β-catenin ameliorates myocardial infarction by preventing YAP-associated apoptosis

Abstract Objective: To explore whether the effect of β-catenin on MI and MI-induced cardiomyocyte apoptosis is YAP-dependent. Methods: The authors established an MI rat model by ligating the anterior descending branch of the left coronary artery, and an MI cell model by treating cardiomyocytes with H2O2. Results: β-catenin downregulation was observed in MI cardiac tissues and in H2O2-treated cardiomyocytes. Lentiviral-CTNNB1 was administered to MI rats to upregulate β-catenin expression in MI cardiac tissue. β-catenin recovery reduced the myocardial infarct area, fibrosis, and apoptotic cell death in MI rats. H2O2 treatment attenuated cell viability and induced cell death in cardiomyocytes, whereas β-catenin overexpression partially reversed these changes. Moreover, H2O2 treatment caused the deactivation of Yes-Associated Protein (YAP), as detected by increased YAP phosphorylation and reduced the nuclear localization of YAP. Upregulation of β-catenin expression reactivated YAP in H2O2-treated cardiomyocytes. Reactivation of YAP was achieved by administration of Mitochonic Acid-5 (MA-5) to H2O2-treated cardiomyocytes, and deactivation of YAP by CIL56 treatment in β-catenin-overexpressing H2O2-treated cardiomyocytes. MA-5 administration increased cell viability and repressed apoptosis in H2O2-treated cardiomyocytes, whereas CIL56 treatment counteracted the effects of β-catenin overexpression on cell survival and apoptosis. Conclusions: The present data indicate that β-catenin and YAP are effective treatment targets for MI, blocking the apoptotic death of cardiomyocytes.

Cytopathic effect of rotavirus NSP4 86-175 protein on rat cardiomyocytes and the possible mechanism / 中华微生物学和免疫学杂志

Objective:To investigate the cytopathic effect of amino acids 86-175 of rotavirus non-structural protein 4 (NSP4 86-175) on rat cardiomyocytes and the possible mechanism. Methods:Rat H9C2 cardiomyocytes were treated with NSP4 86-175 protein. Changes in the growth and morphology of the cells were observed. The activity of LDH in the cell culture medium was detected. Fluo-3AM was used to label intracellular free calcium ions and the concentrations of calcium ions in rat cardiomyocytes with and without NSP4 86-175 treatment were detected by confocal laser microscopy. The expression of Bax, Bcl-2, caspase-3, 78 kDa glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP) and caspase-12 at mRNA level was detected by real-time PCR. The expression of caspase-3, caspase-8, caspase-9, GRP78, CHOP and caspase-12 at protein level was detected by Western blot. Results:Normal cardiomyocytes showed a typical myoblast-like morphology, presenting as spindle-shaped cells with clear boundaries. Obvious cytopathic effect, vacuolar degeneration, shriveled and rounded cells, and cell fragmentation were observed after the treatment with purified NSP4 86-175 protein. The activity of LDH in cell culture medium was enhanced by NSP4 86-175 protein. NSP4 86-175 protein also enhanced the fluorescence of the calcium ions in rat cardiomyocytes, promoted cell apoptosis, up-regulated the expression of apoptotic factors including caspase-3, caspase-8, caspase-9 and Bax-2, and increased the expression of classical markers of endoplasmic reticulum stress such as GRP78, CHOP and caspase-12. Conclusions:NSP4 86-175 had a cytopathic effect on rat cardiomyocytes, which might be related to the induced intracellular calcium overload, endoplasmic reticulum stress, apoptosis and necrosis. These results might be used as theoretical reference for further study on rotavirus infection and myocardial injury.

Liraglutide ameliorates high glucose-induced oxidative stress injury in rat H9c2 cells through modulation of SIRT1 / 中华内分泌代谢杂志

Objective:To investigate the effect of liraglutide(LRG) on high glucose-induced oxidative stress injury in(H9c2) cardiomyocytes and its underlying mechanisms.Methods:A high glucose treatment was applied to H9c2 cells for 24 hours to establish an in vitro model of myocardial cell injury. Different concentrations of liraglutide(10, 100, 1000 nmol/L) were administered for intervention. Cell viability was evaluated using the CCK-8 assay, and changes in cell morphology were observed under an inverted microscope. After 24 hours of liraglutide(100 nmol/L) intervention following high glucose treatment, the levels of lactate dehydrogenase(LDH), superoxide dismutase(SOD), and malondialdehyde(MDA) in the cell supernatant were measured. RT-PCR and Western blotting were used to detect the mRNA and protein levels of silent information regulator factor 1(SIRT1) and forkhead box protein O1(FOXO1). Western blotting was also used to assess the acetylation level of FOXO1 protein. Small interfering RNA(siRNA) technology was employed to silence SIRT1 in H9c2 cells to confirm its role in the study. Results:Compared to the control group, the high glucose group showed decreased cell viability, cell structure damage, increased levels of LDH and MDA in the cell supernatant, decreased SOD levels, aggravated oxidative stress, decreased SIRT1 expression, and increased acetylation level of FOXO1(all P<0.05). Compared to the high glucose group, liraglutide intervention resulted in increased cell viability, improved cardiac cell morphology, reduced oxidative stress levels, increased SIRT1 expression, and decreased acetylation level of FOXO1(all P<0.05). When SIRT1 was downregulated, the protective effects of liraglutide were weakened(all P<0.05). Conclusions:Liraglutide has a protective effect against high glucose-induced oxidative stress injury in H9c2 cells, which may be associated with the upregulation of SIRT1 expression.

