The Selective Serotonin 2A Receptor Antagonist Sarpogrelate Prevents Cardiac Hypertrophy and Systolic Dysfunction via Inhibition of the ERK1/2–GATA4 Signaling Pathway

Drug repositioning has recently emerged as a strategy for developing new treatments at low cost. In this study, we used a library of approved drugs to screen for compounds that suppress cardiomyocyte hypertrophy. We identified the antiplatelet drug sarpogrelate, a selective serotonin-2A (5-HT2A) receptor antagonist, and investigated the drug’s anti-hypertrophic effect in cultured cardiomyocytes and its effect on heart failure in vivo. Primary cultured cardiomyocytes pretreated with sarpogrelate were stimulated with angiotensin II, endothelin-1, or phenylephrine. Immunofluorescence staining showed that sarpogrelate suppressed the cardiomyocyte hypertrophy induced by each of the stimuli. Western blotting analysis revealed that 5-HT2A receptor level was not changed by phenylephrine, and that sarpogrelate suppressed phenylephrine-induced phosphorylation of ERK1/2 and GATA4. C57BL/6J male mice were subjected to transverse aortic constriction (TAC) surgery followed by daily oral administration of sarpogrelate for 8 weeks. Echocardiography showed that 5 mg/kg of sarpogrelate suppressed TAC-induced cardiac hypertrophy and systolic dysfunction. Western blotting revealed that sarpogrelate suppressed TAC-induced phosphorylation of ERK1/2 and GATA4. These results indicate that sarpogrelate suppresses the development of heart failure and that it does so at least in part by inhibiting the ERK1/2–GATA4 signaling pathway.

CircRNA circ-NNT mediates myocardial ischemia/reperfusion injury through activating pyroptosis by sponging miR-33a-5p and regulating USP46 expression

Pyroptosis has been implicated in the pathophysiology of myocardial infarction (MI) in rodents, but its contribution to reperfusion injury in MI patients is unclear. Here, we evaluated pyroptosis in MI patients in vitro and in vivo models of myocardial ischemia/reperfusion (I/R) injury. We also investigated the molecular mechanisms that regulate pyroptosis and myocardial I/R injury in these in vitro and in vivo models. The study showed that MI patients exhibited elevated serum concentrations of the pyroptosis-related pro-inflammatory cytokines IL-1β and IL-18. Increased levels of IL-1β and IL-18 as well as the pyroptosis-related inflammatory caspases (caspase-1 and 11) were detected in cultured cardiomyocytes after anoxia/reoxygenation (A/R) and in cardiac tissues after I/R. Circ-NNT and USP46 were upregulated while miR-33a-5p was downregulated in MI patients, as well as in cultured cardiomyocytes after A/R and cardiac tissues after I/R. Circ-NNT or USP46 knockdown or miR-33a-5p overexpression inhibited the expression of pro-caspase-1, cleaved caspase-1, pro-caspase-11, cleaved caspase-11, IL-1β, and IL-18 in A/R cardiomyocytes and attenuated myocardial infarction in I/R mice. The results from luciferase reporter assays and gene overexpression/knockdown studies indicated that miR-33a-5p directly targets USP46, and circ-NNT regulates USP46 by acting as a miR-33a-5p sponge. Direct association between circ-NNT and miR-33a-5p in cardiomyocytes was confirmed by pull-down assays. In summary, pyroptosis is activated during myocardial I/R and contributes to reperfusion injury. Circ-NNT promotes pyroptosis and myocardial I/R injury by acting as a miR-33a-5p sponge to regulate USP46. This circ-NNT→miR-33a-5p→USP46 signaling axis may serve as a potential target for the development of cardio-protective agents to improve the clinical outcome of reperfusion therapy.

