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Apoptosis is a crucial pathological process in myocardial ischemia/reperfusion injury (MIRI). Verapamil (Ver), normally used to treat hypertension or heart rhythm disorders, also attenuates MIRI. The potential of Ver to inhibit apoptosis and thereby attenuate MIRI remains unclear, as does the mechanism. We established an in vivo mouse ischemia/reperfusion (I/R) model by occlusion of the left anterior descending coronary. To construct a hypoxia/reoxygenation model in vitro, H9c2 cardiomyocytes were immersed in a hypoxic buffer in a hypoxia/anaerobic workstation. Ver significantly improved cardiac function and reduced myocardial infarction size in I/R mice, while decreasing apoptosis. Both in vivo and in vitro, application of Ver activated the JAK2/STAT3 signaling pathway and elevated Bcl-2 expression, while decreasing Bax and cleaved caspase-3 levels. Treatment with AG490, a JAK2 inhibitor, partially counteracted the anti-apoptotic and the cardioprotective effect of Ver. Thus, we conclude that Ver alleviates MIRI by reducing apoptosis via the JAK2/STAT3 signaling pathway activation. These findings provide a novel mechanism of Ver in the treatment of MIRI., Competing Interests: Declaration of Competing Interest The authors of this article declare that they have no known competing financial interests or personal relationships that could possibly influence the work reported in this article., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The occurrence of myocardial ischemia/reperfusion injury is commonly observed during cardiac surgery; however, there remains a dearth of effective therapeutic strategies to mitigate this injury. The a disintegrin and metallopeptidase domain 10 (ADAM10) is a transmembrane protein anchored on the cell membrane surface, and its precise mechanism of action in myocardial ischemia/reperfusion injury remains incompletely understood. This study aims to investigate the impact of ADAM10 on cardiomyocyte injury induced by hypoxia/reoxygenation (H/R) and elucidate the underlying mechanisms. The ADAM10 overexpression plasmid was transfected into H9c2 cells, which were subsequently treated with the Notch signaling pathway inhibitor DAPT and cultured under H/R conditions. Cell proliferation activity was assessed using the CCK-8 assay. The levels of LDH, SOD, and MDA were quantified through colorimetric analysis. The levels of ROS and the rate of apoptosis were measured using flow cytometry. The morphological changes in the nucleus of H9c2 cells were observed by employing Hoechst 33258 staining. The mRNA expression levels of ADAM10, Notch1, NICD, and Hes1 in H9c2 cells were determined using qRT-PCR. The expressions of Notch signaling pathway and apoptosis-related proteins were analyzed by Western blot. Overexpression of ADAM10 provided protection to H9c2 cells against injury induced by H/R, leading to an increase in SOD levels and alleviation of oxidative stress caused by the accumulation of ROS and the decrease of SOD activity. Meanwhile, overexpression of ADAM10 inhibited apoptosis in H9c2 cells exposed to H/R by regulating the expression of apoptosis-related proteins, such as Bax, Bcl-2 and Cleaved-caspase-3. Additionally, overexpression of ADAM10 facilitated the activation of the Notch1 signaling pathway in H9c2 cells exposed to H/R by upregulating the protein expression of Notch1, NICD, and Hes1. However, the protective effect of ADAM10 on H/R-induced H9c2 cells was partially reversed by DAPT. Our findings demonstrate that ADAM10 exerts protective effects in H/R-induced H9c2 cells by suppressing oxidative stress and apoptosis via the activation of the Notch signaling pathway., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Cardiovascular diseases are the leading cause of morbidity and mortality worldwide. Ischemic heart disease is one of the most harmful conditions to cellular structure and function. After reperfusion treatment, a spectrum of adverse effects becomes evident, encompassing altered cell viability, heightened oxidative stress, activated autophagy, and increased apoptosis. Photobiomodulation (PBM) has been utilized in experimental models of cardiac hypoxia to enhance mitochondrial response and ameliorate biochemical changes in injured tissue. However, the effects of PBM on cultured cardiomyocytes subjected to hypoxia/reoxygenation are not yet well established., Method: H9C2 cardiomyocytes were exposed to hypoxia with concentrations of 300 μM CoCl 2 for 24 h, followed by 16 h of reoxygenation through incubation in a normoxic medium. Treatment was conducted using GaAIAs Laser (850 nm) after hypoxia at an intensity of 1 J/cm 2 . Cells were divided into three groups: Group CT (cells maintained under normoxic conditions), Group HR (cells maintained in hypoxia and reoxygenation conditions without treatment), Group HR + PBM (cells maintained in hypoxia and reoxygenation conditions that underwent PBM treatment). Cell viability was analyzed using MTT, and protein expression was assessed by western blot. One-way ANOVA with the Tukey post hoc test was used for data analysis. Differences were significant when p < 0.05., Results: PBM at an intensity of 1 J/cm 2 mitigated the alterations in cell survival caused by hypoxia/reoxygenation. Additionally, it significantly increased the expression of proteins Nrf2, HSP70, mTOR, LC3II, LC3II/I, and Caspase-9, while reducing the expression of PGC-1α, SOD2, xanthine oxidase, Beclin-1, LC3I, and Bax., Conclusion: PBM at intensities of 1 J/cm 2 reverses the changes related to oxidative stress, mitochondrial biogenesis, autophagy, and apoptosis caused by hypoxia and reoxygenation in a culture of cardiomyocytes., Competing Interests: Declaration of competing interest The authors declare that no conflict of interest is relevant to the content of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This research is concentrated on investigating the role and mechanism of miR-652-3p in the protective effects of isoflurane (ISO) against myocardial ischemia-reperfusion (I/R) injury. H9c2 cells underwent pretreatment with varying concentrations of ISO, and subsequently, a hypoxia/reoxygenation (H/R) model was constructed. The levels of miR-652-3p, ISL LIM homeobox 1 (ISL1), and inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) were evaluated through reverse transcription polymerase chain reaction (RT-qPCR). Enzyme-linked immunosorbent assay was employed to investigate concentrations of myocardial injury markers, such as creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI). Cell counting kit-8 was used to evaluate cell viability, while flow cytometry was utilized to measure apoptosis. Additionally, a dual luciferase reporter assay was conducted to validate the targeting relationship between ISL1 and miR-652-3p. Herein, we confirmed that the level of miR-652-3p was gradually increased with prolonged hypoxia; nevertheless, this increase was suppressed by ISO pretreatment (P < 0.05). Additionally, ISO pretreatment prevented the decrease in cell viability, increase in apoptosis, and overproduction of IL-6, TNF-α, CK-MB, and cTnI induced by H/R (P < 0.05). However, the inhibitory effects of ISO were counteracted by the increased levels of miR-652-3p (P < 0.05). ISL1 is a potential target of miR-652-3p. H/R induction suppressed ISL1 levels compared to the control, but ISO treatment increased its expression (P < 0.05). Overexpression of ISL1 inhibited the elimination of the protective effect of ISO on myocardial damage induced by the elevation of miR-652-3p (P < 0.05). The findings of this research confirm that miR-652-3p attenuated the protective effect of ISO on cardiomyocytes in myocardial ischemia by targeting ISL1., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objectives: To investigate the regulatory role of miRNA-224-5p in hypoxia/reoxygenation (H/R) -induced H9c2 cardiomyocyte injury., Methods: Plasma samples were collected from 160 patients with acute myocardial infarction and 80 healthy controls(HC) to measure miRNA-224-5p levels and other biochemical parameters. In cultured H9c2 cells with H/R injury, the effects of transfection with miR-224-5p mimics or a negative control sequence on cell viability, malondialdehyde (MDA) content, and superoxide dismutase 2 (SOD2) and lactate dehydrogenase (LDH) activities were tested. Dual luciferase reporter gene assay was performed to verify the targeting relationship between miR-224-5p and PTEN. Bioinformatics methods were used to analyze the potential mechanisms of the target genes. The expression of miRNA-224-5p in the treated cells was detected with qRT-PCR, the protein expressions of PTEN, Bcl-2, Bax, cleaved caspase-3, SOD2, p-PI3K/PI3K, p-Akt/Ak and p-FoxO1/FoxO1 were determined using Western blotting, and cell apoptosis was analysed with flow cytometry., Results: The levels of blood glucose, C-reactive protein, CK, CK-MB and cTnI were significantly higher in the AMI group compared with the HC group ( P < 0.05). The expression level of miR-224-5p was significantly lowered in patients with STEMI and NSTEMI and in H9c2 cells with H/R injury. The viability of H9c2 cells decreased time-dependently following H/R injury. PTEN was a target gene of miR-224-5p, and the PI3K/Akt pathway was the most significantly enriched pathway. H9c2 cells with H/R injury showed significantly decreased SOD2 activity, increased LDH activity and MDA content, increased cell apoptosis, decreased protein expression levels of p-PI3K, p-Akt, p-FoxO1, SOD2, and Bcl-2, and increased expressions of PTEN, Bax, and cleaved caspase-3. These changes were obviously attenuated by trasnfection of the cells with miR-224-5p mimics prior to H/R exposure., Conclusion: MiR-224-5p overexpression upregulates the expression of the antioxidant gene SOD2 through the PI3K/Akt/FoxO1 axis to relieve H/R-induced oxidative stress and reduce apoptosis of H9c2 cells.