To investigate the effect of Astragaloside Ⅳ on high-glucose-induced cardiomyocytes pyroptosis / 中华老年医学杂志

Objective:To investigate the effect of Astragaloside Ⅳ on high glucose-induced cardiomyocyte pyroptosis.Methods:H9c2 cells were cultured in vitro and divided into control group(5.5 mmol/L glucose), high glucose group(33.3 mmol/L glucose), Astragaloside Ⅳ group(33.3 mmol/L glucose+ 100μmol/L Astragaloside Ⅳ), and NLRP3 inhibitor group(33.3 mmol/L glucose+ 1μmol/L MCC950). Cell counting kit 8(CCK-8)was used to detect the activity of H9c2 cells.Lactate dehydrogenase(LDH)kit was used to detect the content of LDH in cell supernatant.Superoxide anion fluorescent probe(DHE)was used to detect the level of intracellular reactive oxygen species(ROS). Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and Western blot were used to detect the mRNA and protein expression levels of pyroptosis-related genes.Immunofluorescence was used to detect the fluorescence intensity of NLRP3.Enzyme-linked immunosorbent assay(ELISA)was used to detect the level of inflammatory factors in cell supernatant.Results:When the concentration of Astragaloside Ⅳ was 100 μmol/L, it could significantly inhibit the decrease of cardiomyocyte viability induced by high glucose( P<0.01)and reduce LDH release( P<0.01). Compared with the control group, the level of ROS was increased( P<0.01), the mRNA and protein expressions of pyroptosis-related molecules were up-regulated( P<0.01 for all), the fluorescence intensity of NLRP3 was increased( P<0.01), and the levels of inflammatory factors in the cell supernatant were increased in the high glucose group( P<0.01). Compared with the high glucose group, the ROS level was decreased( P<0.01), the mRNA and protein expressions of pyroptosis-related molecules were down-regulated( P<0.05 or P<0.01), the fluorescence intensity of NLRP3 was decreased( P<0.01), and the levels of inflammatory factors in cell supernatant were decreased( P<0.05 or P<0.01)in Astragaloside Ⅳ group and inhibitor group. Conclusions:Astragaloside Ⅳ plays a protective role in high glucose-induced cardiomyocyte injury by inhibiting NLRP3/Caspase-1 signaling pathway and inhibiting pyroptosis.Moreover, it can improve the anti-inflammatory and antioxidant properties in cell models.

Effects of tropomyosin 3 on pyroptosis of cardiomyocytes and fibroblast activation induced by hypoxia/reoxygenation in rats / 中华危重病急救医学

Objective:To explore the role of tropomyosin 3 (TPM3) in hypoxia/reoxygenation (H/R)-induced cardiomyocyte pyroptosis and fibroblast activation.Methods:Rat cardiomyocytes (H9c2 cells) were treated with H/R method to simulate myocardial ischemia/reperfusion (I/R) injury, and cell proliferation activity was evaluated with cell counting kit-8 (CCK8). The expression of TPM3 mRNA and protein was detected by quantitative real-time polymerase chain reaction (RT-qPCR) and Western blotting. H9c2 cells with stable TPM3-short hairpin RNA (shRNA) expression were constructed and treated with H/R (hypoxia for 3 hours, and reoxygenation for 4 hours). The expression of TPM3 was measured by RT-qPCR. The expressions of TPM3, pyroptosis-related proteins including caspase-1, NOD-like receptor protein 3 (NLRP3) and Gasdermin family proteins-N (GSDMD-N) were measured by Western blotting. The expression of caspase-1 was also observed by immunofluorescence assay. The levels of human interleukins (IL-1β, IL-18) in the supernatant were determined by enzyme-linked immunosorbent assay (ELISA) to elucidate the effect of sh-TPM3 on pyroptosis of cardiomyocytes. Rat myocardial fibroblasts were incubated with the above cell supernatant, and the expressions of human collagen Ⅰ, collagen Ⅲ, matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase inhibitor 2 (TIMP2) were detected by Western blotting to determine the effect of TPM3-interfered cardiomyocytes on the activation of fibroblasts under H/R conditions.Results:Compared with the control group, H/R treatment for 4 hours significantly decreased the survival rate of H9c2 cells [(25.81±1.90)% vs. (99.40±5.54)%, P < 0.01], promoted the expression of TPM3 mRNA and protein [TPM3/GAPDH (2 -ΔΔCt): 3.87±0.50 vs. 1, TPM3/β-Tubulin: 0.45±0.05 vs. 0.14±0.01, both P < 0.01], and promoted the expressions of caspase-1, NLRP3, GSDMD-N, and the enhanced release of cytokines IL-1β and IL-18 [cleaved caspase-1/caspase-1: 0.89±0.04 vs. 0.42±0.03, NLRP3/β-Tubulin: 0.39±0.03 vs. 0.13±0.02, GSDMD-N/β-Tubulin: 0.69±0.05 vs. 0.21±0.02, IL-1β (μg/L): 13.84±1.89 vs. 4.31±0.33, IL-18 (μg/L): 17.56±1.94 vs. 5.36±0.63, all P < 0.01]. However, compared with the H/R group, sh-TPM3 significantly weakened the promoting effects of H/R on these proteins and cytokines [cleaved caspase-1/caspase-1: 0.57±0.05 vs. 0.89±0.04, NLRP3/β-Tubulin: 0.25±0.04 vs. 0.39±0.03, GSDMD-N/β-Tubulin: 0.27±0.03 vs. 0.69±0.05, IL-1β (μg/L): 8.56±1.22 vs. 13.84±1.89, IL-18 (μg/L): 9.34±1.04 vs. 17.56±1.94, all P < 0.01]. In addition, the expressions of collagen Ⅰ, collagen Ⅲ, TIMP2, and MMP-2 in myocardial fibroblasts were significantly increased by the cultured supernatants from the H/R group (collagen Ⅰ/β-Tubulin: 0.62±0.05 vs. 0.09±0.01, collagen Ⅲ/β-tubulin: 0.44±0.03 vs. 0.08±0.00, TIMP2/β-tubulin: 0.73±0.04 vs. 0.20±0.03, TIMP2/β-Tubulin: 0.74±0.04 vs. 0.17±0.01, all P < 0.01). However, these boosting effects were weakened by sh-TPM3 (collagen Ⅰ/β-Tubulin: 0.18±0.01 vs. 0.62±0.05, collagen Ⅲ/β-Tubulin: 0.21±0.03 vs. 0.44±0.03, TIMP2/β-Tubulin: 0.37±0.03 vs. 0.73±0.04, TIMP2/β-Tubulin: 0.45±0.03 vs. 0.74±0.04, all P < 0.01). Conclusion:Interference with TPM3 can alleviate H/R-induced cardiomyocyte pyroptosis and fibroblast activation, suggesting that TPM3 may be a potential target of myocardial I/R injury.

