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Cardiac senescence is associated with cardiomyopathy which is a degenerative disease in the aging process of the elderly. The present study investigates using multiple experimental approaches whether the natural flavone acacetin could attenuate myocardial senescence in C57/BL6 mice and H9C2 rat cardiac cells induced by D-galactose. We found that the impaired heart function in D-galactose-induced accelerated aging mice was improved by oral acacetin treatment in a dose-dependent manner. Acacetin significantly countered the increased serum advanced glycation end products, the myocardial telomere length shortening, the increased cellular senescence marker proteins p21 and p53, and the reduced mitophagy signaling proteins PINK1/Parkin and Sirt6 expression in aging mice. In H9C2 rat cardiac cells, acacetin alleviated cell senescence induced by D-galactose in a concentration-dependent manner. Acacetin decreased p21 and p53 expression, up-regulated PINK1/Parkin, LC3II/LC3I ratio, pLKB1, pAMPK and Sirt6, and reversed the depolarized mitochondrial membrane potential in aging cardiac cells. Mitophagy inhibition with 3-methyladenine or silencing Sirt6 abolished the protective effects of acacetin against cardiac senescence. Further analysis revealed that acacetin effect on Sirt6 was mediated by Sirt1 activation and increase of NAD+/NADH ratio. These results demonstrate that acacetin significantly inhibits in vivo and in vitro cardiac senescence induced by D-galactose via Sirt1-mediated activation of Sirt6/AMPK signaling pathway, thereby enhancing mitophagy and preserving mitochondrial function, which suggests that acacetin may be a drug candidate for treating cardiovascular disorders related to aging.

Doxorubicin cardiotoxicity is frequently reported in patients undergoing chemotherapy. The present study investigates whether cardiomyopathy induced by doxorubicin can be improved by the natural flavone acacetin in a mouse model and uncovers the potential molecular mechanism using cultured rat cardiomyoblasts. It was found that the cardiac dysfunction and myocardial fibrosis induced by doxorubicin were significantly improved by acacetin in mice with impaired Nrf2/HO‐1 and Sirt1/pAMPK molecules, which is reversed by acacetin treatment. Doxorubicin decreased cell viability and increased ROS production in rat cardiomyoblasts; these effects are significantly countered by acacetin (0.3‐3 μM) in a concentration‐dependent manner via activating Sirt1/pAMPK signals and enhancing antioxidation (Nrf2/HO‐1 and SOD1/SOD2) and anti‐apoptosis. These protective effects were abolished in cells with silencing Sirt1. The results demonstrate for the first time that doxorubicin cardiotoxicity is antagonized by acacetin via Sirt1‐mediated activation of AMPK/Nrf2 signal molecules, indicating that acacetin may be a drug candidate used clinically for protecting against doxorubicin cardiomyopathy.

Cardiac hypertrophy is a major risk factor for developing heart failure. This study investigates the effects of the natural flavone acacetin on myocardial hypertrophy in cellular level and whole animals. In cardiomyocytes from neonatal rat with hypertrophy induced by angiotensin II (Ang II), acacetin at 0.3, 1, and 3 μM reduced the increased myocyte surface area, brain natriuretic peptide (BNP), and ROS production by upregulating anti-oxidative molecules (i.e. Nrf2, SOD1, SOD2, HO-1), anti-apoptotic protein Bcl-2, and downregulating the pro-apoptotic protein Bax and the inflammatory cytokine IL-6 in a concentration-dependent manner. In addition, acacetin rescued Ang II-induced impairment of PGC-1α, PPARα and pAMPK. These beneficial effects of acacetin were mediated by activation of Sirt1, which was confirmed in cardiac hypertrophy induced by abdominal aorta constriction (AAC) in SD rats. Acacetin prodrug (10 mg/kg, s.c., b.i.d.) treatment reduced the elevated artery blood pressure, improved the increased heart size and thickness of left ventricular wall and the ventricular fibrosis associated with inhibiting myocardial fibrosis and BNP, and reversed the impaired protective signal molecules including PGC-1α, Nrf2, PPARα, pAMPK and Sirt1 of left ventricular tissue. Our results demonstrate the novel pharmacological effect that acacetin ameliorates cardiac hypertrophy via Sirt1-mediated activation of AMPK/PGC-1α signal molecules followed by reducing oxidation, inflammation and apoptosis.

