. Results showed that miR-199b-5p up/down-regulation groups exhibited no obvious differences in the expressions of HSF1 mRNA compared to control group. However, miR-199b-5p up-regulation group recorded lower expressions of HSF1 and VEGF in the level of protein, and reduced cell proliferation and tubule formation. Whereas, miR-199b-5p down-regulation group presented the contrary results. The experiment indicated that miR-199b-5p can regulate proliferation and angiogenesis in mouse MMVECs through the pathway of HSF1/VEGF.

Role of HSF1-upregulated AC6 in ameliorating heart failure in mice.

PURPOSE: Our previous studies discovered that Heat shock factor 1(HSF1) can alleviate pressure overload induced heart failure in mice. However, its molecular mechanisms are yet to be further explained. Many studies have already verified that Adenylyl Cyclase 6 (AC6) can ameliorate heart failure, but it is still unknown whether or not the pathway HSF1 is involved in the process. Our preliminary experiment showed that the expression level of AC6 is positively associated with HSF1. Therefore, in the present study, we aimed to explore whether HSF1 can play its role in ameliorating heart failure by regulating AC6, and how the potential internal mechanisms work. METHODS: We applied the Transverse Aortic Constriction (TAC) for 4 weeks to develop the C57BL/6 mice pressure overload induced heart failure model. First, the mice were divided into TAC group and SHAM group. Changes in the cardiac function and morphology of the mice were observed by an ultrasonic device and Masson staining slices, expressions of AC6 mRNA were observed by RT-QPCR, expressions of HSF1 and proteinkinase A (PKA) were examined by Western Blotting, and the levels of cyclic adenosine monophosphate (cAMP) from aortic blood were measured by ELISA. Second, the TAC group were further divided into subgroups of HSF1 transgene mice, HSF1 knockout mice and wild type mice, followed by the aforesaid observations. RESULTS: In the SHAM group, no obvious variations of cardiac function, AC6 mRNAHSF1, PKA, cAMP and other test results were found among each of the subgroups. Compared to the SHAM group, the TAC group presented clearly weakened heart functions, while, expressions of AC6 mRNA, HSF1, PKA and cAMP all recorded obvious increases. In the TAC group, compared to the WT subgroup, the HSF1 KO subgroup presented decreases in expressions of AC6 mRNA, HSF1, PKA and cAMP, and at the same time, the heart functions were weaker, while, the HSF1 TG subgroup recorded the contrary results. CONCLUSION: In the pressure overload heart failure model, HSF1 can ameliorate heart failure by positively regulating the pathway of AC6/cAMP/PKA.