Apoptosis In H9c2 Cardiomyocytes Research Articles

Hederagenin protects against myocardial ischemia-reperfusion injury via attenuating ALOX5-mediated ferroptosis.

Hederagenin (HDG), a medical herb, is known for its beneficial activities against diverse diseases. The cardioprotective effect of HDG has been preliminarily disclosed, but the efficacy and underlying mechanism by which HDG protects against myocardial ischemia-reperfusion (MI/R) injury have not been elucidated yet. To simulate MI/R injury, the left anterior descending artery was occluded for 30min and then reperfusion for 120min in a rat model, and the cellular model of hypoxia-reoxygenation (H/R) injury was constructed in H9c2 cardiomyocytes. Hematoxylin-eosin, Prussian blue, and 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) staining were conducted to assess the histological injury, iron deposition, and myocardial infarction. Myocardial enzymes and oxidative stress-related factors were detected using their commercial kits. Lipid peroxidation was measured using BODIPY581/591 probe, and iron content was detected. Cell counting kit (CCK)-8, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and flow cytometry assays were performed to assess cell viability and apoptosis. Protein levels were investigated by western blot. The interaction between HDG and 5-lipoxygenase (ALOX5) was verified using molecular docking. Our findings indicated that HDG significantly attenuated myocardial dysfunction by reducing infarction and myocardial injury. HDG significantly attenuated myocardial apoptosis in vitro and in vivo, as well as alleviating oxidative stress via reducing reactive oxygen species (ROS) and maintaining the balance between antioxidant and oxidant enzymes. Meanwhile, HDG inhibited I/R-induced ferroptosis in myocardium and cardiomyocytes, including reducing lipid peroxidation and iron level. Moreover, the binding relationship between HDG and ALOX5 was verified, and HDG could concentration dependently downregulate ALOX5. Furthermore, ALOX5 overexpression eliminated the inhibition of HDG on H/R-induced apoptosis, oxidative stress, and ferroptosis in H9c2 cardiomyocytes. HDG ameliorated myocardial dysfunction and cardiomyocyte injury by reducing apoptosis, oxidative stress, and ferroptosis through inhibiting ALOX5, providing a new perspective on the prevention and treatment of MI/R injury.

Everolimus prevents doxorubicin-induced apoptosis in H9c2 cardiomyocytes but not in MCF-7 cancer cells: Cardioprotective roles of autophagy, mitophagy, and AKT

Cardiotoxicity is a severe side effect of the chemotherapeutic agent doxorubicin (DOX). We recently showed that DOX-induced cardiomyocyte apoptosis and death were attenuated through autophagy pre-induction. Herein, we assessed how the autophagy/mitophagy-inducing antitumor drug everolimus (EVL) affected DOX-induced cytotoxicity in the rat cardiomyocyte cell line H9c2 and human breast cancer cell line MCF-7. Apoptosis was assessed using annexin V assay. Autophagy and mitophagy were assessed using fluorescence assays. Cellular protein levels were determined using western blotting. Pretreatment with EVL (1 nM) before DOX exposure inhibited mammalian target of rapamycin (mTOR) activity, induced autophagy and mitophagy, and activated protein kinase B (AKT) in H9c2 cells. In mitochondria, DOX (1 μM) induced structural damage (decreased membrane potential and release of cytochrome c), increased superoxide levels, decreased apoptosis inhibitor Bcl-2, and increased apoptosis inducer Bax, leading to apoptosis and reduced viability in H9c2 cells. EVL pretreatment suppressed DOX-induced changes. EVL anti-apoptotic effects were inhibited by treatment with MK-2206, a selective AKT inhibitor. Furthermore, EVL suppressed DOX-induced cardiotoxicity through autophagy/mitophagy and AKT activation but did not attenuate DOX-induced apoptosis or reduction in viability in MCF-7 cells. Altogether, EVL can protect cardiomyocytes from DOX-induced apoptosis and toxicity without reducing DOX antitumor effects, allowing safer chemotherapy.

Riboflavin protects against heart failure via SCAD-dependent DJ-1-Keap1-Nrf2 signalling pathway.