Diabetic cardiomyopathy seriously affects the life quality of diabetic patients and can lead to heart failure and death in severe cases. Acacetin was reported to be an anti-oxidant and anti-inflammatory agent in several cardiovascular diseases. However, the effect of acacetin on diabetic cardiomyopathy was not understood. This study was designed to explore the therapeutic effect of acacetin on diabetic cardiomyopathy and the potential mechanism with in vitro and in vivo experimental techniques. In cultured neonatal rat cardiomyocytes and H9C2 cardiac cells, acacetin (0.3, 1, 3 μM) showed effective protection against high glucose-induced injury in a concentration-dependent manner. Acacetin countered high glucose-induced increase of Bax and decrease of Bcl-2, SOD1, and SOD2. In streptozotocin-induced rat diabetic cardiomyopathy model, treatment with acacetin prodrug (10 mg/kg, s.c., b.i.d.) significantly improved the cardiac function and reduced myocardial injury, and reversed the increase of serum MDA, Ang Ⅱ, and IL-6 levels and myocardial Bax and IL-6, and the decrease of serum SOD, indicating that acacetin plays a cardioprotective effect by inhibiting oxidative stress, inflammation, and apoptosis. In addition, both in vitro and in vivo experimental results showed that acacetin increased the expression of PPAR-α and pAMPK, indicating that PPAR-α and pAMPK are potential targets of acacetin for the protection against diabetic cardiomyopathy. This study demonstrates the new application of acacetin for treating diabetic cardiomyopathy.

MiR-30a-5p plays an important role in various cardiovascular diseases, but its effect in atherosclerosis has not been reported. Apolipoprotein E-deficient (Apo E−/−) mice were used to investigate the role of miR-30a-5p in atherosclerosis, and the underlying mechanism was investigated in vivo and in vitro. The fluorescence in situ hybridization test revealed that miR-30a-5p was expressed in Apo E−/− mice lesions. Nevertheless, in RAW264.7 macrophages, the expression of miR-30a-5p was reduced by lipopolysaccharide (LPS) or oxidized low-density lipoprotein. MiR-30a-5p-ago-treated Apo E−/− mice significantly reduced lesion areas in the aorta and aortic root, reduced levels of lipoprotein and pro-inflammatory cytokines, and increased levels of anti-inflammatory cytokines. The ratio of M1/M2 macrophages was decreased in miR-30a-5p-ago-treated Apo E−/− mice and LPS-treated RAW264.7 macrophages by the regulation of Smad-1/2 phosphorylation. MiR-30a-5p reduced lipid uptake in oxidized low-density lipoprotein-treated macrophages by regulating the expression of PPAR-γ, ABCA1, ABCG1, LDLR, and PCSK9. Ubiquitinated ligase NEDD4L was identified as a target of miR-30a-5p. Interestingly, knockdown of NEDD4L decreased the M1/M2 ratio and oxidized low-density lipoprotein uptake in macrophages by inhibiting the ubiquitination of PPAR-γ and phosphorylation of Smad-1/2 and regulating ABCA1, ABCG1, LDLR, and PCSK9. We demonstrated a novel effect and mechanism of miR-30a-5p in atherosclerosis., Graphical abstract, MiR-30a-5p attenuates atherosclerosis and decreases serum lipid and pro-inflammatory cytokines by regulating the polarization of M1/M2 macrophages and lipid metabolism. The ubiquitination ligase NEDD4L is a target of miR-30a-5p, which participates in miR-30a-5p-regulated lipid metabolism and M1/M2 macrophage polarization by mediating the ubiquitination of PPAR-γ and phosphorylation of Smad-1/2.