La miocardiopatía hipertrófica induce cambios en la expresión de la triptófano-5-hidroxilasa, el transportador de serotonina y receptores serotoninérgicos / Hypertrophic cardiomyopathy induces changes in the tryptophan-5-hydroxylase, serotonin transporter and serotonergic receptors expressions

Resumen Introducción: Los cardiomiocitos poseen la maquinaria bioquímica capaz de sintetizar, utilizar y recapturar serotonina. Objetivo: Determinar si la miocardiopatía hipertrófica (MCH) induce cambios en la expresión de la triptófano-5-hidroxilasa (TPH) 1 y 2, el transportador de serotonina (SERT) y los receptores serotoninérgicos (RS). Métodos: Estudio transversal de cinco bloques de tejido de corazones con MCH y cinco bloques de corazones de control. Se obtuvieron cinco cortes de la pared libre del ventrículo izquierdo (PLVI) y del septum interventricular (SIV) de cada bloque, para determinar la expresión de TPH1 y TPH2, SERT y RS con anticuerpos por inmunofluorescencia. La inmunofluorescencia fue evaluada mediante t de WELCH, con nivel de significación de p < 0.05. Resultados: La PLVI y el SIV de los corazones con MCH mostraron aumento de la expresión de TPH1 y TPH2, así como de los receptores 5-HT2A y 5-HT2B en comparación con los controles (p < 0.01). El receptor 5-HT4 y SERT aumentaron en el SIV de los corazones con MCH (p < 0.01). Conclusiones: Se demostró aumento de las expresiones de TPH, SERT y RS en los cardiomiocitos de los corazones con MCH en comparación con los controles, lo cual podría participar en la fisiopatología de la MCH en los humanos.

Abstract Introduction: Cardiomyocytes have a biochemical machinery with the capacity to synthesize, utilize and reuptake serotonin. Objective: To determine whether hypertrophic cardiomyopathy (HCM) induces changes in the expression of tryptophan-5-hydroxylase (TPH) 1 and 2, serotonin transporter (SERT) and serotonergic receptors (SR). Methods: Cross-sectional study of five tissue blocks from hearts with HCM and five controls. Five sections of the left ventricular free wall (LVFW) and interventricular septum (IVS) were obtained from each block to determine the expression of TPH1 and TPH2, SERT and SRs by immunofluorescence with specific antibodies. Immunofluorescence was evaluated by WELCH t-test, with a level of significance of p < 0.05. Results: LVFW and IVS of hearts with HCM showed an increase in the expression of TPH1 and TPH 2 and 5-HT2A and 5-HT2B receptors in comparison with controls (p < 0.01). The 5-HT4 receptor and SERT showed an increase in the IVS of hearts with HCM (p < 0.01). Conclusions: This study demonstrated an increased expression of TPH, SERT and SRs in cardiomyocytes from hearts with HCM in comparison with controls, which could be involved in the pathophysiology of HCM in humans.

Cenário Disfuncional dos Principais Componentes Responsáveis pelo Equilíbrio do Trânsito de Cálcio Miocárdico na Insuficiência Cardíaca Induzida por Estenose Aórtica / The Dysfunctional Scenario of the Major Components Responsible for Myocardial Calcium Balance in Heart Failure Induced by Aortic Stenosis

Resumo Fundamento O remodelamento cardíaco patológico se caracteriza por disfunção diastólica e sistólica, levando à insuficiência cardíaca. Neste contexto, o cenário disfuncional do trânsito de cálcio miocárdico (Ca2+) tem sido pouco estudado. Um modelo experimental de estenose aórtica tem sido extensamente utilizado para aprimorar os conhecimentos sobre os principais mecanismos do remodelamento patológico cardíaco. Objetivo Entender o processo disfuncional dos principais componentes responsáveis pelo equilíbrio do cálcio miocárdico e sua influência sobre a função cardíaca na insuficiência cardíaca induzida pela estenose aórtica. Métodos Ratos Wistar de 21 dias de idade foram distribuídos em dois grupos: controle (placebo; n=28) e estenose aórtica (EaO; n=18). A função cardíaca foi analisada com o ecocardiograma, músculo papilar isolado e cardiomiócitos isolados. No ensaio do músculo papilar, SERCA2a e a atividade do canal de Ca2+ do tipo L foram avaliados. O ensaio de cardiomiócitos isolados avaliou o trânsito de cálcio. A expressão proteica da proteínas do trânsito de cálcio foi analisada com o western blot. Os resultados foram estatisticamente significativos quando p <0,05. Resultados Os músculos papilares e cardiomiócitos dos corações no grupo EaO demonstraram falhas mecânicas. Os ratos com EaO apresentaram menor tempo de pico do Ca2+, menor sensibilidade das miofibrilas do Ca2+, prejuízos nos processos de entrada e recaptura de cálcio pelo retículo sarcoplasmático, bem como disfunção no canal de cálcio do tipo L (CCTL). Além disso, os animais com EaO apresentaram maior expressão de SERCA2a, CCTL e trocador de Na+/Ca2+. Conclusão Insuficiência cardíaca sistólica e diastólica devido à estenose aórtica supravalvular acarretou comprometimento da entrada de Ca2+ celular e inibição da recaptura de cálcio pelo retículo sarcoplasmático devido à disfunção no CCTL e SERCA2a, assim como mudanças no trânsito de cálcio e na expressão das principais proteínas responsáveis pela homeostase de Ca2+ celular.