Objective: This study aimed to investigate the function and mechanism of lncRNA NORAD in Sevoflurane (Sev) protection against myocardial hypoxia-reoxygenation (H/R)., Methods: Preprocess rat cardiomyocytes H9c2 cells with Sev at concentrations of 0.5%, 1.0%, and 1.5%, and subjected them to H/R treatment. qRT-PCR was used to detect levels of NORAD and miR-144-3p. Measure concentrations of the inflammatory cytokines IL-6, TNF-α, and IL-10, as well as cardiac injury markers cTnI, CK-MB, and LDH using ELISA. Assess cell proliferation and apoptosis using CCK-8 and flow cytometry. Perform dual-luciferase reporter assay and RIP assay to validate the targeting relationship between NORAD and miR-144-3p., Results: H/R induced inhibition of cell proliferation, increase in apoptosis, and production of IL-6, TNF-α, CK-MB, LDH, and cTnI were significantly attenuated by Sev. As hypoxic treatment time lengthened, the NORAD levels in myocardial cells showed an increase, with Sev pretreatment being able to suppress the NORAD levels elevation. The overexpression of NORAD notably weakened the cardioprotective effect of Sev. NORAD targetedly binds to miR-144-3p and negatively regulates miR-144-3p. Increased miR-144-3p levels inhibited the antagonistic effect of NORAD on the cardioprotective effects of Sev., Conclusion: The current study confirmed that sevoflurane attenuated H/R-induced cardiomyocyte injury via the NORAD/miR-144-3p axis., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Mitsugumin 53 (MG53) is a membrane repair factor that is associated with acute myocardial infarction. This study aimed to investigate the effects of MG53 on cardiomyocyte injury and the posttranslational modification of MG53., Methods: Cardiomyocyte injury was evaluated by enzyme-linked immunosorbent assay and flow cytometry. The succinylation and ubiquitination levels of MG53 were examined by immunoprecipitation (IP) and western blot. The relationship between MG53 and KAT3B or SIRT7 was assessed by co-IP and immunofluorescence., Results: The results showed that overexpression of MG53 inhibited inflammation response and apoptosis of cardiomyocytes induced by hypoxia/reoxygenation (H/R). Succinylation and protein levels of MG53 were downregulated in H/R-induced cells, which was inhibited by SIRT7 and promoted by KAT3B. SIRT7 aggravated and KAT3B alleviated MG53-mediated cardiomyocyte injury. Moreover, MG53 was succinylated and ubiquitinated at K130., Conclusion: SIRT7 inhibited/KAT3B promoted succinylation of MG53 at K130 sites, which suppressed ubiquitination of MG53 and upregulated its protein levels, thereby alleviating H/R-induced cardiomyocyte injury. The findings suggested that MG53 may be a potential therapy for myocardial infarction.

Acute myocardial infarction is regarded as myocardial necrosis resulting from myocardial ischemia/reperfusion (I/R) damage and retains a major cause of mortality. Neferine, which was extracted from the green embryos of mature seeds of Nelumbo nucifera Gaertn., has been reported to possess a broad range of biological activities. However, its underlying mechanism on the protective effect of I/R has not been fully clarified. A hypoxia/reoxygenation (H/R) model with H9c2 cells closely simulating myocardial I/R injury was used as a cellular model. This study intended to research the effects and mechanism underlying neferine on H9c2 cells in response to H/R stimulation. Cell Counting Kit-8 and lactate dehydrogenase (LDH) release assays were employed to measure cell viability and LDH, respectively. Apoptosis and reactive oxygen species (ROS) were determined by flow cytometry analysis. Oxidative stress was evaluated by detecting malondialdehyde, superoxide dismutase, and catalase. Mitochondrial function was assessed by mitochondrial membrane potential, ATP content, and mitochondrial ROS. Western blot analysis was performed to examine the expression of related proteins. The results showed that hypoxia/reoxygenation (H/R)-induced cell damage, all of which were distinctly reversed by neferine. Moreover, we observed that neferine inhibited oxidative stress and mitochondrial dysfunction induced by H/R in H9c2 that were concomitant with increased sirtuin-1 (SITR1), nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 expression. On the contrary, silencing the SIRT1 gene with its small interferingRNA eliminated the beneficial effects of neferine. It is concluded that neferine preconditioning attenuated H/R-induced cardiac damage via suppressing apoptosis, oxidative stress, and mitochondrial dysfunction, which may be partially ascribed to the activation of SIRT1/Nrf2 signaling pathway., (© 2023 Wiley Periodicals LLC.)

Mesenchymal stem cell-derived conditioned medium (MSC-CM) improves cardiac function, which is partly attributed to the released paracrine factors. Since such cardioprotection is moderate and transient, it is essential that MSC-CM's effective components are optimized to alleviate myocardial injury. To optimize MSC-CM, MSCs were treated with or without lipopolysaccharides (LPSs) for 48 h (serum-free), and the supernatant was collected. Then, LPS-CM (MSC stimulated by LPS) was further treated with LPS remover (LPS Re-CM) or was concentrated with a 10 kDa cutoff filter (10 kDa-CM). Enzyme-linked immunosorbent assay showed that all the pretreatments increased the levels of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin growth factor (IGF) except LPS Re-CM; 10 kDa-CM was superior to the other CMs. Cell Counting Kit-8 displayed that the viability of injured H9c2 cells was enhanced with the increase in the MSC-CM concentration. We also found that the 10 kDa-CM significantly alleviated H9c2 hypoxia/reoxygenation (H/R) injury, as evidenced by the increased Bcl-2/Bax ratio, and decreased the levels of lactate dehydrogenase and cardiac troponin. Transmission electron microscopy (TEM), TdT-mediated dUTP nick-end labelling (TUNEL), and hematoxylin and eosin staining (H&E) confirmed that 10 kDa-CM inhibited H/R-induced H9c2 morphological changes. Proteomic analysis identified 41 differentially expressed proteins in 10 kDa-CM, among which anti-inflammation, proangiogenesis, and antiapoptosis were related to cardiac protection. This study indicates that 10 kDa-CM protects H9c2 cardiomyocytes from H/R injury by preserving most of the protective factors, such as VEGF, HGF, and IGF, in MSC-CM.

Background: Anshen Buxin Liuwei pill (ABLP) is a Mongolian medicinal formula that is composed of six medicinal materials: the Mongolian medicine Bos taurus domesticus Gmelin, Choerospondias axillaris (Roxb.) Burtt et Hill, Myristica fragrans Houtt., Eugenia caryophμllata Thunb., Aucklandia lappa Decne., and Liqui dambar formosana Hance. ABLP is considered to have a therapeutic effect on symptoms such as coronary heart disease, angina pectoris, arrhythmia, depression and irritability, palpitation, and shortness of breath., Methods: H9c2 cardiomyocytes were used to construct a hypoxia/reoxygenation (HR) injury model. CCK-8 assay and Annexin V-FITC cell apoptosis assays were used for cell viability and cell apoptosis determination. The LDH, SOD, MDA, CAT, CK, GSH-Px, Na + -K + -ATPase, and Ca 2+ -ATPase activities in cells were determined to assess the protective effects of ABLP. The mRNA levels of Sirtuin3 (Sirt3) and Cytochrome C (Cytc) in H9c2 cells were determined by quantitative real-time PCR., Results: The results indicate that HR-treated cells began to shrink from the spindle in an irregular shape with some floated in the medium. By increasing the therapeutic dose of ABLP (5, 25, and 50 μg/mL), the cells gradually reconverted in a concentration-dependent manner. The release of CK in HR-treated cells was significantly increased, indicating that ABLP exerts a protective effect in H9c2 cells against HR injury and can improve mitochondrial energy metabolism and mitochondrial function integrity. The present study scrutinized the cardioprotective effects of ABLP against HR-induced H9c2 cell injury through antioxidant and mitochondrial pathways., Conclusions: ABLP could be a promising therapeutic drug for the treatment of myocardial ischemic cardiovascular disease. The results will provide reasonable information for the clinical use of ABLP., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

MicroRNA-7a-5p (miR-7a-5p) is closely related to apoptosis and plays an important role in ischemia/reperfusion (I/R) injury. Whether miR-7a-5p is involved in hypoxia/reoxygenation (H/R)-induced cardiomyocyte apoptosis is unknown. Therefore, this study aims to evaluate the role of miR-7a-5p in cardiomyocyte H9C2 cells in response to H/R stimulation. The results of RT-qPCR demonstrated that the expression level of miR-7a-5p was significantly down-regulated in H/R-treated H9C2 cells. MTT assay revealed that the cell viability was notably decreased in H/R group. Flow cytometric analysis found that the ratio of apoptotic cells was increased markedly following H/R. Enforced miR-7a-5p expression increased cell viability and decreased the apoptotic rate. Western blot analysis revealed that the expressions of pro-apoptotic proteins cleaved caspase-3 and Bax were down-regulated, while the expression of anti-apoptotic protein Bcl-2 was up-regulated in H/R-treated H9C2 cells transfected with miR-7a-5p mimic. On the contrary, miR-7a-5p downexpressing promoted apoptosis in H/R-treated H9C2 cells. Furthermore, the bioinformatics prediction manifested voltage-dependent anion channel 1 (VDAC1) was a potential target for miR-7a-5p, and dual-luciferase reporter assay confirmed that miR-7a-5p targeted VDAC1 3' untranslated regions, which leads to the repressed expressions of VDAC1 mRNA and protein. Knockdown of VDAC1 potentiated the protective effects of miR-7a-5p against H/R-induced cell injury. In conclusion, our results demonstrated that miR-7a-5p is involved in H/R-induced cardiomyocyte apoptosis through targeting VDAC1. MiR-7a-5p/VDAC1 axis might be utilized as hopeful biomarkers to reveal the potential mechanism of myocardial I/R injury., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Numerous studies have demonstrated that long non‑coding RNAs (lncRNAs) serve an important regulatory role in ischemic injury of cardiomyocytes. lncRNA small nucleolar RNA host gene 1 (SNHG1) could effectively protect cardiomyocytes against various injuries. However, the role of SNHG1 in ischemic cardiomyocyte injury is unclear. It was hypothesized that SNHG1 may have a protective effect on cardiomyocyte injury induced by hypoxia/reoxygenation (H/R) by sponging microRNA (miRNA/miR). The purpose of the present study was to explore the role and molecular mechanism of SNHG1 in ischemic cardiomyocyte injury. A H9c2 cardiomyocyte H/R model was established. The expression levels of SNHG1 in cardiomyocytes treated with H/R were detected using reverse transcription‑quantitative PCR. A luciferase reporter assay was used to analyze the associations among SNHG1, miR‑16‑5p and GATA binding protein 4 (GATA4). Chromatin immunoprecipitation experiments were performed to analyze the interaction between SNHG1 and GATA4. Cell Counting Kit‑8, enzyme‑linked immunosorbent assay, terminal deoxynucleotidyl‑transferase‑mediated dUTP nick end labeling and western blotting experiments were used to detect cell activity, lactate dehydrogenase release, apoptosis and apoptosis‑related proteins (Bcl‑2, Bax, Cleaved caspase‑3 and Cleaved caspase‑9), respectively. The expression levels of SNHG1 were downregulated in cardiomyocytes treated with H/R. Overexpression of SNHG1 had a protective effect on cardiomyocyte injury induced by H/R. In addition, SNHG1 could regulate the expression levels of GATA4 via sponging of miR‑16‑5p. Further experiments revealed that GATA4 could bind to the promoter region of SNHG1 and subsequently regulated the expression levels of SNHG1, indicating the important role of the positive feedback loop of SNHG1/miR‑16‑5p/GATA4 in cardiomyocyte ischemic injury. To conclude, the present study revealed the protective effect of the SNHG1/miR‑16‑5p/GATA4 positive feedback loop on cardiomyocyte injury induced by H/R and provided a potential therapeutic target for ischemic cardiomyocyte injury.