Background Multiple miRNAs were reported to be associated with sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) in mediating the process of heart failure. However, whether these miRNAs are relevant to chronic heart failure (CHF) is rarely reported. Methods This study was designed to explore a new biomarker from the SERCA2a related candidate miRNAs for CHF. A total of 195 blood samples from 84 CHF patients, 86 non-heart failure patients and 25 healthy subjects were collected. The circulating expression of SERCA2a related candidate miRNAs (let-7a-5p, miR-24-3p, miR-25-3p, miR-133a-3p, miR-137a-3p, miR-140-3p, miR-141-3p, miR-142-3p, miR-148a-3p, and miR-153-3p) were detected with qRT-PCR. The area under curve value, correlations between miRNAs and NT-proBNP were applied to determine the relationship between CHF and the candidate miRNAs. Results The results showed among the 10 candidate miRNAs, only let-7a-5p, miR-25-3p, and miR-140-3p were significantly increased of CHF patients. However, only miR-140-3p showed the high area under the curve (AUC) value (ROC = 0.8873) and strong positive correlations (r = 0.5113) between miR-140-3p levels and NT-proBNP concentration. The level of miR-140-3p in LVEF 45%. However, there was no significant difference between hypertension and non-hypertension of the levels of miR-140-3p in CHF patients. Interestingly, the level of miR-140-3p was higher in NYHA IV CHF patients than in NYHA II or III. More important, the miR-140-3p level also declined when the heart function of NYHA IV patients was improved after treatment. Conclusions These results indicated circulation levels of miR-140-3p is related to the severity of chronic heart failure, and miR-140-3p may be an important biomarker for heart failure with reduced ejection fraction.

Oxidative stress has a considerable influence on endothelial cell dysfunction and atherosclerosis. Acacetin, an anti‐inflammatory and antiarrhythmic, is frequently used in the treatment of myocarditis, albeit its role in managing atherosclerosis is currently unclear. Thus, we evaluated the regulatory effects of acacetin in maintaining endothelial cell function and further investigated whether the flavonoid could attenuate atherosclerosis in apolipoprotein E deficiency (apoE−/−) mice. Different concentrations of acacetin were tested on EA.hy926 cells, either induced or non‐induced by human oxidized low‐density lipoprotein (oxLDL), to clarify its influence on cell viability, cellular reactive oxidative stress (ROS) level, apoptotic ratios and other regulatory effects. In vivo, apoE−/− mice were fed either a Western diet or a chow diet. Acacetin pro‐drug (15 mg/kg) was injected subcutaneously two times a day for 12 weeks. The effects of acacetin on the atherosclerotic process, plasma inflammatory factors and lipid metabolism were also investigated. Acacetin significantly increased EA.hy926 cell viability by reducing the ratios of apoptotic and necrotic cells at 3 μmol/L. Moreover, 3 μmol/L acacetin clearly decreased ROS levels and enhanced reductase protein expression through MsrA and Nrf2 pathway through phosphorylation of Nrf2 and degradation of Keap1. In vivo, acacetin treatment remarkably attenuated atherosclerosis by increasing reductase levels in circulation and aortic roots, decreasing plasma inflammatory factor levels as well as accelerating lipid metabolism in Western diet‐fed apoE−/− mice. Our findings demonstrate the anti‐oxidative and anti‐atherosclerotic effects of acacetin, in turn suggesting its potential therapeutic value in atherosclerotic‐related cardiovascular diseases (CVD).

Our recent study showed that bradykinin increases cell cycling progression and migration of human cardiac c-Kit+ progenitor cells by activating pAkt and pERK1/2 signals. This study investigated whether bradykinin-mediated Ca2+ signalling participates in regulating cellular functions in cultured human cardiac c-Kit+ progenitor cells using laser scanning confocal microscopy and biochemical approaches. It was found that bradykinin increased cytosolic free Ca2+ ( Cai2+ ) by triggering a transient Ca2+ release from ER IP3Rs followed by sustained Ca2+ influx through store-operated Ca2+ entry (SOCE) channel. Blockade of B2 receptor with HOE140 or IP3Rs with araguspongin B or silencing IP3R3 with siRNA abolished both Ca2+ release and Ca2+ influx. It is interesting to note that the bradykinin-induced cell cycle progression and migration were not observed in cells with siRNA-silenced IP3R3 or the SOCE component TRPC1, Orai1 or STIM1. Also the bradykinin-induced increase in pAkt and pERK1/2 as well as cyclin D1 was reduced in these cells. These results demonstrate for the first time that bradykinin-mediated increase in free Cai2+ via ER-IP3R3 Ca2+ release followed by Ca2+ influx through SOCE channel plays a crucial role in regulating cell growth and migration via activating pAkt, pERK1/2 and cyclin D1 in human cardiac c-Kit+ progenitor cells.