Abstract Background Maladaptive cardiac remodelling is characterized by diastolic and systolic dysfunction, culminating in heart failure. In this context, the dysfunctional scenario of cardiac calcium (Ca2+) handling has been poorly studied. An experimental model of aortic stenosis has been extensively used to improve knowledge about the key mechanisms of cardiac pathologic remodelling. Objective To understand the dysfunctional process of the major components responsible for Ca2+ balance and its influence on cardiac function in heart failure induced by aortic stenosis. Methods Male 21-day-old Wistar rats were distributed into two groups: control (sham; n= 28) and aortic stenosis (AoS; n= 18). Cardiac function was analysed by echocardiogram, isolated papillary muscle, and isolated cardiomyocytes. In the papillary muscle assay, SERCA2a and L-type Ca2+ channel activity was evaluated. The isolated cardiomyocyte assay evaluated Ca2+ handling. Ca2+ handling protein expression was analysed by western blot. Statistical significance was set at p <0.05. Results Papillary muscles and cardiomyocytes from AoS hearts displayed mechanical malfunction. AoS rats presented a slower time to the Ca2+ peak, reduced Ca2+ myofilament sensitivity, impaired sarcoplasmic reticulum Ca2+ influx and reuptake ability, and SERCA2a and L-type calcium channel (LTCC) dysfunction. Moreover, AoS animals presented increased expression of SERCA2a, LTCCs, and the Na+/Ca2+ exchanger. Conclusion Systolic and diastolic heart failure due to supravalvular aortic stenosis was paralleled by impairment of cellular Ca2+ influx and inhibition of sarcoplasmic reticulum Ca2+ reuptake due to LTCC and SERCA2a dysfunction, as well as changes in Ca2+ handling and expression of the major proteins responsible for cellular Ca2+ homeostasis.

Study on protective effects of methyl ferulate on mitochondria of H 9c2 cardiomyocytes injured by hypoxia / 中国药房

OBJECTIVE To investigate the e ffects of methyl ferulate （MF） on the mitochondrial function of H 9c2 cardiomyocytes after hypoxia-induced injury. METHODS H9c2 cardiomyocytes were divided into normal group （no administration，no modeling ），hypoxia model group （modeling alone ），MF high-dose ，medium-dose and low-dose groups （40， 20，10 μmol/L）and positive control drug group （cyclosporin A ，1 μmol/L）. After drug pretreatment and inducing hypoxia-induced injury，the levels of lactate dehydrogenase （LDH），malondialdehyde（MDA），creatine kinase （CK）and adenosine triphosphate （ATP）were tested. The intracellular reactive oxygen species （ROS），mitochondrial membrane potential （MMP），the opening of mitochondrial membrane permeability transition pore （mPTP） were detected with flow cytometry. RESULTS Compared with hypoxia model group ，the levels of LDH ，MDA，CK and ROS fluorescence intensity were decreased significantly in MF high-dose，medium-dose and low-dose groups ，while the level of ATP was increased significantly （P＜0.01 or P＜0.05）. The red/ green fluorescence intensity ratio of MMP and the green fluorescence intensity of mPTP were increased significantly （P＜0.01 or P＜0.05）. CONCLUSIONS MF can reverse the levels of biochemical indexes in H 9c2 cardiomyocyte after hypoxia-induced injury，keep MMP stable ，reduce the opening of mPTP ，and has an obvious protective effect on the mitochondrial function of H9c2 cardiomyocytes injured by hypoxia ，and this protective effect is dose-dependent.

Basic biological characteristics of lncRNA B230352I09 and its role in the process of myocardial injury / 中华急诊医学杂志

Objective:To explore the basic biological characteristics of lncRNA B230352I09 and its role in the process of myocardial injury.Methods:We analyzed the biological characteristics of lncRNA B230352I09 on the UCSC website and predicted the possible binding protein of lncRNA B230352I09 by the catRAPID. Real-time fluorescence quantitative (RT) PCR method was applied to detect the expression of lncRNA B230352I09 in heart tissues at different time points (0, 1, 3, 7d) within 7 days after birth, the organs distribution and expression of lncRNA B230352I09 in neonatal mouse and the expression pattern of lncRNA B230352I09 in the heart of mice with myocardial injury. In addition, we constructed hypoxia model by culturing primary cardiomyocytes to detect the effect of lncRNA 230352I09 overexpression on hypoxic cardiomyocyte apoptosis by Hoechst staining kit, the effect of lncRNA B230352I09 overexpression on ROS content of hypoxic cardiomyocyte by DCFDA probe and changes in mitochondrial membrane potential of hypoxic cardiomyocytes by JC-1 Fluorescent probes.Results:Full-length of mouse B230352I09 was 663bp, located in the chr7:123031415-123066439 forward strand. RBBP6 gene was adjacent to B230352I09, which may be the target of lncRNA B230352I09 by catrapid prediction analysis. With the development of the heart, the expression level of lncRNA B230352I09 showed a gradual downward trend. The main expression organs of lncRNA B230352I09 in 1-day-old mice were heart, brain, kidney and liver. In heart tissue, lncRNA B230352I09 expression in non-cardiomyocytes was significantly less than in cardiomyocytes [ (1.0± 0.03) vs. (9.2± 3.29), P=0.013]. After myocardial injury, the expression level of lncRNA B230352I09 showed an increasing trend compared with the normal developing mice, but there was no statistical significance. Hoechst staining showed that lncRNA B230352I09 could inhibit the apoptosis of hypoxic cardiomyocytes. Detecting the content of ROS in cardiomyocytes showed that compared with the hypoxia group, the generation of ROS was significantly reduced in the lncRNA B230352I09 overexpression group ([(3.8±0.71) vs. (1.65±0.56), P=0.015]). JC-1 fluorescent probe was used to detect the mitochondrial membrane potential, and the results showed that the mitochondrial membrane potential of cardiomyocytes in the lncRNA B230352I09 overexpression group was significantly higher than that in the hypoxia group. Conclusions:In heart tissue, lncRNA B230352I09 was mainly expressed in cardiomyocytes. LncRNA B230352I09 has a protective effect in the process of myocardial injury in mice, mainly by inhibiting apoptosis of cardiomyocytes, reducing ROS production, and protecting mitochondrial membrane potential of cardiomyocytes.