Forkhead box protein P1 (Foxp1) exerts an extensive array of physiological and pathophysiological impacts on the cardiovascular system. However, the exact function of myocardial Foxp1 in myocardial ischemic reperfusion injury (MIRI) stays largely vague. The hypoxia reoxygenation model of H9c2 cells (the rat ventricular myoblasts) closely mimics myocardial ischemia-reperfusion injury. This report intends to research the effects and mechanisms underlying Foxp1 on H9c2 cells in response to hypoxia (12 h)/reoxygenation (4 h) (HR) stimulation. Expressions of Foxp1 and Phosphatidylinositol 3-kinase interacting protein 1 (Pik3ip1) were both upregulated in ischemia/reperfusion (IR)/HR-induced injury. Stimulation through HR led to marked increases in cellular apoptosis, mitochondrial dysfunction, and superoxide generation in H9c2 cells, which were rescued with knockdown of Foxp1 by siRNA. Silence of Foxp1 depressed expression of Pik3ip1 directly activated the PI3K/Akt/eNOS pathway and promoted nitric oxide (NO) release. Moreover, the knockdown of Foxp1 blunted HR-induced enhancement of reactive oxygen species (ROS) generation, thus alleviating excessive persistence of mitochondrial permeability transition pore (mPTP) opening and decreased mitochondrial apoptosis-associated protein expressions in H9c2 cells. Meanwhile, these cardioprotective effects can be abolished by LY294002, NG-nitro-L-arginine methyl ester (L-NAME), and Atractyloside (ATR), respectively. In summary, our findings indicated that knockdown of Foxp1 prevented HR-induced encouragement of apoptosis and oxidative stress via PI3K/Akt/eNOS signaling activation by targeting Pik3ip1 and improved mitochondrial function by inhibiting ROS-mediated mPTP opening. Inhibition of Foxp1 may be a promising therapeutic avenue for MIRI.

For isolation of neonatal rat cardiomyocytes (NRCM) the ventricular muscles of neonatal rats were treated with different digestive solutions: 0.06% trypsin (method I), 0.08% collagenase II (method II), 0.06% trypsin and 0.08% collagenase II for stepwise digestion (methods III and IV). After enzymatic dissociation of the tissue, the complete medium was added to stop this process. The cells suspensions obtained by methods I-III were collected and centrifuged. In contrast, the novel and improved method IV did not use centrifugation. Instead, various methods of adhesion were employed to separate non-myocardial cells. The isolation methods were compared by the quantity, survival rate, morphology, spontaneous pulsation rate, purity, and vitality of NRCM. These assessments showed that isolation method IV is a simple, efficient, and convenient way to obtain NRCM for culturing., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Sevoflurane (Sevo) prevents hypoxia/reoxygenation (H/R)-induced cardiomyocytes apoptosis. MiR-27a-3p expression is up-regulated in Sevo-treated hippocampal neurons., Objective: This study explored whether the effect of Sevo on cardiomyocytes was mediated by miR-27a-3p., Methods: The cardiomyocytes were cultured under H/R condition or pre-treated with Sevo, and further transfected with miR-27a-3p inhibitor or treated with an autophagy inhibitor 3-methyladenine (3-MA). Then the cell morphology was observed under an optical microscope. The cell viability and apoptosis were measured by MTT and flow cytometry. Expressions of miR-27a-3p, apoptosis-related, and autophagy-related factors were determined by western blot or RT-qPCR., Key Findings: Sevo improved the abnormal morphology, promoted the cell viability and the expressions of Bcl-2 and miR-27a-3p, but reduced the apoptosis and Bax and C-caspase-3 levels of H/R-induced cardiomyocytes. MiR-27a-3p inhibitor had an effect opposite to Sevo on the cardiomyocytes and further counteracted the effect of Sevo on the H/R-induced cardiomyocytes. Downregulation miR-27a-3p increased the expression of Beclin 1 and the ratio of LC3B-II to LC3B-I in H/R-induced cardiomyocytes. Furthermore, 3-MA had an opposite effect to miR-27a-3p inhibitor and further counteracted the effect of the miR-27a-3p inhibitor on H/R-induced cardiomyocytes., Conclusion: Sevo inhibited the apoptosis of H/R-induced cardiomyocytes via regulating miR-27a-3p-mediated autophagy., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The protective effect and mechanism of isoflurane on myocardial injury was investigated by constructing in vitro hypoxia/reoxygenation (HR) cell model. HR cell models were established in vitro and treated with isoflurane (ISO). qRT-PCR was used to detect the relative expression of miR-18a-5p. CCK-8 kit and flow cytometry were performed to evaluate cell proliferation and apoptosis. The myocardial injury related markers, such as Cκ-MB, cTnI and LDH were detected by ELISA. Luciferase reporter gene assay was used to verify the interaction between miR-18a-5p and target genes. The expression of miR-18a-5p was significantly increased in hypoxic cardiomyocytes compared with control group (P < 0.001). Meanwhile, cardiomyocytes in the HR group showed inhibition of proliferation, a significant increase in cell apoptosis and in myocardial injury indicators, such as Cκ-MB, cTnI and LDH (P < 0.001). However, 1% ISO treatment alleviated myocardial cell injury induced by HR. Transfection of miR-18a-5p under ISO reduced the protective effect of 1% ISO against myocardial cell damage. Luciferase report gene assay confirmed that CCND2 might be the target gene of miR-18a-5p. In the in vitro cell model of myocardium, ISO alleviated cardiomyocyte injury caused by hypoxia/reoxygenation by down-regulating the expression of miR-18a-5p., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

We have focused on the underlying role of miR-1224 in cardiomyocyte injury stimulated by hypoxia/reoxygenation (H/R). In the current study, the rat cardiomyocyte cell line H9C2 was used to construct a H/R cell model to validate the cardioprotective effects of miR-1224. Data from the dual-luciferase assay revealed that the glutathione peroxidase 4 (GPX4) was a direct target of miR-1224. Expression of miR-1224, determined using qRT-PCR, was remarkably increased while that of GPX4 protein, evaluated via western blotting, was significantly decreased in cardiomyocytes in response to H/R exposure. ROS generation, superoxide dismutase (SOD) activity, concentrations of malondialdehyde (MDA) and 4-hydroxy aldehydes (4-HNE), and H9C2 cell apoptosis were further evaluated following overexpression of miR-1224 or silencing of GPX4 in H9C2 cells. H9C2 cells under H/R conditions displayed increased synthesis of ROS, along with overexpression of miR-1224 and downregulation of GPX4. SOD activity was significantly decreased while concentrations of MDA and 4-HNE were markedly increased under H/R injury conditions. In addition, miR-1224 mimic or GPX4 siRNA plasmids dramatically enhanced H/R-mediated apoptosis, Bax expression and caspase-3 activity, with a concomitant reduction in Bcl-2 expression. Conversely, inhibition of miR-1224 exerted suppressive effects on oxidative stress and apoptosis in H9C2 cells under H/R conditions. Interestingly, silencing of GPX4 attenuated the negative effects of miR-1224 inhibition. Our results suggested that inhibition of miR-1224 caused resistance to H/R and diminished oxidative stress in vitro through targeting of GPX4., (Copyright © 2021 Elsevier Inc. All rights reserved.)

We aimed to investigate the protective role and mechanism of dexmedetomidine (DEX) on H9c2 cardiomyocytes after hypoxia/reoxygenation (H/R) injury. Six experimental groups were designed as follows: normal control group (group C), H/R group, H/R + DEX group, H/R + gastrodin group, H/R + Ex527 (SIRT1 inhibitor) group, and H/R + DEX + Ex527 group. Lactate dehydrogenase (LDH) activity and the levels of oxidative stress-related enzymes such as malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) were measured using corresponding commercial kits. Cell counting kit (CCK)-8 assay was used to detect cell survival rate while flow cytometry and caspase 3/7 activity were used to determine cell apoptosis, respectively. Western blot was used to detect the expression of silent information regulator 1 (SIRT1), C/EBP homologous protein (CHOP), cleaved-caspase-12/3 and pro-caspase-12/3 in each group. From our findings, when compared with H/R, H/R + Ex527 and H/R + DEX + Ex527 groups, DEX pretreatment of cells in H/R + DEX group significantly increased cell survival rate, and simultaneously reduced LDH activity, oxidative stress and the apoptosis rate of H9c2 cells with H/R injury. Moreover, DEX up-regulated SIRT1 expression level and down-regulated the levels of endoplasmic reticulum (ER) stress-related markers such as CHOP, cleaved-caspase-12 and cleaved-caspase-3, respectively. Ex527 could completely block DEX-induced upregulated expression of SIRT1, and partially blocked the DEX-induced downregulated expression levels of CHOP, cleaved-caspase-12 and cleaved-caspase-3. These results proved that DEX reversed H/R injury-induced oxidative stress and ER stress-dependent apoptosis of cardiomyocytes via SIRT1/CHOP signaling pathway.