Aim: To explore circulating miRNA-302 family members for acute heart failure (AHF) diagnosis. Methods: Three groups of subjects, in other words, AHF patients, AHF free patients and healthy controls were recruited. Circulating levels of miR-302 family members were measured and analyzed for AHF diagnosis. Results: Plasma miR-302s except miR-302f were significantly elevated in AHF patients. MiR-302b-3p had the highest area under curve value of 0.87. There were strong positive correlations between miR-302s and NT-proBNP levels. MiR-302b-3p levels were significantly higher in left ventricular ejection fraction ≤45% and New York Heart Association class IV patients compared with left ventricular ejection fraction >45% and New York Heart Association class II patients, respectively. Conclusion: Levels of circulating miR-302s, miR-302b-3p in particular, could be potentially applied for AHF diagnosis and the differentiation of disease severity.

BACKGROUND AND PURPOSE Recent studies showed that clemizole has a potential therapeutic effect for hepatitis C infection and also potently inhibits TRPC5 channels. The aim of the present study was to investigate whether clemizole blocks cardiac K+ currents and affects cardiac repolarization. EXPERIMENTAL APPROACH Whole-cell patch technique was employed to examine the effects of clemizole on hERG channel current, IKs and Kv1.5 current in HEK 293 cell expression systems as well as on ventricular action potentials of guinea pig hearts. The ex vivo guinea pig hearts were used to determine the effect on electrocardiogram. KEY RESULTS Clemizole decreased hERG current by blocking both open and closed states of the channel in a concentration-dependent manner (IC50: 0.07 μM). The S631A, S636A, Y652A and F656V hERG mutant channels reduced the inhibitory effect of clemizole (IC50: 0.82, 0.89, 1.49 and 2.98 μM, respectively), suggesting that clemizole is a pore blocker of hERG channels. Clemizole also moderately decreased IKs and hKv1.5 current. Moreover, clemizole increased ventricular action potential duration of guinea pig hearts and the QTc interval in perfused ex vivo guinea pig hearts in a concentration dependent manner (0.1-1.0 μM). CONCLUSION AND IMPLICATIONS Our results demonstrate the first evidence that clemizole potently blocks hERG channels, moderately inhibits cardiac IKs, delays cardiac repolarization, and thereby prolongs QT intervals. Thus, caution should be taken when clemizole is used as a TRPC5 channel blocker or for treating hepatitis C infection.

Background Our recent study demonstrated that the nonselective cation current mediated by the transient receptor potential canonical 1 (TRPC1) channel is activated by endothelin-1 (ET-1) in human atrial myocytes; however, the related signal molecules involved are unknown. Objective The purpose of this study was to investigate how the TRPC1 channel is regulated by ET-1 and whether it is upregulated in human atria from patients with atrial fibrillation (AF). Methods Whole-cell patch technique and molecular biology techniques were used in the study. Results The ET-1–evoked TRPC1 current was inhibited by the ET-1 type A (ETA) receptor antagonist BQ123 and the ET-1 type B (ETB) receptor antagonist BQ788 as well as the protein kinase C inhibitor chelerythrine. ETA receptor–mediated TRPC1 channel activity was selectively inhibited by the phosphoinositide-3-kinase inhibitor wortmannin, while ETB receptor–mediated TRPC1 activity was inhibited by the phospholipase C inhibitor U73122. The messenger RNAs and proteins of the TRPC1 channel and ETA receptor, but not the ETB receptor, were significantly upregulated in atria from patients with AF. The basal TRPC1 current increased in AF myocytes, and the response to ET-1 was greater in AF myocytes than in sinus rhythm myocytes. ET-1 induced a delayed repolarization in 20% of AF myocytes. Conclusion These results demonstrate for the first time that TRPC1 activation by ET-1 is mediated by protein kinase C through the distinct phospholipids pathways phosphoinositide-3-kinase and phospholipase C and that the TRPC1 channel and ETA receptor are upregulated in AF atria, which are likely involved in atrial electrical remodeling in patients with AF.