Mechanism of Aconiti Kusnezoffii Radix Processed with Chebulae Fructus Against H9c2 Cardiomyocyte Toxicity Based on TRPV1 Channel / 中国实验方剂学杂志

ObjectiveTo explore the role of transient receptor potential vanilloid 1 （TRPV1） channel in reducing cardiomyocyte toxicity of Aconiti Kusnezoffii Radix processed with Chebulae Fructus. MethodH9c2 cardiomyocytes cultured in vitro were used as a model to assess cell viability by methyl thiazolyl tetrazolium （MTT） assay， the expression of TRPV1 mRNA was detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and the leakage rate of lactate dehydrogenase （LDH）， the changes of nucleus， reactive oxygen species （ROS）， mitochondrial membrane potential and Ca2+ contents were detected by enzyme linked immunosorbent assay （ELISA）. ResultCompared with the blank group， when the concentration was ≥0.5 g·L-1， the cell viability was significantly decreased （P<0.01）， the leakage rate of LDH， the release of ROS and Ca2+ were increased， the mitochondrial membrane potential was decreased， and the nucleus was pyknosis or even broken in raw Aconiti Kusnezoffii Radix and Aconiti Kusnezoffii Radix processed with Chebulae Fructus groups. When the concentration was ≥0.5 g·L-1， compared with the same mass concentration of raw Aconiti Kusnezoffii Radix group， the cell viability increased significantly （P<0.01）， the leakage rate of LDH， the release of ROS and Ca2+ decreased， the mitochondrial membrane potential increased， and the nuclear morphology improved in Aconiti Kusnezoffii Radix processed with Chebulae Fructus group. Application of the same mass concentration of raw Aconiti Kusnezoffii Radix to H9c2 cardiomyocytes pretreated with the TRPV1 inhibitor BCTC significantly increased cell viability， decreased leakage rate of LDH， ROS and Ca2+ release， increased mitochondrial membrane potential and improved nuclear pyknosis compared with untreated H9c2 cardiomyocytes. Application of the same mass concentration of Aconiti Kusnezoffii Radix processed with Chebulae Fructus to H9c2 cardiomyocytes pretreated with BCTC decreased cell viability， increased LDH leakage rate， ROS and Ca2+ release， reduced mitochondrial membrane potential compared with untreated H9c2 cardiomyocytes. Real-time PCR results showed that both raw Aconiti Kusnezoffii Radix and Chebulae Fructus decoction could increase the expression of TRPV1 mRNA in cardiomyocytes in a concentration dependent manner. ConclusionRaw Aconiti Kusnezoffii Radix can induce cardiomyocyte apoptosis and cardiotoxicity by activating TRPV1 channel， while Aconiti Kusnezoffii Radix processed with Chebulae Fructus can attenuate the toxicity through TRPV1 channel， which may be related to the synergistic effect of acid components in Chebulae Fructus and alkaloids in Aconiti Kusnezoffii Radix on TRPV1 channel.

Ginsenoside Rg1 Reduces Cardiotoxicity While Increases Cardiotonic Effect of Aconitine in vitro / 中国结合医学杂志

OBJECTIVE@#To explore the synergic mechanism of ginsenoside Rg1 (Rg1) and aconitine (AC) by acting on normal neonatal rat cardiomyocytes (NRCMs) and pentobarbital sodium (PS)-induced damaged NRCMs.@*METHODS@#The toxic, non-toxic, and effective doses of AC and the most suitable compatibility concentration of Rg1 for both normal and damaged NRCMs exposed for 1 h were filtered out by 3- (4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide, respectively. Then, normal NRCMs or impaired NRCMs were treated with chosen concentrations of AC alone or in combination with Rg1 for 1 h, and the cellular activity, cellular ultrastructure, apoptosis, leakage of acid phosphatase (ACP) and lactate dehydrogenase (LDH), intracellular sodium ions [Na+], potassium ions [K+] and calcium ions [Ca2+] levels, and Nav1.5, Kv4.2, and RyR2 genes expressions in each group were examined.@*RESULTS@#For normal NRCMs, 3000 µ mol/L AC significantly inhibited cell viability (P<0.01), promoted cell apoptosis, and damaged cell structures (P<0.05), while other doses of AC lower than 3000 µ mol/L and the combinations of AC and Rg1 had little toxicity on NRCMs. Compared with AC acting on NRCMs alone, the co-treatment of 3000 and 10 µ mol/L AC with 1 µ mol/L Rg1 significantly decreased the level of intracellular Ca2+ (P<0.01 or P<0.05), and the co-treatment of 3000 µ mol/L AC with 1 µ mol/L Rg1 significantly decreased the level of intracellular Ca2+ via regulating Nav1.5, RyR2 expression (P<0.01). For damaged NRCMs, 1500 µ mol/L AC aggravated cell damage (P<0.01), and 0.1 and 0.001 µ mol/L AC showed moderate protective effect. Compared with AC used alone, the co-treatment of Rg1 with AC reduced the cell damage, 0.1 µ mol/L AC with 1 µ mol/L Rg1 significantly inhibited the level of intracellular Na+ (P<0.05), 1500 µ mol/L AC with 1 µ mol/L Rg1 significantly inhibited the level of intracellular K+ (P<0.01) via regulating Nav1.5, Kv4.2, RyR2 expressions in impaired NRCMs.@*CONCLUSION@#Rg1 inhibited the cardiotoxicity and enhanced the cardiotonic effect of AC via regulating the ion channels pathway of [Na+], [K+], and [Ca2+].

Palmitic acid suppresses autophagy in neonatal rat cardiomyocytes via the cGAS-STING-IRF3 pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of palmitic acid (PA) on autophagy in neonatal rat cardiomyocytes (NRCMs) and explore the underlying mechanism.@*METHODS@#NRCMs were isolated and cultured for 24 h before exposure to 10% BSA and 0.1, 0.3, 0.5, or 0.7 mmol/L PA for 24 h. After the treatments, the expressions of Parkin, PINK1, p62, LC3Ⅱ/ LC3Ⅰ, cGAS, STING and p-IRF3/IRF3 were detected using Western blotting and the cell viability was assessed with CCK8 assay, based on which 0.7 mmol/L was selected as the optimal concentration in subsequent experiments. The effects of cGAS knockdown mediated by cGAS siRNA in the presence of PA on autophagy-related proteins in the NRCMs were determined using Western blotting, and the expressions of P62 and LC3 in the treated cells were examined using immunofluorescence assay.@*RESULTS@#PA at different concentrations significantly lowered the expressions of Parkin, PINK1, LC3 Ⅱ/LC3 Ⅰ and LC3 Ⅱ/LC3 Ⅰ+Ⅱ (P < 0.05), increased the expression of p62 (P < 0.05), and inhibited the viability of NRCMs (P < 0.05). Knockdown of cGAS obviously blocked the autophagy-suppressing effect of PA and improved the viability of NRCMs (P < 0.05).@*CONCLUSION@#PA inhibits autophagy by activating the cGAS-STING-IRF3 pathway to reduce the viability of NRCMs.