Expression and roles of N-type Ca channel in cardiaomyocytes

Background Voltage dependent Ca channels are divided to L-, T-, N-, P/Q-, and R-types, and N-type Ca channel (NCC) is mainly expressed in nerve terminal and regulates neurotransmitter release. Recently, NCC has been reported to express in adrenal gland and renal tubular cells. We examined whether NCC is expressed in cardiac myocytes and if so, the roles of this channel. Methods Expression of NCC mRNA and protein in cardiomyocytes were assessed by quantitative real time PCR and Western blot analysis using neonatal rat cultured cardiomyocytes, infant, and adult rat hearts. Expression site of NCC in cardiomyocytes was examined by confocal imaging of immunofluorescent staining. The roles of NCC in physiological Ca transient in neonatal myocytes were examined using fluorescence imaging of Fluo4, an intracellular Ca indicator. To examine the effects of pathological condition, such as heart failure and ischemia-reperfusion, on NCC expression, cultured cardiomyocytes were treated with norepinephrine (10 μmol/L, 24 hours) or subjected to 5 hours of hypoxia followed by 30 minutes of reoxygenation. In addition, adult rats were subjected to myocardial infarction by ligating the left anterior coronary artery. Lethal myocyte injury was examined by LDH activity in culture medium and myocyte apoptosis was examined by nuclear staining with DAPI and caspase 3 activity. To clarify the roles of NCC in neonatal myocytes in these pathological conditions, we examine the effect of ω-conotoxin, a selective NCC blocker. Results NCC mRNA and protein were expressed in neonatal cardiomyocytes. Immunocytochemical staining showed NCC was expressed in myocyte plasma membrane. During physiological spontaneous beating, ω-conotoxin did not affect beating rate and intra cellular Ca transient, suggesting that the roles of NCC on physiological beating are little. After birth level of NCC mRNA expression in cardiac tissue gradually decreased within 2 weeks and low level of mRNA expressed continuously in adult cardiac tissue. However, in pathological condition, mRNA and protein levels of NCC in non-infarcted region were increased 4 weeks after myocardial infarction. In addition, hypoxia-reoxygenation and norepinephrine administration increased LDH release and myocyte apoptosis in association with increase in NCC expression in neonatal cultured myocytes. ω-conotoxin significantly suppressed hypoxia/reoxygenation- and norepinephrine-induced LDH release and caspase 3 activation. Conclusion NCC is expressed in neonatal cardiac myocytes and the expression level was decreased after birth. Pathological condition, such as ischemic heart disease and heart failure, upregulated NCC expression in cardiomyocytes and NCC exacerbated lethal myocyte injury, while roles of NCC in physiological beating are little. FUNDunding Acknowledgement Type of funding sources: None.

Abstract P381: Ecklonia Stolonifera Okamura Extract Suppresses Hypertrophic Responses In Cardiomyocytes And Development Of Heart Failure By Inhibiting P300-HAT Activity

Introduction: Heart failure is the leading cause of death in the world. Cardiomyocyte hypertrophy is observed during the development of heart failure, suggesting that its inhibition is a potential target for the prevention and treatment of heart failure. In this study, we screened a natural compound library using cultured cardiomyocytes and found that Ecklonia stolonifera Okamura extract (ESE) suppressed cardiomyocyte hypertrophy. ESE, a perennial brown alga, has been reported to have various bioactive effects, such as antioxidant and anti-inflammatory activity, but its effect on heart failure is still unclear. Therefore, we investigated whether ESE has an inhibitory effect on cardiomyocyte hypertrophic response and on the progression of heart failure in post-myocardial infarction (MI) rats. Methods and Results: First, primary cultured cardiomyocytes from neonatal rats were treated with ESE and then stimulated with phenylephrine (PE) for 48 hours. ESE (1000 μg/mL) significantly suppressed PE-induced increases in cardiomyocyte surface area, hypertrophic response gene transcription, and acetylation of histone H3K9. An in vitro p300-HAT assay indicated that ESE directly inhibited p300-HAT activity (IC50: 505 μg/mL). Next, one week after the ligation of the left anterior descending artery, rats with moderate MI (left ventricular fractioning shorting (LVFS) <40%) were randomly assigned to three groups: vehicle (saline) (n=9), ESE (0.3 g/kg) (n=10), or ESE (1 g/kg) (n=10). Daily oral administration was repeated for 8 weeks. After treatment, LVFS was significantly higher in the ESE (1 g/kg) group (23.3 ± 0.7%, p<0.05) than in the vehicle group (16.6 ± 1.3%). Next, the hearts were isolated and histological analysis, evaluation of gene transcription, and measurement of histone H3K9 acetylation. were performed. ESE treatment significantly suppressed MI-induced increases both in myocardial cell diameter and in the mRNA levels of hypertrophic response genes. ESE also inhibited MI-induced perivascular fibrosis and the acetylation of histone H3K9. Conclusion: These results suggest that ESE suppresses both hypertrophic responses in cardiomyocytes and the development of heart failure by inhibiting p300-HAT activity. Further studies are needed to clarify the effectiveness of ESE for heart failure therapy.

Abstract P454: The Interaction Of P300 With Brg1 Acetylated The Histone H3 Globular Domain In Heart Failure

Background: Epigenetic regulatory mechanisms such as histone post-translational modifications are involved in the development of heart failure. Although the acetylation of tail domains, such as H3K9, has been extensively studied, that of H3K122, the globular domain, has received much less attention. Acetylation of the globular domain directly activates transcription by destabilizing histone-DNA binding. However, the acetylation of these domains during the transition from left ventricular hypertrophy (LVH) to heart failure (HF) remains unknown. Methods and Results: Primary cultured cardiomyocytes prepared from neonatal rats were treated with phenylephrine (PE). PE increased the acetylation of H3K9 and H3K122. The acetylation of H3K9 and H3K122 on the promoters of ANF and BNP, which are hypertrophic reaction genes, was increased in cardiomyocyte hypertrophy. To investigate whether the transcriptional coactivator p300 is involved in the acetylation of H3K9 and H3K122, p300 knockdown was used. p300 knockdown suppressed PE-induced cardiomyocyte hypertrophy and the acetylation of H3K9 and H3K122. In Dahl-salt sensitive rats, in vivo chromatin-immunoprecipitation assays revealed that the acetylation of H3K9 on the promoter of the hypertrophic response genes was significantly increased in LVH, but the acetylation of H3K122 was not increased in LVH. However, H3K122 acetylation was significantly increased in HF. On the other hand, there was no difference in the amount of recruitment of p300 in LVH and HF. Interestingly, immunoprecipitation-WB showed that binding of p300 with BRG1, a key component of the SWI/SNF complex, was enhanced in HF. The recruitment of BRG1 increased significantly in HF compared to LVH. Moreover, PFI-3, a BRG1 inhibitor, significantly suppressed a PE-induced increase in cardiomyocyte surface area, the mRNA levels of ANF and BNP, and the acetylation of H3K9 and H3K122 in cultured cardiomyocytes. Conclusion: This study demonstrates that the acetylation of H3K122 is enhanced via the interaction of p300 and BRG1 in heart failure, providing novel insights into the epigenetic regulatory mechanism governing transcriptional activity in these processes.