Our recent studies have shown that flavin adenine dinucleotide (FAD) exerts cardiovascular protective effects by supplementing short-chain acyl-CoA dehydrogenase (SCAD). The current study aimed to elucidate whether riboflavin (the precursor of FAD) could improve heart failure via activating SCAD and the DJ-1-Keap1-Nrf2 signalling pathway. Riboflavin treatment was given to the mouse transverse aortic constriction (TAC)-induced heart failure model. Cardiac structure and function, energy metabolism and apoptosis index were assessed, and relevant signalling proteins were analysed. The mechanisms underlying the cardioprotection by riboflavin were analysed in the cell apoptosis model induced by tert-butyl hydroperoxide (tBHP). In vivo, riboflavin ameliorated myocardial fibrosis and energy metabolism, improved cardiac dysfunction and inhibited oxidative stress and cardiomyocyte apoptosis in TAC-induced heart failure. In vitro, riboflavin ameliorated cell apoptosis in H9C2 cardiomyocytes by decreasing reactive oxygen species (ROS). At the molecular level, riboflavin significantly restored FAD content, SCAD expression and enzymatic activity, activated DJ-1 and inhibited the Keap1-Nrf2/HO1 signalling pathway in vivo and in vitro. SCAD knockdown exaggerated the tBHP-induced DJ-1 decrease and Keap1-Nrf2/HO1 signalling pathway activation in H9C2 cardiomyocytes. The knockdown of SCAD abolished the anti-apoptotic effects of riboflavin on H9C2 cardiomyocytes. DJ-1 knockdown hindered SCAD overexpression anti-apoptotic effects and regulation on Keap1-Nrf2/HO1 signalling pathway in H9C2 cardiomyocytes. Riboflavin exerts cardioprotective effects on heart failure by improving oxidative stress and cardiomyocyte apoptosis via FAD to stimulate SCAD and then activates the DJ-1-Keap1-Nrf2 signalling pathway.

Effect and mechanism of Fisetin on myocardial damage induced by Patulin.

The aim of this study is to investigate whether fisetin can effectively reduce the myocardial damage induced by patulin. This study also aims to reveal the mechanism and target of fisetin in inhibiting myocardial damage. Network pharmacology was used to screen the targets of fisetin on myocardial damage and the regulatory network of active ingredients-drug targets was constructed. GO and KEGG enrichment analyses were performed to screen out the key pathways and targets of fisetin on myocardial damage. Patulin induced apoptosis in H9c2 cardiomyocytes to verify the key targets. The mechanism of fisetin in inhibiting myocardial damage was determined. FIS can reduce the apoptosis of cardiomyocytes by protecting cardiomyocytes from PAT injury. According to the results of network pharmacology analysis, combined with enzyme activity detection and WB experiment, it was found that the mechanism of FIS to reduce myocardial damage may be related to the P53 signaling pathway, Caspase3/8/9 and Bax/Bcl-2. FIS plays a protective role in PAT-induced myocardial damage. On the one hand, FIS inhibits the protein overexpression of P53, Caspase-9 and Bax. On the other hand, FIS enhances the protein expression of Bcl-2.

Apurinic/apyrimidinic endonuclease 1 regulates palmitic acid-mediated apoptosis in cardiomyocytes via endoplasmic reticulum stress

Cardiomyocyte apoptosis caused by fat metabolism disorder plays an essential role in the pathogenesis of diabetic cardiomyopathy (DCM). Apurinic/apyrimidinic endonuclease 1 (APE1) has multiple functions, including regulating redox and DNA repair. However, the role of APE1 in the pathogenesis of DCM remains unclear. To investigate the mechanism of APE1 on high-fat induced apoptosis in H9C2 cells, we treated H9C2 cells with palmitic acid (PA) as an apoptosis model caused by hyperlipidemia. We found that PA reduced the viability and increased apoptosis of H9C2 cells by inducing up-regulation of APE1 protein and endoplasmic reticulum (ER) stress. APE1 knockdown enhanced PA-induced apoptosis, and ER stress and overexpression of APE1 demonstrated the opposite effect. Furthermore, APE1 regulated PA-induced apoptosis via ER stress. The APE1 mutant (C65A, lack of redox regulation) loses its protective effect against ER stress and apoptosis. These findings indicate that APE1 protects PA-induced H9C2 cardiomyocyte apoptosis through ER stress via its redox-regulated function. This study provided new insights into the therapy for DCM.