Our previous study reported that microRNA (miR)‑30a‑5p upregulation under hypoxia postconditioning (HPostC) exert a protective effect on aged H9C2 cells against hypoxia/reoxygenation injury via DNA methyltransferase 3B‑induced DNA hypomethylation at the miR‑30a‑5p gene promoter. This suggests that miR‑30a‑5p may be a potential preventative and therapeutic target for ischemic heart disease in aged myocardium. The present study aimed to investigate the underlying mechanisms of miR‑30a‑5p transcription in aged myocardium in ischemic heart disease. Cardiomyocytes were treated with 8 mg/ml D‑galactose for 9 days, and then exposed to hypoxic conditions. Cell viability was determined using a cell viability assay. Expression levels of histone deacetylase 2 (HDAC2), LC3B‑II/I, beclin‑1 and p62 were detected via reverse transcription‑quantitative PCR and western blotting. Chromatin immunoprecipitation‑PCR and luciferase reporter assays were performed to evaluate the effect of c‑Myc binding and activity on the miR‑30a‑5p promoter in senescent cardiomyocytes following HPostC. It was found that HPostC enhanced the acetylation levels of H3K14 at the miR‑30a‑5p gene promoter in senescent cardiomyocytes, which attributed to the decreased expression of HDAC2. In addition, c‑Myc could positively regulate miR‑30a‑5p transcription to inhibit senescent cardiomyocyte autophagy. Mechanically, it was observed that increased H3K14 acetylation level exposed to romidepsin facilitated c‑Myc binding to the miR‑30a‑5p gene promoter region, which led to the increased transcription of miR‑30a‑5p. Taken together, these results demonstrated that HDAC2‑mediated H3K14 hyperacetylation promoted c‑Myc binding to the miR‑30a‑5p gene promoter, which contributed to HPostC senescent cardioprotection.

Glutaredoxin 2 (GRX2) plays a cytoprotective role under various pathological conditions. However, whether GRX2 plays a role during myocardial ischemia-reperfusion injury has not been fully elucidated. In this work, we aimed to explore the detailed role and mechanism of GRX2 in modulating hypoxia/reoxygenation (H/R)-induced cardiac injury in vitro. H/R treatment resulted in a significant increase in GRX2 expression in cardiomyocytes. GRX2 knockdown enhanced the sensitivity of cardiomyocytes to H/R-induced apoptosis, oxidative stress, and inflammation, while GRX2 up-regulation exerted a cardioprotective role in H/R-injured cardiomyocytes. Further investigations revealed that GRX2 up-regulation enhanced the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling associated with upregulation of the phosphorylation of Akt and glycogen synthase kinase-3β (GSK-3β). Akt inhibition markedly abolished GRX2-mediated activation of Nrf2, while GSK-3β inhibition reversed GRX2-knockdown-mediated inhibition of Nrf2. In addition, Nrf2 inhibition markedly abrogated GRX2-mediated protective effects against H/R-induced apoptosis, oxidative stress and inflammation. Overall, this work indicates that GRX2 protects cardiomyocytes from H/R-induced apoptosis, oxidative stress, and inflammation by enhancing Nrf2 activation via modulation of the Akt/GSK-3β axis. Our study highlights a potential relevance of GRX2 in myocardial ischemia-reperfusion injury; it may serve as an attractive target for cardioprotection., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Mitochondrial reactive oxygen species (mtROS)-induced apoptosis has been suggested to contribute to myocardial ischemia/reperfusion injury. Interleukin 35 (IL-35), a novel anti-inflammatory cytokine, has been shown to protect the myocardium and inhibit mtROS production. However, its effect on cardiomyocytes upon exposure to hypoxia/reoxygenation (H/R) damage has not yet been elucidated. The present study aimed to investigate the potential protective role and underlying mechanisms of IL-35 in H/R-induced mouse neonatal cardiomyocyte injury. Mouse neonatal cardiomyocytes were challenged to H/R in the presence of IL-35, and we found that IL-35 dose dependently promotes cell viability, diminishes mtROS, maintains mitochondrial membrane potential, and decreases the number of apoptotic cardiomyocytes. Meanwhile, IL-35 remarkably activates mitochondrial STAT3 (mitoSTAT3) signaling, inhibits cytochrome c release, and reduces apoptosis signaling. Furthermore, co-treatment of the cardiomyocytes with the STAT3 inhibitor AG490 abrogates the IL-35-induced cardioprotective effects. Our study identified the protective role of IL-35 in cardiomyocytes following H/R damage and revealed that IL-35 protects cardiomyocytes against mtROS-induced apoptosis through the mitoSTAT3 signaling pathway during H/R., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

This study proposed to investigate the function of miR-19a/ACSL axis in hypoxia/reoxygenation (H/R)-induced myocardial injury and determine whether metformin exerts its protective effect via miR-19a/ACSL axis. Firstly, bioinformatics analysis of data from Gene Expression Omnibus (GEO) database indicated that miR-19a was downregulated in patients with myocardial infarction (MI) compared to that in control group. H/R model was constructed with AC16 cells in vitro. qRT-PCR assay revealed that miR-19a was downregulated in H/R-treated AC16 cells. Then, CCK-8 assay demonstrated that upregulation of miR-19a significantly alleviated H/R-induced decline of cell viability. Moreover, bioinformatics prediction, western blotting and dual-luciferase reporter assays were performed to check the target genes of miR-19a, and ACSL1 was determined as a downstream target gene of miR-19a. Besides, the analysis based on Comparative Toxicogenomics Database (CTD) suggested that metformin targeting ACSL1 can be used as a potential drug for further research. Biological function experiments in vitro revealed that H/R markedly declined the viability and elevated the apoptosis of AC16 cells, while metformin can significantly mitigate these effects. Furthermore, overexpression of miR-19a significantly strengthened the beneficial effect of metformin on H/R-induced AC16 cells injury, which can be reversed by upregulation of ACSL1. In conclusion, metformin can alleviate H/R-induced cells injury via regulating miR-19a/ACSL axis, which lays a foundation for identifying novel targets for myocardial I/R injury therapy., (Copyright © 2021 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) and autophagy exert an important role in hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury. The current study aimed to explore the role of miRNA and autophagy in H/R-induced cardiomyocyte injury. Cardiomyocyte H9c2 was exposed to H/R to simulate H/R injury in vitro. The differentially expressed miRNAs were identified using quantitative RT-PCR (qPCR). Lactate dehydrogenase (LDH) activity was assayed to assess H/R injury. The role of miRNA and autophagy in regulating the viability and cell apoptosis was evaluated using cell counting kit-8 (CCK-8) assay, flow cytometry (FCM), and western blot. The autophagy activation was assessed through testing the number of light chain 3 (LC3) puncta and LC3-II expression using western blot and immunofluorescence analysis. In the present study, we found that the miR-542-5p expression and the autophagy activation were significantly increased in H9c2 cells after H/R injury. Functionally, forced expression of miR-542-5p further aggravated H/R injury in H9c2 cells, whereas miR-542-5p inhibition alleviated H/R injury as measured by the cell viability, LDH activity and cell apoptosis. miR-542-5p repressed autophagy activation, whereas miR-542-5p inhibition facilitated autophagy activation in H9c2 cells exposed to H/R as measured by the LC3 puncta number, LC3II, and p62 protein level. Especially, autophagy inhibition by specific inhibitor partially lessened the role of miR-542-5p inhibitor in alleviating H/R injury. Finally, the autophagy-related 7 (ATG7) was identified as a novel target gene of miR-542-5p in H9c2 cells. The current data suggest that miR-542-5p/autophagy pathway might be a potential target for the treatment of H/R-related heart diseases.

Presently, optimal proportions and synergistic mechanisms of component-based Chinese medicines are critical for developing novel strategies to treat cardiovascular diseases (CVDs). A new multi-objective optimization algorithm based on uniform design (UD) and stepwise regression (SR) modeling is proposed to find the synergistic effect of orientin (Ori), quercitrin (Que) and vitexin (Vit), the three effective components from Polygonum orientale L., using the H9c2 cells injury induced by hypoxia/reoxygenation (H/R). The optimal proportion of these three components was calculated by simulated annealing (SA). In this research, the excellent combination named OQV-e (Ori: Que: Vit =12.55 μM: 39.99 μM: 19.99 μM) could exert significant cardioprotection against the H9c2 cells injury induced by H/R through increasing cell viability, decreasing leakage rate of lactate dehydrogenase (LDH) and the level of nitric oxide (NO). Moreover, western blot analysis revealed that OQV-e could activate autophagy by inhibiting the p-JNK/JNK signaling pathway, which showed that the method (UD-SR-SA) was a feasible strategy. Mathematical system modeling may be a considerable approach for the powerful mathematical analysis of the complex pharmacological effects of component-based Chinese medicines from herbal medicines, which might greatly enhance the efficiency to find new modern Chinese drugs for CVDs based on Chinese herbal medicine (CHM) with affirmative therapeutic effect., (Copyright © 2020. Published by Elsevier Masson SAS.)