Efeito Cardioprotetor da Suplementação Materna com Resveratrol sobre a Toxicidade Induzida por Doxorrubicina em Cardiomiócitos de Neonatos / Cardioprotective Effect of Maternal Supplementation with Resveratrol on Toxicity Induced by Doxorubicin in Offspring Cardiomyocytes

Resumo Fundamento A doxorrubicina (DOX) é frequentemente usada para tratar muitos tipos de cânceres, apesar da cardiotoxicidade dose-dependente. Como alternativa, o resveratrol é um polifenol que tem demonstrado efeitos cardioprotetores em vários modelos de disfunção cardíaca. Objetivo Este estudo investigou se o tratamento com resveratrol em ratas gestantes protege contra toxicidade induzida por doxorrubicina em cardiomiócitos da ninhada. Métodos Ratas Wistar (n-8) receberam sresveratrol como suplemento alimentar durante a gestação. No nascimento da ninhada, os corações (9-11) foram usados para se obter a cultura primária de cardiomiócitos. A cardiotoxicidade induzida por DOX e os efeitos da suplementação com resveratrol foram avaliados por marcadores de stress oxidativo, tais como oxidação da diclorofluoresceína diacetato, diminuição da atividade de enzimas antioxidantes, e oxidação do teor total de grupos sulfidrila, além da avaliação da viabilidade celular, geração de danos ao DNA, bem como a resposta de reparo aos danos ao DNA. Um valor de p <0,05 foi considerado estatisticamente significativo. Resultados Os cardiomiócitos de neonatos de ratas que receberam suplemento resveratrol apresentaram um aumento (p <0,01) na viabilidade das células, e diminuição (p <0,0001) de células apoptóticas/necróticas após o tratamento com DOX, o que está correlacionado às atividades de enzimas antioxidantes e produção de diclorofluoresceína. Além disso, o resveratrol protegeu os cardiomiócitos de danos ao DNA induzidos por DOX, apresentando uma diminuição (p <0,05) nas quebras de DNA induzidas por stress oxidativo, avaliadas pela atividade de enzimas reparadoras do DNA endonuclease III e formamidopirimidina glicosilase. A suplementação com resveratrol aumentou (p <0,05) a expressão da proteína reparadora Sirt6 nos cardiomiócitos dos filhotes. Conclusão Essa pesquisa indica que a suplementação com resveratrol durante o período gestacional tem um efeito cardioprotetor no coração da ninhada contra a toxicidade induzida por DOX, o que pode se dever a sua função antioxidante, e o aumento na resposta de danos ao DNA.

Abstract Background Doxorubicin (DOX) is frequently used to treat many types of cancers, despite its dose-dependent cardiotoxicity. Alternatively, resveratrol is a polyphenol that has shown useful cardioprotective effects in many heart dysfunction models. Objective This study investigated whether resveratrol treatment in pregnant rats protects against doxorubicin-induced toxicity in offspring cardiomyocytes. Methods Wistar rats (n=8) were supplemented with dietary resveratrol during pregnancy. Upon the offspring's birth, hearts (9-11) were used to obtain the primary culture of cardiomyocytes. DOX-induced cardiotoxicity and the effects of resveratrol supplementation were evaluated by oxidative stress markers, such as dichlorofluorescein diacetate oxidation, decrease in the activity of antioxidant enzymes, and oxidation of total sulfhydryl content, in addition to cell viability evaluation, DNA damage generation, and DNA damage repair response. A value of p<0.05 was considered statistically significant. Results Neonatal cardiomyocytes from resveratrol supplemented rats exhibiting an increase (p<0.01) in cell viability and lower (p<0.0001) apoptotic/necrotic cells after DOX treatment, which correlates with the activities of antioxidant enzymes and dichlorofluorescein production. Moreover, resveratrol protected cardiomyocytes from DOX-induced DNA damage, showing a decrease (p<0.05) in DNA breaks induced by oxidative stress, evaluated by the activity of DNA-repair enzymes endonuclease III and formamidopyrimidine glycosylase. Supplementation with resveratrol increased (p<0.05) the expression of the repair protein Sirt6 in the cardiomyocytes of the pups. Conclusion This research indicates that supplementation with resveratrol during the gestational period has a notable cardioprotective effect on the offspring's heart against DOX-induced toxicity, which may well be due to its antioxidant function, and the increase in the DNA damage repair response.

Effects of Quercetin on the Expression of Ang Ⅱ-induced Myocardial Contractile Protein of Rats through ACE 2- Ang-（1-7）-Mas Axis / 中国药房