Isoalantolactone Regulates the Activity of Mitogen-Activated Protein Kinases and Nuclear-Factor Erythroid 2-Related Factor 2 Pathways to Attenue Angiotensin II-Induced Cardiomyocyte Hypertrophy and Oxidative Stress

While isoalantolactone is a sesquiterpene lactone known for its anti-inflammatory and antioxidant activities, its role in cardiac hypertrophy is unknown. This study aimed to explore the role and mechanism of isoalantolactone in cardiomyocyte hypertrophy using angiotensin II-treated H9C2 cells as a model. The Western blot analysis was used to evaluate changes in the cardiac hypertrophy marker proteins atrial natriuretic peptide and brain natriuretic peptide and the activity of nuclear-factor erythroid 2-related factor 2/mitogen-activated protein kinase signaling pathway. Rhodamine-phalloidin staining was used to measure the surface area of cardiomyocytes. Apoptosis and intracellular reactive oxygen species levels were measured by flow cytometry. The results showed that isoalantolactone inhibited angiotensin II-induced H9C2 cell hypertrophy and cardiomyocyte apoptosis and reduced intracellular reactive oxygen species and cytochrome C levels in a dose-dependent manner. Furthermore, isoalantolactone upregulated the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 and inhibited extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and p38 (a stress-stimulated kinase) activation. Collectively, the findings suggest that isoalantolactone can enhance the antioxidant capacity of H9C2 cells, reduce apoptosis, and inhibit cardiac hypertrophy, and these protective effects may be related to the regulation of nuclear-factor erythroid 2-related factor 2 and mitogen-activated protein kinase pathway activity by isoalantolactone.

MicroRNA-21-3p/Rcan1 signaling axis affects apoptosis of cardiomyocytes of sepsis rats.

Accumulating evidence has reported the role of microRNA-21-3p (miR-21-3p) in sepsis, and our objective was to discuss the effect of the miR-21-3p/regulator of calcineurin 1 (Rcan1) axis on cardiomyocyte apoptosis of septic rats. miR-21-3p and Rcan1 expression in myocardial tissues of lipopolysaccharide (LPS)-treated rats and LPS-treated H9c2 cardiomyocytes was determined. The influences of downregulating miR-21-3p or upregulating Rcan1 in cardiomyocyte apoptosis of LPS-treated rats and LPS-treated H9c2 cardiomyocytes were then evaluated. The target relation between Rcan1 and miR-21-3p was verified. It was observed that miR-21-3p was elevated and Rcan1 was reduced in LPS-treated rats and LPS-treated H9c2 cardiomyocytes. Downregulating miR-21-3p or upregulating Rcan1 could suppress cardiomyocyte apoptosis of LPS-treated rats and LPS-treated H9c2 cardiomyocytes. Based on the dual-luciferase activity assay, miR-21-3p was directly targeted Rcan1 in H9c2 cardiomyocytes. In the rescue experiment, the LPS+miR-21-3p inhibitor+si-Rcan1 group enhanced the apoptosis of H9c2 cardiomyocytes in comparison to the LPS+miR-21-3p inhibitor+si-NC group. Together, our findings identify that the miR-21-3p/Rcan1 axis may affect apoptosis of cardiomyocytes in sepsis, which provides a new idea for understanding the potential mechanism of sepsis.

Mangiferin Protects Against Angiotensin-II-Enhanced Hypertrophic Markers and Apoptosis in H9c2 Cardiomyocytes.

Hypertrophic cardiomyopathy accompanies numerous cardiovascular diseases, and the intervention of cardiac hypertrophy is an important issue to prevent detrimental consequences. Mangiferin (MGN) is a glucosylxanthone found in Mangifera indica, which exhibits anti-oxidant and anti-inflammatory properties. Various studies have demonstrated the cardioprotective potential of MGN, but the mechanisms behind its beneficial effects have not been fully revealed. Here, angiotensin-II (Ang-II) was used to induce cardiac hypertrophy, and we examined cell size, expression of hypertrophy markers (e.g., ANP, BNP, and [Formula: see text]-MHC), and oxidative stress (e.g., the ratio of NADPH/NADP[Formula: see text], the expression of p22phox and p67phox, and ROS and SOD production) of cardiomyocytes. Moreover, we assessed the activation of mitogen-activated protein kinase (MAPK) signaling (e.g., p38 and ERK) and the NF-[Formula: see text]Bp65/iNOS axis. Additionally, an annexin V/PI assay was employed to evaluate whether MGN administration can attenuate Ang-II-elicited apoptosis. Lastly, the expression of Ang-II type 1 receptor (AT1) was measured to confirm its involvement in MGN-mediated protection. Our results showed that treatment with MGN attenuated the Ang-II-induced cell size, expression of hypertrophy markers, and oxidative stress in cardiomyocytes. MGN also abrogated the activation of MAPK signaling and the NF-[Formula: see text]Bp65/iNOS axis. Additionally, MGN prevented apoptosis and downregulated the elevation of AT1 in cardiomyocytes that had been exposed to Ang-II. Altogether, these results demonstrated the potential of using MGN to ameliorate the Ang-II-associated cardiac hypertrophy, which may be due to its anti-oxidant and anti-inflammatory effects through suppression of MAPK signaling and the NF-[Formula: see text]Bp65/iNOS axis.