Myocardial injury is still a serious condition damaging the public health. Clinically, myocardial injury often leads to cardiac dysfunction and, in severe cases, death. Reperfusion of the ischemic myocardial tissues can minimize acute myocardial infarction (AMI)-induced damage. MicroRNAs are commonly recognized in diverse diseases and are often involved in the development of myocardial ischemia/reperfusion injury. However, the role of miR-431 remains unclear in myocardial injury. In this study, we investigated the underlying mechanisms of miR-431 in the cell apoptosis and autophagy of human cardiomyocytes in hypoxia/reoxygenation (H/R). H/R treatment reduced cell viability, promoted cell apoptotic rate, and down-regulated the expression of miR-431 in human cardiomyocytes. The down-regulation of miR-431 by its inhibitor reduced cell viability and induced cell apoptosis in the human cardiomyocytes. Moreover, miR-431 down-regulated the expression of autophagy-related 3 (ATG3) via targeting the 3'-untranslated region of ATG3. Up-regulated expression of ATG3 by pcDNA3.1-ATG3 reversed the protective role of the overexpression of miR-431 on cell viability and cell apoptosis in H/R-treated human cardiomyocytes. More importantly, H/R treatments promoted autophagy in the human cardiomyocytes, and this effect was greatly alleviated via miR-431-mimic transfection. Our results suggested that miR-431 overexpression attenuated the H/R-induced myocardial damage at least partly through regulating the expression of ATG3., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The high-mortality rate of cardiovascular diseases (CVDs) is associated with the myocardial ischemia and reperfusion (I/R). Recent investigations have revealed that microRNAs (miRNAs) exert vital functions in the apoptosis of cardiomyocyte cell. Nevertheless, the potential role of miR-30a-5p in the regulation of cardiomyocyte cell apoptosis needs to be illuminated. In the current study, we observed that hypoxia/reoxygenation (H/R) remarkably raised the level of miR-30a-5p but reduced the expression of E2F transcription factor 3 (E2F3) in H9c2 cardiomyocytes. In vivo, miR-30a-5p was found to be significantly upregulated in the hearts of rats following I/R. Downregulation of miR-30a-5p using anti-miR-30a-5p decreased H9c2 cardiomyocytes apoptosis caused by H/R and promoted the proliferation of H9c2 inhibited by H/R. Moreover, E2F3 was a possible target gene of miR-30a-5p and upregulation of miR-30a-5p reduced the expression level of E2F3 in H9c2 cardiomyocytes. We further identified that E2F3 silencing reversed the effect of anti-miR-30a-5p on the proliferation and apoptosis in H/R treated H9c2 cells. These studies suggested that downregulation of miR-30a-5p attenuated the impact of H/R on H9c2 cardiomyocytes through targeting E2F3., (© 2020 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia on behalf of Kaohsiung Medical University.)

Purpose: Citrate has a positive effect on improving the pathophysiological changes of cardiomyocytes such as cardiac failure and auricular fibrillation. However, the underlying mechanism remains still unclear. Methods: Rat cardiomyocytes were used to establish hypoxia/reoxygenation (H/R) cell model. Citrate was conduct to pretreat with cardiomyocytes, and microRNA-142-3p (miR-142-3p) knockdown and overexpression were used to determine the underlying mechanism of their functions in cardiomyocytes. Cell viability and apoptosis were respectively detected by CCK-8 and flow cytometry. Protein and mRNA levels were determined by Western blot and qRT-PCR. Luciferase reporter assay and Targetscan were performed to study the regulation of miR-142-3p and Rac1. Results: The level of miR-142-3p was down-regulated in H/R model, but up-regulated in cardiomyocytes following citrate treatment. Citrates attenuated H/R injury induced miR-142-3p level and cell viability, and also inhibited H/R injury induced apoptosis, LDH, MDA and autophagy. Cell viability was improved, and autophagy was suppressed by miR-142-3p mimic, while inhibitor had opposite results. Compared with H/R + miR-142-3p inhibitor group, cell viability was higher, and apoptosis and autophagy were lower in Cit + H/R + miR-142-3p inhibitor group. Furthermore, Rac1 was target gene of miR-142-3p, and decreased by citrate, in comparison with H/R + miR-142-3p inhibitor group. Conclusion: Taken together, our findings indicated that citrate ameliorates H/R injury-induced cardiomyocytes autophagy by regulating miR-142-3p/Rac1 aix.

Cardiomyocyte apoptosis induced by hypoxia and ischemia plays important roles in heart dysfunction after acute myocardial infarction (AMI). However, the mechanism of apoptosis induction remains unclear. A previous study reported that Y-box protein 1 (YB1) is upregulated after myocardial hypoxia/reoxygenation or ischemia/reperfusion (H/R or I/R, respectively) injury; however, whether YB1 is associated with H/R-induced cardiomyocyte apoptosis is completely unknown. In the present study, we investigated the roles of YB1 in H/R-induced cardiomyocyte apoptosis and the possible underlying molecular mechanisms. In vitro, H/R treatment upregulated the YB1 expression in H9C2 cells, whereas YB1 knockdown inhibited H/R-induced cardiomyocyte apoptosis and induced H9C2 cell proliferation via Src homology region 2 domain-containing phosphatase 1 (SHP-1)-mediated activation of signal transducer and activator of transcription 3 (STAT3). In vivo, YB1 knockdown ameliorated AMI, reducing infarct size, cardiomyocyte apoptosis, and oxidative stress, via SHP-1-mediated inactivation of STAT3. Additionally, YB1 knockdown inhibited H/R- or I/R-induced oxidative stress in vitro and in vivo. H/R and I/R increase YB1 expression, and YB1 knockdown ameliorates AMI injury via SHP-1-dependent STAT3 inactivation., (© 2020 Wiley Periodicals, Inc.)

Moderate hypothermia plays a major role in myocardial cell death as a result of hypoxia/reoxygenation (H/R) injury. However, few studies have investigated the molecular mechanisms of hypothermic cardioprotection. Several responses to stress and other cell functions are regulated by post‑translational protein modifications controlled by small ubiquitin‑like modifier (SUMO). Previous studies have established that high SUMOylation of proteins potentiates the ability of cells to withstand hypoxic‑ischemic stress. The level to which moderate hypothermia affects SUMOylation is not fully understood, as the functions of SUMOylation in the heart have not been studied in depth. The aim of the present study was to investigate the effect of moderate hypothermia (33˚C) on the protective functions of SUMOylation on myocardial cells. HL‑1 and H9c2 cells were treated with the hypoxia‑mimetic chemical CoCl2 and complete medium to simulate H/R injury. Hypothermia intervention was then administered. A Cell Counting kit‑8 assay was used to analyze cell viability. Mitochondrial membrane potential and the generation of reactive oxygen species (ROS) were used as functional indexes of mitochondria dysfunction. Bcl‑2 and caspase‑3 expression levels were analyzed by western blotting. The present results suggested that moderate hypothermia significantly increased SUMO1 and Bcl‑2 expression levels, as well as the mitochondrial membrane potential, but significantly decreased the expression levels of caspase‑3 and mitochondrial ROS. Thus, moderate hypothermia may enhance SUMOylation and attenuate myocardial H/R injury. Moreover, a combination of SUMOylation and moderate hypothermia may be a potential cardiovascular intervention.

Objective: LncRNA Fendrr plays an important role in cardiac development, but its role in myocardial ischaemia-reperfusion (I/R) injury remains unclear. P53 has been shown to be an important regulator of apoptosis and is involved in myocardial I/R-induced apoptosis. This study aims at investigating whether Fendrr affects hypoxia/reoxygenation (H/R)-induced cardiomyocyte apoptosis through p53., Methods: The left anterior descending coronary artery of the rat was ligated for 30 min and then reperfusion for 120 min by releasing the suture. Neonatal rat ventricular myocytes (NRVM) and rat cardiac cell line H9c2 were cultured for 6 h in hypoxia (95% N 2 and 5% CO 2 ), followed by reoxygenation (95% air and 5% CO 2 ) for 6 h. Transfection were performed in cells. Apoptosis was detected by flow cytometry. Moreover, RNA pull-down, RNA immunoprecipitation, ubiquitination assay, GST pull-down assay and co-immunoprecipitation were used to detect the regulation of Fendrr on p53 protein., Key Findings: Fendrr was decreased in I/R-induced myocardium and H/R-induced cardiomyocyte, and overexpression of Fendrr inhibited H/R-induced NRVM or H9c2 cells apoptosis. Further research found that the 1381-2100 nt of Fendrr bound to p53 protein and Fendrr promoted t direct binding of p53 to Cop1. The inhibition of Fendrr reduced the binding of E3 ubiquitin-protein ligase constitutive photomorphogenesis protein 1 (COP1) to p53 and reduced the ubiquitination of p53. Furthermore, the inhibition of Fendrr on H/R-induced NRVM or H9c2 cells apoptosis could be reversed by overexpression of p53., Conclusions: Fendrr can inhibit H/R-induced cardiomyocyte apoptosis, which is partly through promoting the ubiquitination and degradation of p53 by increasing the binding of Cop1 and p53., (© 2020 Royal Pharmaceutical Society.)

Endoplasmic reticulum (ER) oxidase 1α (ERO1α) is a glycosylated flavoenzyme that is located on the luminal side of the ER membrane, which serves an important role in catalyzing the formation of protein disulfide bonds and ER redox homeostasis. However, the role of ERO1α in myocardial hypoxia/reoxygenation (H/R) injury remains largely unknown. In the present study, ERO1α expression levels in H9C2 cardiomyocytes increased following H/R, reaching their highest levels following 3 h of hypoxia and 6 h of reoxygenation. In addition, H/R induced apoptosis, and significantly increased expression levels of ER stress (ERS) markers 78 kDa glucose‑regulated protein and C/EBP homologous protein. Moreover, the genetic knockdown of ERO1α using short hairpin RNA suppressed cell apoptosis, caspase‑3 activity, expression levels of cleaved caspase‑12 and cytochrome c in the cytoplasm. Overall, this suggested that ERO1α knockdown may protect against H/R injury. The ERS activator tunicamycin (TM) was used to counteract the ERO1α‑induced reduction in ERS; however, the percentage of apoptotic cells and the level of mitochondrial damage did not change. In conclusion, the results from the present study suggested that ERO1α knockdown may protect H9C2 cardiomyocytes from H/R injury through inhibiting intracellular ROS production and increasing intracellular levels of Ca2+, suggesting that ERO1α may serve an important role in H/R.