OBJECTIVE：To in vestigate the effects of quercetin （Que）on the expressio n of angiotensin Ⅱ（AngⅡ）-induced myocardial contractile proteins of primary rats through angiotensin-converting enzyme 2-angiotensin-（1-7）-Mas （ACE2-Ang- （1-7）-Mas）axis. METHODS ：Cardiac tissue of rats aged 1-2 d were collected ，and primary cardiomyocytes were isolated and cultured. The gene silencing model of cardiomyocytes ACE2 was constructed. Experiments were divided into 12 groups. Among them，AngⅡ group，AngⅡ+ small interference RNA （siRNA）group，and Ang Ⅱ+ A 779 group were the model groups ；AngⅡ+ losartan group was positive control group ；AngⅡ+Que40 group，AngⅡ+Que80 group，AngⅡ+siRNA+Que40 group，AngⅡ+ siRNA+Que80 group，AngⅡ+A779+Que40 group and Ang Ⅱ+A779+Que80 group were the experimental groups ；blank group and siRNA group were set up. Ang Ⅱ concentration was 1×10－6 mol/L；siRNA final concentration was 50 nmol/L；Que concentration was 40 and 80 μmol/L；A779（Mas receptor inhibitor ）concentration was 1 μmol/L；losartan concentration was 1×10－4 mol/L. mRNA and protein expression of ACE 2，Ang-（1-7） and Mas in primary cardiomyocytes were detected ；the expressions of myocardial contractile proteins were also determined ，such as Na +/Ca2+ exchange channel （NCX），calcium pump （SERCA2a）， phosphoprotein （PLB）. RESULTS ：Compared with Ang Ⅱ group，mRNA expression of Mas was increased significantly in Ang Ⅱ + Que 80 group （P＜0.05）；mRNA expression of ACE2 and Mas were increased significantly in Ang Ⅱ + CZ0210-01） losartan group （P＜0.05）. Compared with Ang Ⅱ group，the 851136165@qq.com protein expression of ACE 2 and Ang- （1-7） were increased significantly in Ang Ⅱ+ Que 40 group（P＜0.05）；compared with Ang Ⅱ + siRNA group ，the protein expression of Ang-（1-7）were increased significantly in Ang Ⅱ+ siRNA+Que 40 group（P＜0.05）；compared with Ang Ⅱ+A779 group，the protein expression of Ang- （1-7）were increased significantly in Ang Ⅱ+A779+ Que 40 group（P＜0.05）. Compared with Ang Ⅱ group，the protein expression of NCX was decreased in Ang Ⅱ+Que40 group（P＜0.05），protein expression of NCX was reduced in Ang Ⅱ+ losartan group （P＜0.05）；compared with Ang Ⅱ+A779 group，the protein expression of NCX was decreased in Ang Ⅱ+A779+ Que80 group （P＜0.05）. CONCLUSIONS ：Que improves the expression of Ang Ⅱ -induced ACE 2-Ang-（1-7）-Mas axis in cardiomyocyte model to some extent ，so as to regulate myocardial contractile protein.

Testosterone, cardiomyopathies, and heart failure: a narrative review / 亚洲男科学杂志(英文版)

Testosterone exerts an important regulation of cardiovascular function through genomic and nongenomic pathways. It produces several changes in cardiomyocytes, the main actor of cardiomyopathies, which are characterized by pathological remodeling, eventually leading to heart failure. Testosterone is involved in contractility, in the energy metabolism of myocardial cells, apoptosis, and the remodeling process. In myocarditis, testosterone directly promotes the type of inflammation that leads to fibrosis, and influences viremia with virus localization. At the same time, testosterone exerts cardioprotective effects that have been observed in different studies. There is increasing evidence that low endogenous levels of testosterone have a negative impact in some cardiomyopathies and a protective impact in others. This review focuses on the interrelationships between testosterone and cardiomyopathies and heart failure.

Effects of ophiopogonin D on fatty acid metabolic enzymes in cardiomyocytes / 中国中药杂志

To explore the effect of ophiopogonin D on main fatty acid metabolic enzymes in human cardiomyocyte AC-16,so as to provide reference for cardiovascular protection mechanism and safe clinical application of Ophiopogon japonicus.CCK-8 (cell counting kit-8) was used to detect the effect of different concentrations of ophiopogonin D on the viability of cardiomyocytes.Meanwhile,the effect of different concentrations of ophiopogonin D on the morphology and quantity of cardiomyocytes was observed under microscope.The effect of ophiopogonin D on the mRNA expression of CYP2J2,CYP4F3,CYP4A11,CYP4A22 and CYP4F2 in cardiomyocytes was detected by RT-PCR.Western blot was used to detect the protein expression of CYP4F3 in different concentrations of ophiopogonin D.Compared with the control group,low-concentration ophiopogonin D had no effect on the viability of cardiomyocytes.However,ophiopogonin D with a concentration of higher than 20μmol·L~(-1)could promote the viability.Under the microscope,ophiopogonin D with a concentration of below 100μmol·L~(-1)had no significant effect on the morphology and number of cardiomyocytes.RT-PCR results showed that compared with the control group,5μmol·L~(-1)ophiopogonin D could slightly up-regulate mRNA expressions of CYP2J2 and CYP4F3,while high-concentration ophiopogonin D (10 and 20μmol·L~(-1)) could significantly induce mRNA expressions of CYP2J2and CYP4F3 in a dose-dependent manner (P<0.05).The same concentration of ophiopogonin D had a little effect on the mRNA expressions of CYP4A11,CYP4A22 and CYP4F2.Western blot results showed that 20μmol·L~(-1)ophiopogonin D could significantly induce the protein expression of CYP4F3 in a dose-dependent manner (P<0.05).Based on the above results,ophiopogonin D (less than100μmol·L~(-1)) has no effect on the viability of AC-16 cardiomyocytes.Ophiopogonin D (less than 100μmol·L~(-1)) can selectively induce the expressions of CYP2J2 and CYP4F3,regulate the metabolic pathway of fatty acid signaling molecules,and thus protecting the cardiovascular system.

Study on Improvement Effects of Polygonum orientale Flower Extract on Hypoxia-reoxygenation Injury of H 9c2 Cardiomyocytes / 中国药房