The protective effect of thiolutin on doxorubicin-induced H9c2 cardiomyocyte injury.

The use of doxorubicin (DOX) may contribute to cardiotoxicity, limiting its clinical application. Thiolutin (THL) has been found to exert protective roles in various biological activities, while its effects on DOX-induced cardiotoxicity are still uncovered. Cell counting kit 8 assay was utilized to detect cell viability and half maximal inhibitory concentration of THL in H9c2 cardiomyocytes. The level of lactate dehydrogenase (LDH), adenosine triphosphate (ATP), interleukin (IL)-18 and IL-1 beta (IL-1β) were measured using the corresponding detection kits, and flow cytometry determined cell apoptosis rate. The reactive oxygen species (ROS) accumulation was evaluated by utilizing immunofluorescence or flow cytometry assay. The protein levels of NLR family Pyrin domain 3 (NLRP3), pro-Caspase1, cleaved-Caspase1, gasdermin D (GSDMD) and cleaved-GSDMD (GSDMD-N) in H9c2 cells were detected by immunoblotting assay. The treatment of THL reduced H9c2 cell viability in a gradient-dependent manner. THL treatment reversed the DOX-induced inhibition of proliferation, decrease of ATP, up-regulation of LDH, IL-18, IL-1β and production of ROS, activation of NLRP3 and inflammasome-mediated pyroptosis in H9c2 cells. Additionally, NLRP3 knockdown abolished the effects of THL in DOX-treated H9c2 cells remarkably. This investigation proved that THL notably ameliorated DOX-induced apoptosis, oxidative stress, and pyroptosis in H9c2 cardiomyocytes. Besides, THL effectively inactivated DOX-induced NLRP3 inflammasome in H9c2 cells. These findings revealed a promising drug to assist DOX in its anti-cancer effects and protect the heart of patients.

MicroRNA‑194‑5p attenuates hypoxia/reoxygenation‑induced apoptosis in H9C2 cardiomyocytes by inhibiting the over‑activation of RAC1 protein.

Ras‑related C3botulinum toxin substrate1 (RAC1), a member of the Rac family of guanosine triphosphate phosphohydrolases, has been suggested to be a regulator of myocardial injury during ischemia and reperfusion (I/R). Whether microRNAs (miRs) are involved in the regulation of the aforementioned process remains to be elucidated. In the present study, an invitro model of H9C2 cardiomyocytes was used to establish the overexpression of RAC1 following hypoxia and reoxygenation (H/R). Overexpression of RAC1 in H/R‑cultured cardiomyocytes could lead to cellular accumulation of reactive oxygen species (ROS) and facilitate the induction of apoptosis of H9C2 cardiomyocytes during H/R. Subsequent bioinformatic analysis indicated that RAC1 was the target of miRNA‑194‑5p. Further experiments showed that miR‑194‑5p attenuated the accumulation of cellular ROS and alleviated the induction of apoptosis of H9C2 cardiomyocytes caused by H/R, which was accompanied by the reduction in the expression levels of the RAC1 protein. Taken together, these results indicated that upregulation of miR‑194‑5p may function as a self‑regulated cardioprotective response against RAC1‑mediated ROS accumulation and cardiomyocyte apoptosis. Exogenous administration of miR‑194‑5p may be a novel target to ameliorate I/R injury‑induced myocardial apoptosis.

A novel peptide HSP-17 ameliorates oxidative stress injury and apoptosis in H9c2 cardiomyocytes by activating the PI3K/Akt pathway.