Pum2 is a ribonucleic acid binding protein that controls target mRNA turnover. It has been reported to be potentially associated with cardiac fibrosis. However, little is known about the role of Pum2 in cardiac disease. In this study, we found that Pum2 was upregulated in the rat heart tissue subjected to ischemia/reperfusion procedure and cultured neonatal rat ventricular cardiomyocytes (NRVMs) with hypoxia/reoxygenation (H/R) treatment. Further, knockdown of Pum2 showed a beneficial effect on H/R treated NRVMs through decreasing caspase 3-associated apoptosis, whereas overexpression of Pum2 increased H/R-induced NRVMs apoptosis. Moreover, our results demonstrated that Sirt1 was identified as the target of Pum2-mediated mRNA decay in cardiomyocytes, and two Pum2 binding elements were found in the 3'-untranslated region of Sirt1 mRNA. Additionally, overexpression of Pum2 prompted the acetylation of LKB1 by decreasing Sirt1's mRNA level, which in turn repressed the activity of AMPK pathway in both normoxic and H/R-treated NRVMs. Finally, our data indicated that the pro-apoptotic effect of Pum2 was dependent on Sirt1 and AMPK. Collectively, our results provide the evidence that Pum2-mediated Sirt1 mRNA decay plays a detrimental role in H/R-induced cardiomyocytes injury., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Neuregulin-1 (NRG-1)/erythroblastic leukaemia viral oncogene homologues (ErbB) pathway activation plays a crucial role in regulating the adaptation of the adult heart to physiological and pathological stress. In the present study, we investigate the effect of recombined human NRG-1 (rhNRG-1) on mitochondrial biogenesis, mitochondrial function, and cell survival in neonatal rat cardiac myocytes (NRCMs) exposed to hypoxia/reoxygenation (H/R). The results of this study showed that, in the H/R-exposed NRCMs, mitochondrial biogenesis was impaired, as manifested by the decrease of the expression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and mitochondrial membrane proteins, the inner membrane (Tim23), mitofusin 1 (Mfn1), and mitofusin 2 (Mfn2). RhNRG-1 pretreatment effectively restored the expression of PGC-1α and these membrane proteins, upregulated the expression of the anti-apoptosis proteins Bcl-2 and Bcl-xL, preserved the mitochondrial membrane potential, and attenuated H/R-induced cell apoptosis. Blocking PGC-1 expression with siRNA abolished the beneficial role of rhNRG-1 on mitochondrial function and cell survival. The results of the present study strongly suggest that NRG-1/ErbB activation enhances the adaption of cardiomyocytes to H/R injury via promoted mitochondrial biogenesis and improved mitochondrial homeostasis. SIGNIFICANCE OF THE STUDY: The results of this research revealed for the first time the relationship between neuregulin-1 (NRG-1)/erythroblastic leukaemia viral oncogene homologues (ErbB) activation and mitochondrial biogenesis in neonatal cardiomyocytes and verified the significance of this promoted mitochondrial biogenesis in attenuating hypoxia/reoxygenation injury. This finding may open a new field to further understand the biological role of NRG-1/ErbB signalling pathway in cardiomyocyte., (© 2020 John Wiley & Sons Ltd.)

Background: Apoptosis, reactive oxidative stress (ROS) and inflammation act as the pivotal pathogenesis of myocardial ischemia/reperfusion (I/R) injury (MIRI). Our prior study and other investigation have demonstrated the participations of src homology 2 (SH2) B adaptor protein 1 (SH2B1) in ischemic injury and cardiac hypertrophy; whereas, the involvements of SH2B1 in MIRI and underlying mechanisms are completely unknown., Method: In present study, MIRI model in vivo was induced by 30 min of ligation of LAD coronary artery and 24 h of reperfusion, and primary cultured cardiomyocytes were challenged with 2 h of hypoxia followed by 4 h of reoxygenation (H/R) to mimic MIRI in vitro. Adenovirus encoding for SH2B1 or GFP were pre-transfected into myocardium prior to MIRI both in vivo and in vitro. The myocardial damage, cardiac function, apoptosis, ROS and inflammation were evaluated systematically. Immunofluorescence staining and western blotting were alternatively performed to detect protein expression., Results: The results exhibited that H/R or I/R significantly reduced SH2B1 in cardiomyocytes, followed by impaired cell survival and function, which were strongly reversed after the adenovirus-mediated SH2B1 up-regulation. Meanwhile, I/R- and H/R-elevated inflammation, apoptosis and ROS were also alleviated by SH2B1 up-regulation. A mechanistic study suggested that the protective contributions of SH2B1 on H/R-suffered cardiomyocytes were based on the activation of the PI3K/AKT pathway. The abolishment of the PI3K/AKT via a pharmacological inhibitor (LY294002) repressed anti-H/R capabilities of SH2B1., Conclusion: Therefore, SH2B1 prevents cardiomyocytes from inflammation, apoptosis and ROS in MIRI partially through the PI3K/AKT-dependent avenues. It may provide a novel therapeutic target for the treatment of MIRI., Competing Interests: Declaration of competing interest The authors declare that there is no duality of interest associated with this manuscript., (Copyright © 2019. Published by Elsevier B.V.)

Myocardial infarction represents one of the severe cardiovascular diseases and is one of the high-risk factors for mortality, and ischemia/reperfusion (I/R) injury is one of the risk factors that contribute to the high mortality of myocardial infarction. MicroRNAs have been proven as key regulators in various diseases including myocardial infarction. The present study was sought to determine the role of miR-703 in the myocardial I/R injury and to explore detailed mechanisms. Hypoxia/reoxygenation (H/R) treatment repressed cell viability, increased cytotoxicity and pyroptosis in mouse cardiomyocytes. More importantly, we found that miR-703 was suppressed in mouse cardiomyocytes upon H/R stimulation. Restoration of miR-703 expression in mouse cardiomyocytes counteracted the H/R-induced cytotoxicity and pyroptosis in mouse cardiomyocytes; and the effects of miR-703 inhibition on cell viability and pyroptosis were similar to that of H/R treatment in mouse cardiomyocytes. In a further investigation, we found that NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) was targeted and repressed by miR-703 in mouse cardiomyocytes. NLRP3 knockdown also attenuated H/R-induced cytotoxicity and pyroptosis in mouse cardiomyocytes. In the mechanistic perspective, NLRP3 enforced expression disrupted the protective effects of miR-703 restoration on the H/R-induced cytotoxicity and pyroptosis in mouse cardiomyocytes. Our results for the first time demonstrate the protective actions of miR-703 in the H/R-induced mouse cardiomyocyte injury. More importantly, miR-703 protects against H/R-induced cardiomyocyte injury via inhibiting the NLRP3/caspase-1-mediated pyroptosis.

Objective: This study aimed to determine whether proinflammatory cytokines have an effect on myocardial cells (MCs) and hepatocytes during myocardial ischemia to induce cyclic AMP-responsive element-binding protein H (CREBH) cleavage, activate the acute phase response in the liver, and cause a superimposed injury in MCs., Methods: In this study, a hepatocyte-MC transwell co-culture system was used to investigate the relationship between myocardial hypoxia/reperfusion injury and CREBH cleavage. MCs and hepatocytes of neonatal rats were obtained from the ventricles and livers of Sprague-Dawley rats, respectively. MCs were inoculated into the lower chamber of transwell chambers for 12 hours under hypoxia. Levels of the endoplasmic reticulum stress protein glucose-regulated protein 78 in MCs, CREBH in hepatocytes, inflammatory factor (tumor necrosis factor-α and interleukin-6) levels, and cell viability were evaluated. The effect of CREBH knockdown was also studied using a CREBH-specific short hairpin RNA (Ad-CREBHi)., Results: We found that proinflammatory cytokines affect MCs and hepatocytes during myocardial ischemia to induce CREBH cleavage, activate the acute phase response in the liver, and cause superimposed injury in MCs., Conclusions: Expression of CREBH aggravates myocardial injury during myocardial ischemia.

Myocardial ischemia/reperfusion (I/R) injury is a major complication of reperfusion therapy in myocardial infarction. Ischemic myocardium produces a variety of peptides. We recently identified PDRPS7 as a novel peptide in cardiomyocytes that can be induced by hypoxia. However, the role of PDRPS7 is unknown. Here, we investigated the effects of PDRPS7 on hypoxia/reoxygenation (H/R)-induced injury in rat cardiomyoblast H9c2 cells and NRCMs. We found that PDRPS7 improved cell survival and attenuated lactate dehydrogenase leakage following H/R in H9c2 cells and NRCMs. PDRPS7 also alleviated H/R-induced pulsation reduction in NRCMs. Moreover, H/R-induced cell apoptosis was decreased in the presence of PDRPS7. H/R-induced reactive oxygen species generation was reduced by PDRPS7; in addition, PDRPS7 did not impact H 2 O 2 -induced cell injury. Signaling analysis demonstrated that H/R increased the phosphorylation levels of JNKs, ERKs, and p38 mitogen-activated protein kinases. However, PDRPS7 only attenuated H/R-induced JNK phosphorylation, but not phosphorylation of ERKs and p38. PDRPS7 protected cardiomyocytes from apoptosis by inhibiting JNK phosphorylation and c-Jun phosphorylation pathways, markedly upregulating anti-apoptotic Bcl-2 expression and inhibiting that of pro-apoptotic Bax and cleaved caspase-3. Importantly, pharmacological activation of JNKs diminished the protective effect of PDRPS7 in terms of cell survival against H/R stimulation. In summary, our study identified PDRPS7 as a novel cardioprotective peptide against H/R challenge and this action was mediated, at least in part, through inactivation of JNKs., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Background: Catechin protects heart from myocardial ischaemia/reperfusion (MI/R) injury. However, whether catechin inhibits H/R-induced myocardial cell apoptosis is largely unknown., Objective: This study aims to investigate the underlying mechanism of catechin in inhibiting the apoptosis of H/R-induced myocardial cells., Methods: LncRNA MIAT expression was detected by qRT-PCR. Cell viability of H9C2 cells was detected using CCK-8 assay. The apoptosis of H9C2 cells was detected by flow cytometry. The interaction between CREB and MIAT promoter regions was confirmed by dual-luciferase reporter gene assay and ChIP assay., Results: In MI/R rats, catechin improved heart function and down-regulated lncRNA MIAT expression in myocardial tissue. In H/R-induced H9C2 cells, catechin protected against cell apoptosis, and lncRNA MIAT overexpression attenuated this protective effect of catechin. We confirmed that transcription factor CREB could bind to MIAT promoter region, and catechin suppressed lncRNA MIAT expression through up-regulating CREB. Catechin improved mitochondrial function and relieved apoptosis through promoting Akt/Gsk-3β activation. In addition, MIAT inhibited Akt/Gsk-3β activation and promoted cell apoptosis in H/R-induced H9C2 cells. Finally, we found catechin promoted Akt/Gsk-3β activation through inhibiting MIAT expression in H/R-induced H9C2 cells., Conclusion: Catechin relieved H/R-induced myocardial cell apoptosis through regulating CREB/lncRNA MIAT/Akt/Gsk-3β pathway., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: lncUCA1 is abundantly expressed in the heart, indicating it may be important in maintaining normal myocardial function. However, the underlying mechanism of lncUCA1 in heart disease, particularly myocardial infarction (MI), is still in its infancy., Methods: LncUCA1 and miR-143 expression were measured in hearts of MI models. Overexpression and knockdown of lncUCA1 in neonatal rat cardiomyocytes were performed to confirm the effects of lncUCA1 in hypoxia-induced apoptosis., Results: The expression of lncUCA1 decreased but miR-143 increased inversely in MI heart. Overexpressing lncUCA1 protected cardiomyocytes from H/R induced apoptosis via inhibiting miR-143, which regulates apoptosis by targeting MDM2/p53 pathway. While silencing lncUCA1 caused miR-143 upregulation and H/R-induced apoptosis increase. Moreover, miR-143 was proved to be a competitive target of lncUCA1., Conclusions: lncUCA1 might protect cardiomyocyte against H/R induced apoptosis by suppressing miR-143 and modulated the following downstream MDM2/p53 signaling pathway, indicating the therapeutic potential of targeting lncUCA1 for MI., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background and Purpose: Intracellular calcium concentration ([Ca2+]i) overload occurs in myocardial ischemia and -reperfusion. The augmentation of the late sodium current (INaL) causes intracellular Na+ accumulation and subsequent [Ca2+]i overload via the reverse mode of the Na+/Ca2+ exchange current (reverse-INCX), which can lead to arrhythmia and cardiac dysfunction. Thus, inhibition of INaL is a potential therapeutic approach for ischemic heart disease. The aim of this study was to investigate the effects of thyroid hormone on augmented INaL, reverse-INCX, altered action potential duration (APD), and [Ca2+]i concentration in hypoxia/reoxygenation (H/R)-induced ventricular myocytes in vitro., Methods: The transient Na+ current (INaT), INaL, reverse-INCX, and APs were recorded using a whole-cell patch-clamp technique in neonatal mouse ventricular myocytes. [Ca2+]i concentration alteration were, respectively, observed by confocal microscopy and flow cytometry., Results: Triiodothyronine (T3) pretreatment decreased the INaL in a concentration-dependent manner. H/R injury aggravated the INaL, INaT, and reverse-INCX in cardiomyocytes and increased the continuous accumulation of [Ca2+]i (p < 0.05). The application of T3 prior to H/R injury significantly decreased the increased INaL, INaT, and reverse-INCX and blunted the [Ca2+]i increase. Furthermore, T3 pretreatment shortened the APD induced by H/R injury., Conclusion: T3 inhibited H/R-increased INaL and reverse INCX augmentation, shortened the APD, and diminished [Ca2+]i overload, indicating a potential therapeutic use of T3 as an INaL inhibitor to maintain Ca2+ homeostasis and protect cardiomyocytes against H/R injury., (© 2019 S. Karger AG, Basel.)