OBJECTIVE：To study the improvement effects and mechanism of Polygonum orientale flower extract on hypoxia- reoxygenation injury of H 9c2 cardiomyocytes. METHODS ：H9c2 cardiomyocytes were divided into normal control group ，model group and low- ，medium- and high-concentrations groups of P. orientale flower extract （20，40，80 μg/mL）. Except for normal control group ，other groups were given 800 μmol/L CoCl2 to induce hypoxia-reoxygenation injury model. Cell apoptosis was observed. The levels of Ca 2+（in cytoplasm ），mitochondrial membrane potential （MMP），ATP enzyme （Na+-K+-ATP enzyme ，Ca2+-Mg2+-ATP enzyme） activities， the ratio of cytochrome c （Cyto c ）， protein in cytosol to mitochondria ，phosphorylation levels of reperfusion injury salvage kinase （RISK） signaling pathwayrelated protein [protein kinase B （Akt）and extracellular signal regulated kinase 1/2（ERK1/2）] as well as protein expression of HIF- 1 α were detected respectively. In addition，the cells were divided into normal control group ，model group and P. orientale flower extract group （80 μ g/mL），PI3K inhibitor LY294002+CoCl2 group（15 μmol/L LY294002+80 μmol/L ，LY294002+P. orientale flower extract group （15 μmol/L LY294002+80 μg/mL P. orientale flower extract ），MEK inhibitor PD98059+CoCl2 group（25 μmol/L PD98059+800 μmol/L CoCl2），PD98059+P. orientale flower extract group （25 μmol/L PD98059+80 μg/mL P. orientale flower extract ）. After cultured by the same method ，the phosphorylation levels of Akt protein and ERK1/2 protein in the cells were measured to verify the activation of P. orientale flower extract to RISK signaling pathway. RESULTS：Compared with model group ，nuclear pyknosis and the number of apoptotic bodies were reduced in different concentrations groups of P. orientale flower extract. ROS level ，Ca2+ level（except for low-concentration group ），MMP，ratio of Cyto c in cytoplasm to Cyto c in mitochondria ，protein expression of HIF- 1α were decreased significantly（P＜0.05 or P＜0.01）； the activity of ATP enzyme （except for the low-concentration group ），Akt protein and ERK 1/2 protein phosphorylation level were significantly increased （P＜0.01）. After treated with PI 3K inhibitor LY 294002 and MEK inhibitor PD 98059，Akt protein and ERK 1/2 protein phosphorylation level in cadiomyocyte were decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS ：P. orientale flower extract can improve hypoxia-reoxygenation injury of H 9c2 cardiomyocytes，the mechanism of which may be associated with inhibiting cardiomyocyte apoptosis ，improving ATPase activity ，protecting mitochondria ，regulating RISK signaling pathway related proteins and HIF- 1α protein expression.

Change of gene expression profiles in human cardiomyocytes and macrophages infected with SARS / 中南大学学报(医学版)

OBJECTIVES@#Coronavirus disease 2019 (COVID-19) is an acute respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 can damage the myocardium directly, or activate the immune system, trigger a cytokine storm, and cause inflammatory cells to infiltrate the myocardial tissue and damage the myocardium. This study is based on the sequencing data to analyze the changes in gene expression of cardiomyocytes and macrophages after SARS-CoV-2 infection, and explore the potential effects of SARS-CoV-2 on the heart and immune system.@*METHODS@#The public data set GSE151879 was retrieved. The online software Network Analyst was used to preprocess the data, and the differentially expressed genes (DEGs) [log@*RESULTS@#After data standardization, the data quality was excellent and it can ensure reliable results. Myocardial cell infection with SARS-CoV-2 and gene expression spectrum were changed significantly, including a total of 484 DEGs in adult cardiomyoblasts, a total of 667 DEGs in macrophages, and a total of 1 483 DEGs in human embryo source of cardiomyopathy. The Stum, mechanosensory transduction mediator homolog (STUM), dehydrogenase/reductase 9 (DHRS9), calcium/calmodulin dependent protein kinase II beta (CAMK2B), claudin 1(CLDN1), C-C motif chemokine ligand 2 (CCL2), TNFAIP3 interacting protein 3 (TNIP3), G protein-coupled receptor 84 (GPR84), and C-X-C motif chemokine ligand 1 (CXCL1) were identical in expression patterns in 3 types of cells. The protein-protein interaction suggested that CAMK2B proteins may play a key role in the antiviral process in 3 types of cells; and silicon dioxide (SiO@*CONCLUSIONS@#CAMK2B, CLDN1, CCL2, and DHRS9 genes play important roles in the immune response of cardiomyocytes against SARS-CoV-2. SiO

Mechanism of Guhong Injection against Hypoxia/Reoxygenation Injury of H9c2 Cardiomyocytes Based on PI3K/AKT Signaling Pathway / 中国药学杂志

OBJECTIVE: To explore the protectiveness of Guhong injection (GHI) on H9c2 cardiomyocytes injured by hypoxia/reoxygenation (H/R) based on PI3K/AKT signaling pathway. METHODS: H9c2 cardiomyocytes were cultured and the optimal GHI doses were screened by CCK-8. The cells were randomly subjected into 8 groups: control group, model group, low, medium and high GHI dose groups (30, 60 and 90 μL•mL-1 respectively), positive drug group (verapamil injection 7.5 μL•mL-1), GHI+LY294002 (PI3K inhibitor) group and LY294002 group. Cells were established H/R model with hypoxia for 4 h and reoxygenation for 16 h excepted the control group. The CK-MB, LDH, MDA content and SOD activities in each group were detected by Elisa, and the expression levels of p-PI3K, PI3K, p-AKT, AKT and GSK-3β in each group were detected by Western blot. RESULTS: Compared each drug-treated group with the model group, LDH and CK-MB contents were decreased (P<0.05), MDA content was decreased (P<0.01), and SOD activity was increased (P<0.01). Meanwhile, the expression levels of p-PI3K/PI3K, p-AKT/AKT, and GSK-3β protein were elevated (P<0.01). Compared with the model group, LDH, CK-MB, MDA and SOD activity in LY294002 group were no significant difference, while the expression levels of p-PI3K/PI3K, p-AKT/AKT and GSK-3β protein decreased (P<0.01). CONCLUSION: GHI could represent anti-oxidative and anti-apoptotic effect which reduced the damage of injured H9c2 myocardial cells caused by H/R. These effects might be related to the PI3K/AKT signaling pathway.

Research Advances on Non-Clinical Evaluation in Vitro of Drug-Induced Arrhythmias / 中国药学杂志

Drug-induced arrhythmia is one of the main causes of failure in drug development, and it is also a major cause of drug withdrawal, therefore, accurate prediction of drug-induced arrhythmia in the non-clinical research stage is the best way to reduce cost. Literature was retrieved by formally searching PubMed, Metstr, CNKI and Baidu Scholar, 1 479 published articles were found through search method, 63 full-text articles were included. After reviewed the relevant literatures, the advantages and disadvantages of the different experimental cells and the related evaluation methods are assessed, in order to provide reference for toxicity evaluation.