Oxidative stress and cell apoptosis play pivotal roles in the pathogenesis of doxorubicin (DOX)-induced myocardial injury. Heat shock protein-derived peptide (HSP-17) is a peptide which is low-expressed in DOX treated mouse heart tissue. It has high bioactivity and interspecies sequence consistency, and is predicted to have myocardial protective effect. Firstly, we added 1 µM DOX to H9c2 cell culture medium for 24 hours to construct the myocardial cytotoxicity model. Then we detected the effect of HSP-17 on DOX induced H9c2 cardiomyocyte injury by measuring cell viability and lactate dehydrogenase (LDH) level. In addition, reactive oxygen species (ROS) and tetraethylbenzimidazolylcarbocyanine iodide kits are used to evaluate the effect of the HSP-17 peptide on DOX-induced oxidative stress injury to cardiomyocytes, and the detection of apoptosis related proteins and flow cytometry were applied to detect the level of apoptosis. Furthermore, the protein expression levels [phosphorylated Akt (p-Akt) and phosphorylated PI3K (p-PI3K)] of the PI3K/Akt pathway were also detected by western blotting. We found that the HSP-17 peptide can increase cell viability, protect mitochondrial potential, reduce LDH levels, and reduce ROS and cardiomyocyte apoptosis. In addition, we also observed that HSP-17 upregulated the expression level of p-Akt, and LY294002, a typical inhibitor of PI3K/Akt, was found to eliminate the protective roles of HSP-17. In conclusion, this study demonstrated that the HSP-17 peptide protected H9c2 cells against oxidative stress and apoptosis via PI3K/Akt pathway activation, which provides a new idea for the treatment of DOX-induced myocardial injury.

Effects of Irradiation Combined with Trastuzumab on Myocardial Cell Line H9c2 In Vitro

<h3>Purpose/Objective(s)</h3> The current clinical guidelines do not make clear restrictions or affirmation on whether chest radiotherapy can be delivered currently with trastuzumab. We aim to analyze the effect of trastuzumab combined with dose gradient X-ray irradiation on H9c2 cardiomyocytes and detect the relevant mechanism. <h3>Materials/Methods</h3> Trastuzumab was given at the same time with X-ray irradiation of H9c2 cardiomyocytes with dose gradient (0, 2, 5, 10, 15gy). Flow cytometry was used to detect the proportion of apoptosis, a cell counting kit was used to detect the viability of cells, and WB was used to detect apoptosis related proteins for analyzing the damage effect of irradiation combined with trastuzumab on cardiomyocytes. To analyze the mechanism of trastuzumab aggravating myocardial radiotoxicity by detecting ROS content, Akt protein phosphorylation and DNA damage. <h3>Results</h3> 1. Flow cytometry and WB suggested that irradiation could promote the apoptosis of H9c2 cardiomyocytes, which was positively correlated with the dose of irradiation; after trastuzumab treatment, apoptosis was further improved. 2. A cell counting kit assay showed that irradiation combined with trastuzumab could superimpose the inhibitory effect on cardiomyocyte activity. 3. Compared with irradiation alone, irradiation combined with trastuzumab significantly inhibited the phosphorylation of Akt molecules. 4. Trastuzumab could promote the aggregation of ROS in cells by Flow cytometry, and enhance DNA damage induced by irradiation by the formation of fluorescence focus of γ - H2AX. <h3>Conclusion</h3> 1. Radiation injury to isolated cardiomyocytes was dose-dependent. Meanwhile, trastuzumab aggravated the toxicity of irradiation on cardiomyocytes. 2. Trastuzumab may aggravate myocardial radiation injury by inhibiting Akt phosphorylation, promoting ROS accumulation in cells and promoting DNA damage.

Atractylenolide III Attenuates Apoptosis in H9c2 Cells by Inhibiting Endoplasmic Reticulum Stress through the GRP78/PERK/CHOP Signaling Pathway.