DJ-1 was recently reported to be involved in the cardioprotection of hypoxic preconditioning (HPC) against hypoxia/reoxygenation (H/R)-induced oxidative stress damage, by preserving mitochondrial complex I activity and, subsequently, inhibiting mitochondrial reactive oxygen species (ROS) generation. However, the molecular mechanism by which HPC enables mitochondrial translocation of DJ-1, which has no mitochondria-targeting sequence, to preserve mitochondrial complex I, is largely unknown. In this study, co-immunoprecipitation data showed that DJ-1 was associated with glucose-regulated protein 75 (Grp75), and this association was significantly enhanced after HPC. Immunofluorescence imaging and Western blot analysis showed that HPC substantially enhanced the translocation of DJ-1 from cytosol to mitochondria in H9c2 cells subjected to H/R, which was mimicked by DJ-1 overexpression induced by pFlag-DJ-1 transfection. Importantly, knockdown of Grp75 markedly reduced the mitochondrial translocation of DJ-1 induced by HPC and pFlag-DJ-1 transfection. Moreover, HPC promoted the association of DJ-1 with mitochondrial complex I subunits ND1 and NDUFA4, improved complex I activity, and inhibited mitochondria-derived ROS production and subsequent oxidative stress damage after H/R, which was also mimicked by pFlag-DJ-1 transfection. Intriguingly, these effects of HPC and pFlag-DJ-1 transfection were also prevented by Grp75 knockdown. In conclusion, these results indicated that HPC promotes the translocation of DJ-1 from cytosol to mitochondria in a Grp75-dependent manner and Grp75 is required for DJ-1-mediated protection of HPC on H/R-induced mitochondrial complex I defect and subsequent oxidative stress damage.

This study aims to investigate the role of targeting lncRNA myocardial infarction-associated transcript (MIAT) in protection against hypoxia/reoxygenation (H/R) injury in H9c2 cells in vitro and myocardial ischemia/reperfusion (I/R) injury in vivo by regulating expression of NF-kB and p53 upregulated modulator of apoptosis (PUMA). H9C2 cells were infected with lentivirus expressing the short-hairpin RNA direct against human MIAT gene (Lv-MIAT shRNA) or lentivirus expressing scrambled control (Lv-NC shRNA) or PUMA siRNA or p65 siRNA or their control siRNA respectively. Then the H9c2 cells were infected with Lv-shRNA to 2 hours of hypoxia (H) and 24 hour of reoxygenation (R). 100 ul of Lv-MIAT shRNA (1 × 10 8 PFU) or Lv-NC shRNA was transfected into mouse hearts, then the hearts were subjected to I/R (1h/72 h). We discovered targeting MIAT remarkably enhanced H9c2 cell viability, decreased H/R-induced cell apoptosis and LDH leakage and significantly decreased I/R-induced myocardial infarct size, reduced myocardial apoptosis and enhanced the heart function. Targeting MIAT downregulated p65 nuclear translocation, NF-κB activity and anti-apoptotic protein cleaved-caspase-3, Bax, and upregulated anti-apoptotic protein Bcl-2 induced by H/R or I/R. Our study suggests that targeting MIAT may protect against H9c2 cardiomyoblasts H/R injury or myocardial I/R injury via inhibition of cell apoptosis, mediated by NF-κB and PUMA signal pathway.

MicroRNA (miR) plays an integral role in cardiovascular diseases. M-iR-423-5p is aberrantly expressed in patients with myocardial infarction and heart failure. The aim of the present study was to study the roles and mechanisms of miR-423-5p in hypoxia/reoxygenation (H/R) mediated cardiomyocytes injury. H9C2 cells were transfected with negative control, miR-423-5p mimic, and inhibitor for 48 hr, followed by exposed to H/R condition. Cell apoptosis rate, caspase 3/7 activities, Bax and cleaved-caspase 3 (c-caspase 3) protein levels were assayed by flow cytometry, Caspase-Glo 3/7 Assay kit, western blot analysis, respectively. Furthermore, the mitochondrial membrane potential, adenosine triphosphate (ATP) content, reactive oxygen species (ROS) production, and Drp1 expression were also investigated. Furthermore, the dual-luciferase reporter assay was used to evaluate the relationship between miR-423-5p and Myb-related protein B (MYBL2). The roles of miR-423-5p in wnt/β-catenin were assessed by western blot analysis. The results revealed that H/R triggered miR-423-5p expression. Overexpression of miR-423-5p promoted cardiomyocyte apoptosis, enhanced the activities of caspase 3/7, upregulated the expression of Bax and c-caspase 3. miR-423-5p upregulation caused the loss of mitochondrial membrane potential and the reduction of ATP content, the augment of ROS production and Drp1 expression. However, the opposite trends were observed upon suppression of miR-423-5p. In addition, miR-423-5p could target the 3' untranslated region of MYBL2. miR-423-5p depletion led to the activation of the wnt/β-catenin signaling pathway via targeting MYBL2. Knockdown of MYBL2 was obviously reversed the roles of miR-423-5p in apoptosis and mitochondrial dysfunction. Taken together, miR-423-5p suppression reduced H/R-induced cardiomyocytes injury through activation of the wnt/β-catenin signaling pathway via targeting MYBL2 in cardiomyocytes., (© 2019 Wiley Periodicals, Inc.)

Objective: MiR-499 has been reported to be expressed only in cardiomyocytes, and its expression would increase after acute myocardial infarction (AMI). miR-499 plays a role in the process of cardiomyocytes injury induced by hypoxia/reoxygenation (H/R), however, it still remains unclear., Results: Hypoxia inhibited miR-499-5p expression and H/R induced apoptosis. SOX6 was a target gene of miR-499-5p, and high expression of miR-499-5p inhibited the expression of SOX6. MiR-499-5p reduced H9c2 cells injury by inhibiting the expression of SOX6, overexpression of which could reverse the effect of miR-499-5p on H9c2 cells. MiR-499-5p inhibited the levels of LDH and MDA, while overexpression of miR-499-5p inhibited H/R-induced cell apoptosis. MiR-499-5p could up-regulate the level of Bcl-2 and down-regulate the expression levels of Bax and caspase-3. However, SOX6 partially reversed these effects of miR-499-5p., Conclusion: We proved that miR-499-5p inhibited H/R-induced cardiomyocytes injury by targeting SOX6. Our results suggested that miR-499-5p/SOX6 pathway may present a potential therapeutic target for the treatment of AMI.