β-Hydroxybutyrate, a Ketone Body, Potentiates the Antioxidant Defense via Thioredoxin 1 Upregulation in Cardiomyocytes

Thioredoxin 1 (Trx1) is a major antioxidant that acts adaptively to protect the heart during the development of diabetic cardiomyopathy. The molecular mechanism(s) responsible for regulating the Trx1 level and/or activity during diabetic cardiomyopathy is unknown. β-hydroxybutyrate (βHB), a major ketone body in mammals, acts as an alternative energy source in cardiomyocytes under stress, but it also appears to be involved in additional mechanisms that protect the heart against stress. βHB upregulated Trx1 in primary cultured cardiomyocytes in a dose- and a time-dependent manner and a ketogenic diet upregulated Trx1 in the heart. βHB protected cardiomyocytes against H2O2-induced death, an effect that was abolished in the presence of Trx1 knockdown. βHB also alleviated the H2O2-induced inhibition of mTOR and AMPK, known targets of Trx1, in a Trx1-dependent manner, suggesting that βHB potentiates Trx1 function. It has been shown that βHB is a natural inhibitor of HDAC1 and knockdown of HDAC1 upregulated Trx1 in cardiomyocytes, suggesting that βHB may upregulate Trx1 through HDAC inhibition. βHB induced Trx1 acetylation and inhibited Trx1 degradation, suggesting that βHB-induced inhibition of HDAC1 may stabilize Trx1 through protein acetylation. These results suggest that βHB potentiates the antioxidant defense in cardiomyocytes through the inhibition of HDAC1 and the increased acetylation and consequent stabilization of Trx1. Thus, modest upregulation of ketone bodies in diabetic hearts may protect the heart through the upregulation of Trx1.

Effect of miRNA-223-3p Targeting Stromal Interaction Molecule 1 on Proliferation and Apoptosis of Hypoxia/Reoxygenation-Applied H9c2 Cardiomyocytes

This study was established to determine the effect of miRNA-223-3p on the proliferation and apoptosis of hypoxia/reoxygenation-applied H9c2 cardiomyocytes and the associated mechanisms. A hypoxia/reoxygenation (H/R) model was established, with normal cells also used as a control. miRNA-NC, miRNA-223-3p, anti-miRNA-NC, and anti-miRNA-223-3p plasmids were transfected into normally cultured cardiomyocytes, defined as the miRNA-NC, miRNA-223-3p, anti-miRNA-NC, and anti-miRNA-223-3p groups. In addition, miRNA-223-3p was co-transfected into normally cultured cardiomyocytes with pcDNA3.1 and pcDNA3.1-STIM1 plasmids, followed by treatment with H/R for cells in the miR-NC and miR-223-3p groups, defined as the H/R+miRNA-NC, H/R+miRNA-223-3p, H/R+miRNA-223-3p+pcDNA3.1, and H/R+miRNA-223-3p+pcDNA3.1-STIM1 groups. A liposome method was adopted for assessing transfection. qRT-PCR was used to detect miRNA-223-3p expression, while western blotting was used to detect protein expression. MTT assay was used to detect cell viability, flow cytometry to detect apoptosis, and dual luciferase reporter gene assay to detect fluorescence activity. After H/R treatment, miR-223-3p, cyclin D1, and Bcl-2 expression of cardiomyocytes decreased, p21 and Bax expression significantly increased, cell activity decreased, and the apoptosis rate increased. miRNA-223-3p achieved the targeted regulation of STIM1 expression. miRNA-223-3p overexpression promoted the H/R-induced cardiomyocyte proliferation and inhibited cardiomyocyte apoptosis. STIM1 overexpression reversed the proliferation-promoting and apoptosis-inhibiting effects of miRNA-223-3p on cardiomyocytes treated with H/R. The findings show that miRNA-223-3p overexpression promotes H/R-induced cell proliferation, inhibits apoptosis, and protects H/R-induced cardiomyocytes from injury, via a mechanism probably associated with STIM1 expression. miRNA-223-3p thus provides a new target for treating cardiomyocyte injury.