The objective of this study was to determine the effect of atractylenolide III (ATL-III) on endoplasmic reticulum stress (ERS) injury, H9c2 cardiomyocyte apoptosis induced by tunicamycin (TM), and the GRP78/PERK/CHOP signaling pathway. Molecular docking was applied to predict the binding affinity of ATL-III to the key proteins GRP78, PERK, IREα, and ATF6 in ERS. Then, in vitro experiments were used to verify the molecular docking results. ERS injury model of H9c2 cells was established by TM. Cell viability was detected by MTT assay, and apoptosis was detected by Hoechst/PI double staining and flow cytometry. Protein expression levels of GRP78, PERK, eIF2α, ATF4, CHOP, Bax, Bcl-2, and Caspase-3 were detected by Western blot. And mRNA levels of GRP78, CHOP, PERK, eIF2α, and ATF4 were detected by RT-qPCR. Moreover, the mechanism was further studied by using GRP78 inhibitor (4-phenylbutyric acid, 4-PBA), and PERK inhibitor (GSK2656157). The results showed that ATL-III had a good binding affinity with GRP78, and the best binding affinity was with PERK. ATL-III increased the viability of H9c2 cells, decreased the apoptosis rate, downregulated Bax and Caspase-3, and increased Bcl-2 compared with the model group. Moreover, ATL-III downregulated the protein and mRNA levels of GRP78, CHOP, PERK, eIF2α, and ATF4, consistent with the inhibition of 4-PBA. ATL-III also decreased the expression levels of PERK, eIF2α, ATF4, CHOP, Bax, and Caspase-3, while increasing the expression of Bcl-2, which is consistent with GSK2656157. Taken together, ATL-III could inhibit TM-induced ERS injury and H9c2 cardiomyocyte apoptosis by regulating the GRP78/PERK/CHOP signaling pathway and has myocardial protection.

Effects of PUMA knockout on the apoptosis of H9C2 cardiomyocytes induced by high glucose

To investigate the effects of p53 upregulated modulator of apoptosis (PUMA) on the apoptosis of H9C2 cardiomyocytes induced by high glucose and its mechanisms. H9C2 cardiomyocytes were treated with 5.5mmol/L (control group) or 35 mmol/L glucose (HG group) for 6 h, 12 h, 24 h or 48 h respectively to induce apoptosis, each group sets 5 multiple wells. Apoptosis was tested by TUNEL assay. PUMA mRNA was measured by RT-PCR and protein expression was measured by Western blot assay. The mitochondrial membrane potential was detected by JC-1 method. The expressions of cleaved caspase-3 and cytochrome C (Cyt C) protein in mitochondria and cytoplasm were determined by Western blot assay. H9C2 cardiomyocytes were randomly divided into four groups, control group (5.5 mmol/L), HG (35 mmol/L) group, HG+si-scramble group(si-scramble treatment for 24 h, then 35 mmol/L high glucose treatment for 24 h) and HG-si-PUMA group (si-PUMA treatment for 24 h, then 35mmol/L high glucose treatment for 24 h). Si-PUMA was transfected into cardiomyocytes and the effects of PUMA on high glucose-induced apoptosis were studied. Compared with the control group, high glucose increased cardiomyocyte apoptosis and enhanced PUMA mRNA and protein expressions significantly (P＜0.05 or P＜0.01). Cell injury and increased PUMA expression were time-dependent and there was no significant difference between the high glucose 24 h group and the high glucose 48h group. The following experiment used high glucose 24 h as the stimulation time. The cardiomyocytes transfected with si-PUMA to inhibit PUMA expression had decreased apoptotic rate and cleaved caspase-3, increased mitochondria membrane potential and decreased Cyt C release (P＜0.05 or P＜0.01). There were no significant differences between the HG+si-scramble group and the high glucose group (P＞0.05). PUMA mediates high glucose-induced cardiomyocyte apoptosis suggesting PUMA may be an important target gene of diabetic cardiomyopathy.

Pinoresinol diglucoside ameliorates H/R-induced injury of cardiomyocytes by regulating miR-142-3p and HIF1AN.

This study is aimed to investigate the effect of pinoresinol diglucoside (PDG) in ameliorating myocardial ischemia-reperfusion injury (MIRI).Hypoxia/reperfusion (H/R)-induced H9c2 cardiomyocytes were used to establish an in-vitro ischemia-reperfusion injury model of cardiomyocytes. Cells were treated with 1 μmol/L of PDG. Reactive oxygen species (ROS) level was detected by a 2',7'-dichlorofluorescein-diacetateassay. The release of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) was examined by enzyme-linked immunosorbent assay. The viability and apoptosis of H9c2 cells were probed by MTT assay and flow cytometry. Besides this, Western blot and quantitative real-time PCR were used to detect microRNA-142-3p (miR-142-3p) and hypoxia-inducible factor 1 subunit alpha inhibitor (HIF1AN) expression levels. The binding sequence between miR-142-3p and HIF1AN 3'-untranslated region was validated by a dual-luciferase reporter gene assay.PDG treatment significantly reduced the level of ROS, LDH, and CK-MB, promoted viability, and inhibited the apoptosis of H9c2 cells. PDG treatment promoted miR-142-3p expression and inhibited HIF1AN expression in H9c2 cells. MiR-142-3p overexpression enhanced the effects of PDG on ROS, LDH, CK-MB levels, cell viability, and apoptosis in H9c2 cardiomyocytes, while overexpression of HIF1AN reversed the above effects.PDG ameliorates H/R-induced injury of cardiomyocytes by regulating miR-142-3p and HIF1AN.