Remifentanil postconditioning (RPC) has been shown to provide potent cardioprotection against ischemia/reperfusion (I/R) injury, but the underlying mechanism has not been fully elucidated. The current study was designed to investigate whether RPC protects cardiomyocytes against I/R injury through enhancement of autophagic flux. H9c2 cardiomyocytes were exposed to hypoxia/reoxygenation (H/R) to mimic myocardial I/R injury in vitro. Autophagosome formation was evaluated by detecting of light chain 3 (LC3) puncta number and LC3Ⅱ levels using immunofluorescence and western blotting, respectively. Additionally, dual fluorescent staining of LC3 and lysosomal-associated membrane protein 2, a lysosomal marker protein, were used to detect autolysosome formation. Moreover, autophagic flux integrity was tracked using changes in LC3Ⅱ and p62 levels. Lastly, myocardial injury was detected by Hoechst 33342 and propidium iodide staining and MTT assay. The results showed that RPC increased autophagosome formation and promoted autophagosome-lysosome fusion, thereby improving autophagic flux in H9c2 cells. Reversal of these effect by bafilomycin A1 or chloroquine co-administration at reoxygenation onset indicated that RPC improved the impaired autophagic flux following H/R injury. Induction of autophagy was associated with increased cell viability and decreased apoptosis. Autophagy inhibition with bafilomycin A1 or chloroquine and ATG7shRNA significantly abolished RPC-induced cardioprotection. In conclusion, our finding that RPC can protect cardiomyocytes against H/R injury through enhancement of autophagic flux suggests a new mechanism for myocardial protection of opioid postconditioning., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: To investigate the effect of NACHT-LRR-PYD- containing proteins 3 (NLRP3) mediated pyroptosis in myocardial cells undergoing hypoxia/deoxygenation (H/R) injury. Methods: In order to observe whether H/R-treatment could cause pyroptosis, H9c2 cells were divided into 2 groups randomly using the lottery method: control group(without H/R-treatment) and H/R group (in which the H9c2 cells were underwent H/R-treatment). In order to clarify the role of pyroptosis in H/R-injury, H9c2 cells were divided into 4 groups randomly using the lottery method: control group(in which the H9c2 cells were cultivated with normal medium); YVAD group(in which the H9c2 cells were pretreated with z-Val-Ala-Asp(Ome)-fluoromethylketone (Z-YVAD-FMK) 20 μm for 4 hours, then replaced with normal medium); H/R group(H9c2 cells underwent H/R-treatment); YVAD+H/R group (in which the H9c2 cells were pretreated with 20 μm Z-YVAD-FMK for 4 hours before H/R-treatment). To determine whether H/R-induced cell pyroptosis is associated with NLRP3, H9c2 cells were divided into 4 groups randomly using the lottery method: control group (in which cells were transfected with a control nonspecific siRNA); si-NLRP3 group (in which cells were transfected with NLRP3-targeting siRNA); H/R group(in which cells were transfected with a control nonspecific siRNA before H/R-treatment); si-NLRP3+H/R group(in which the H9c2 cells were transfected with NLRP3-targeting siRNA before H/R-treatment). Pore formation on cell membrane was detected by propidium iodide (PI) staining. Cell viability was detected by CCK8 reagent. The protein expression of Caspase-1, interleukin-1β (IL-1β) and NLRP3 was detected by Western blot. Results: (1) The positive rate of PI staining ((26.46±5.15)% vs. (1.69±0.73)%, P< 0.01), expression of NLRP3 (0.57±0.16 vs. 0.23±0.06, P< 0.01), expression of Caspase-1 (1.07±0.13 vs. 0.37±0.08, P< 0.01), and expression of IL-1β (0.38±0.08 vs. 0.16±0.05, P< 0.01) were significantly higher in H/R group than in control group. (2)The cell vitality was significantly higher in YVAD+H/R group than in H/R group ((87.31±9.05)% vs. (73.30±7.19)%, P< 0.05).The positive rate of PI staining was significantly decreased in YVAD+H/R group than in H/R group ((18.12±4.36)% vs. (26.45±4.60)%, P< 0.05). The expression of Caspase-1 (0.72±0.12 vs. 1.07±0.15, P< 0.05) and IL-1β(0.29±0.07 vs. 0.39±0.06, P< 0.05) were significantly lower in YVAD+H/R group than in H/R group. (3) The cell vitality was significantly increased in si-NLRP3+H/R group than in H/R group ((85.46±7.71)% vs. (72.41±5.53)%, P< 0.05). The positive rate of PI staining was significantly lower in si-NLRP3+H/R group than in H/R group ((18.22±4.20)% vs. (26.73±3.26)%, P< 0.05). The expression of Caspase-1(0.87±0.07 vs. 1.15±0.15, P< 0.05) and IL-1β(0.41±0.07 vs. 0.58±0.10, P< 0.05) were significantly decreased in si-NLRP3+H/R group than in H/R group. Conclusion: NLRP3 mediated pyroptosis is involved in H/R injury of myocardial cells.

Cardiomyocyte apoptosis plays an important role in ischemia/reperfusion (I/R)-induced myocardial injury. Autophagy is suggested to be widely implicated in regulating cell survival and death. The cardioprotection of sevoflurane postconditioning has been long recognized, but the underlying mechanisms are not well understood. This study aims to investigate whether the cardioprotective effects of sevoflurane are associated with autophagy regulation. An in vitro hypoxia/reoxygenation (H/R) model was established in human induced pluripotent stem cell-derived cardiomyocytes. The results showed that autophagy was activated in cardiomyocytes upon to H/R conditions, followed by increased LC3B puncta. Sevoflurane treatment or autophagy inhibition markedly attenuated H/R-induced cardiomyocyte apoptosis. However, the effect of sevoflurane was reversed by autophagy induction. Moreover, sevoflurane significantly blocked H/R-induced autophagosome formation and autophagic flux. Mechanistically, we found that sevoflurane regulated H/R-induced autophagy through mTOR-independent mechanism. Sevoflurane inhibited the increase in PI3KC3 phosphorylation and Beclin-1/PI3KC3 complex formation under H/R conditions. Taken together, these results demonstrate that sevofluran ameliorates H/R-induced cardiomyocyte apoptosis by autophagy inhibition via reducing Beclin-1/PI3KC3 formation and PI3KC3 activity. This novel mechanism may help to better understand the functional role of sevoflurane for the treatment of cardiac I/R injury.

Our previous study showed that Sphingosine-1-phosphate (S1P) could protect cardiomyocytes against hypoxia/reoxygenation (H/R) injury via the JAK-STAT pathway and maintain normal myocardial mitochondria integrity in vivo. However, it is not known yet whether S1P can relieve mitochondrial dysfunction via the mitochondrial apoptotic pathway and its detailed mechanism remains to be investigated. The aim of this study was to demonstrate the mitochondrial protective effects of S1P in a cardiomyocyte H/R injury model. In the present study, we established a H/R model in H9c2 cells. Cell viability was determined by the MTT assay, and apoptosis was evaluated by annexin V-FITC/PI staining. Mitochondrial calcium ion concentration, mitochondrial membrane potential (ΔΨm), opening of the mitochondrial permeability transition pore (mPTP), and release of cytochrome C were detected by laser confocal microscopy. The results showed that S1P inhibited the decrease in cell viability induced by H/R injury and reduced apoptosis. Confocal microscopy showed that S1P prevented loss of ΔΨm, relieved mitochondrial calcium overload, and inhibited opening of the mPTP and release of cytochrome C. The STAT3 inhibitor STATTIC can reverse the antiapoptotic effects of S1P and block the effect of S1P on mitochondria. Taken together, our results indicate that S1P protects cardiomyocytes against H/R injury by relieving mitochondrial dysfunction via the STAT3 pathway., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Exogenously administered B-type natriuretic peptide (BNP) has been shown to provide cardioprotection against various heart diseases. However, the underlying mechanisms remain elusive. This study explores whether BNP exerts its cardioprotection against hypoxia/reoxygenation (H/R) injury under high glucose/high fat (HG/HF) conditions in cardiac H9c2 cells and uncovers the underlying mechanisms. Our data revealed that BNP significantly increased the cell viability and decreased the release of lactate dehydrogenase (LDH) and creatine kinase (CK), with a maximal effect at the BNP concentration of 10 -7  mol/L. In addition, by analyzing the activation of cleaved caspase-3 and by Annexin V-FITC/PI staining, we showed that BNP attenuated H/R-induced cell apoptosis in HG/HF conditions. Western blot analysis showed enhanced phosphorylation of protein kinase RNA (PKR)-like endoplastmic reticulum (ER) kinase (PERK) and eukaryotic initiation factor 2α (eIF2α)(one of the three main signaling pathways in endoplastmic reticulum (ER) stress), and increased expression of GRP78 and CHOP proteins (ER stress-related proteins) in H9c2 cells which underwent H/R in HG/HF conditions. Treatment with BNP or 8-Br-cGMP (an analog of cGMP) reversed this activation. However, this effect was significantly weakened by KT-5823, a selective cGMP-dependent protein kinase G (PKG) inhibitor. In addition, similar to BNP, treatment with a specific inhibitor of ER stress tauroursodeoxycholic acid (TUDCA) protected the cells against H/R injury exposed to HG/HF conditions. In conclusion, these findings demonstrated that BNP effectively protected cells against H/R injury under HG/HF conditions by inhibiting the ER stress via activation of the cGMP-PKG signaling pathway., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Rhein, an anthraquinone compound isolated from rhubarb, has been shown to protect the pancreatic β cells from hyperglycemia induced apoptosis in our previous studies., Purpose: In the present study, we examined whether rhein can protect myocardial cells against ischemia reperfusion (I/R)-induced apoptosis and investigated the underlying mechanism., Methods: We used an in vitro model of myocardial hypoxia/reoxygenation (H/R) injury. H9c2 cells were incubated with rhein for 1 h and then subjected to hypoxia for 6 h, followed by reoxygenation for 2 h. Cells viability, apoptosis and ROS were assayed for the treated cells. AKT, p-AKT, GSK3β, p- GSK3β, P38 and p-P38 proteins were analyzed using Western blotting. PI3K/AKT inhibitor, LY294002, and GSK3β siRNA were also used to determine the signaling pathways involved in the protection by rhein., Results: Rhein increased viability, decreased apoptosis and ROS production, of the cells that were exposed to H/R. Rhein also increased the phosphorylation of AKT and GSK3β, an effect that was eliminated by LY294002. GSK3β silencing by siRNA showed similar effect as LY294002. The p-P38 level was upregulated by H/R and downregulated in the presence of rhein; however, the p-P38 downregulation was completely abolished by GSK3β silencing., Conclusion: Rhein protects myocardial H9c2 cells against hypoxia/reoxygenation induced injury via AKT/ GSK3β/p38 pathway., (Copyright © 2018. Published by Elsevier GmbH.)