The inhibition of transient outward potassium current (Ito) is the major ionic mechanism for quinidine to treat Brugada syndrome; however, quinidine is inaccessible in many countries. The present study compared the inhibitory effect of the nonselective β-adrenergic blocker carvedilol with quinidine on human Kv4.3 (hKv4.3, encoding for Ito) channel and action potential notch using a whole-cell patch technique in HEK 293 cell line expressing K C N D 3 as well as in ventricular epicardial myocytes of rabbit hearts. It was found that carvedilol and quinidine inhibited hKv4.3 current in a concentration-dependent manner. The IC50 of carvedilol was 1.2 μM for inhibiting hKv4.3 charge area, while the IC50 of quinidine was 2.9 μM (0.2 Hz). Both carvedilol and quinidine showed typical o p e n channel blocking properties (i.e. decreasing the time to peak of activation and increasing the inactivation of hKv4.3), negatively shifted the V1/2 of activation and inactivation, and slowed the recovery from inactivation of the channel. Although carvedilol had weaker in use- and rate-dependent inhibition of hKv4.3 peak current than quinidine, its reduction of the charge area was more than quinidine at all frequencies (0.2–3.3 Hz). Moreover, the inhibitory effect of carvedilol on action potential notch was greater than quinidine. These results provide the novel information that carvedilol, like quinidine, significantly inhibits hKv4.3 and action potential notch, suggesting that carvedilol is likely an alternative drug for preventing malignant ventricular arrhythmias in patients with Brugada syndrome in countries where quinidine is unavailable.

The present study investigates the potential signal pathway of acacetin in cardioprotection against ischemia/reperfusion injury using an in vitro hypoxia/reoxygenation model in primary cultured neonatal rat cardiomyocytes and H9C2 cardiomyoblasts. It was found that acacetin (0.3–3 μM) significantly decreased the apoptosis and reactive oxygen species production induced by hypoxia/reoxygenation injury in cardiomyocytes and H9C2 cardiomyoblasts via reducing the pro-apoptotic proteins Bax and cleaved-caspase-3 and increasing the anti-apoptotic protein Bcl-2. In addition, acacetin not only suppressed the release of pro-inflammatory cytokines TLR-4 and IL-6 induced by hypoxia/reoxygenation injury, but also increased the secretion of anti-inflammatory cytokine IL-10. Moreover, acacetin increased Nrf2 and HO-1 in a concentration-dependent manner, and rescued SOD1 and SOD2 reduction induced by hypoxia/reoxygenation insult. These beneficial effects of acacetin disappeared in cells with silenced Nrf2, suggesting that Nrf2 activation participates in the cardioprotective effect of acacetin against hypoxia/reoxygenation insult. However, acacetin-induced Nrf2 activation was not observed in cells with silenced AMPK and in ventricular tissues of rat hearts treated with the AMPK inhibitor Compound C and subjected to ischemia/reperfusion injury. Our results demonstrate for the first time that AMPK-mediated Nrf2 activation is involved in the cardiomyocytes protection of acacetin against hypoxia/reoxygenation injury by activating a series of intracellular signals involved in anti-oxidation, anti-inflammation, and anti-apoptosis.

Our recent study showed that bradykinin increases cell cycling progression and migration of human cardiac c‐Kit+ progenitor cells by activating pAkt and pERK1/2 signals. This study investigated whether bradykinin‐mediated Ca2+ signalling participates in regulating cellular functions in cultured human cardiac c‐Kit+ progenitor cells using laser scanning confocal microscopy and biochemical approaches. It was found that bradykinin increased cytosolic free Ca2+ (Cai2+) by triggering a transient Ca2+ release from ER IP3Rs followed by sustained Ca2+ influx through store‐operated Ca2+ entry (SOCE) channel. Blockade of B2 receptor with HOE140 or IP3Rs with araguspongin B or silencing IP3R3 with siRNA abolished both Ca2+ release and Ca2+ influx. It is interesting to note that the bradykinin‐induced cell cycle progression and migration were not observed in cells with siRNA‐silenced IP3R3 or the SOCE component TRPC1, Orai1 or STIM1. Also the bradykinin‐induced increase in pAkt and pERK1/2 as well as cyclin D1 was reduced in these cells. These results demonstrate for the first time that bradykinin‐mediated increase in free Cai2+ via ER‐IP3R3 Ca2+ release followed by Ca2+ influx through SOCE channel plays a crucial role in regulating cell growth and migration via activating pAkt, pERK1/2 and cyclin D1 in human cardiac c‐Kit+ progenitor cells.

Clemizole, a histamine HWhole-cell patch techniques was used to examine the effects of clemizole on hERG channel current, IClemizole decreased hERG current by blocking both open and closed states of the channel in a concentration-dependent manner (ICOur results provide the first evidence that clemizole potently blocks hERG channels, moderately inhibits cardiac I