Protective effect of salidroside on high fat-induced apoptosis in H9c2 cardiomyocytes through AMPK/mTOR/p70S6K pathway

The study explored the effect of salidroside(SAL) on high fat-induced apoptosis in H9 c2 cardiomyocytes based on AMPK/mTOR/p70 S6 K pathway.H9 c2 cardiomyocytes were cultured in vitro and the lipotoxicity model of H9 c2 cardiomyocytes was constructed by 0.2 mmol·L~(-1) palmitic acid(PA) treatment for 24 hours.The cells were divided into control group, PA group, and SAL group(20 μmol·L~(-1)).Cell proliferation was detected with cell proliferation kit I(MTT) assay after SAL and PA treatment.Dihydroethidium(DHE) probe, Annexin V-FITC/PI kit, and JC-1 probe were used to estimate reactive oxygen species(ROS) level, cell apoptosis, and mitochondrial membrane potential(MMP) change, respectively.The expression levels of p-AMPK/AMPK, p-mTOR/mTOR, p-p70 S6 K/p70 S6 K and apoptosis-related proteins Bax, Bcl-2, and cleaved caspase-3 were investigated with Western blot.The mRNA levels of AMPK, mTOR and p70 S6 K were determined by quantitative reverse transcription-polymerase chain reaction(qRT-PCR).RESULTS:: showed that compared with control group, PA group had decreased cell proliferation ability, MMP, Bcl-2 protein expression and AMPK protein and mRNA expression, while increased ROS level, Bax and cleaved caspase-3 protein expression, and mTOR and p70 S6 K mRNA and protein expression, and the difference was statistically significant(P&lt;0.05, P&lt;0.01).Compared with PA group, SAL improved cell proliferation ability, MMP level, Bcl-2 protein expression, and AMPK mRNA and protein expression, while down-regulated ROS level, cell apoptosis, Bax and cleaved caspase-3 protein expression, and mTOR and p70 S6 K mRNA and protein expression, and the difference was statistically significant(P&lt;0.05, P&lt;0.01).In conclusion, SAL exerted protective effects on high fat-induced lipotoxicity of H9 c2 cardiomyocytes, alleviated the oxidative stress injury and reduced cell apoptosis via regulating AMPK/mTOR/p70 S6 K signaling pathway.

Low-intensity pulsed ultrasound promotes cell viability and inhibits apoptosis of H9C2 cardiomyocytes in 3D bioprinting scaffolds via PI3K-Akt and ERK1/2 pathways.

The aim of this study was to investigate whether low-intensity pulsed ultrasound (LIPUS) promotes myocardial cell viability in three-dimensional (3D) cell-laden gelatin methacryloyl (GelMA) scaffolds. Cardiomyoblasts (H9C2s) were mixed in 6% (w/v) GelMA bio-inks and printed using an extrusion-based 3D bioprinter. These scaffolds were exposed to LIPUS with different parameters or sham-irradiated to optimize the LIPUS treatment. The viability of H9C2s was measured using Cell Counting Kit-8 (CCK8), cell cycle, and live and dead cell double-staining assays. Western blot analysis was performed to determine the protein expression levels. We successfully fabricated 3D bio-printed cell-laden GelMA scaffolds. CCK8 and live and dead cell double-staining assays indicated that the optimal conditions for LIPUS were a frequency of 0.5MHz and an exposure time of 10min. Cell cycle analysis showed that LIPUS promoted the entry of cells into the S and G2/M phases from the G0/G1 phase. Western blot analysis revealed that LIPUS promoted the phosphorylation and activation of ERK1/2 and PI3K-Akt. The ERK1/2 inhibitor (U0126) and PI3K inhibitor (LY294002) significantly reduced LIPUS-induced phosphorylation of ERK1/2 and PI3K-Akt, respectively, which in turn reduced the LIPUS-induced viability of H9C2s in 3D bio-printed cell-laden GelMA scaffolds. A frequency of 0.5MHz and exposure time of 10min for LIPUS exposure can be adapted to achieve optimized culture effects on myocardial cells in 3D bio-printed cell-laden GelMA scaffolds via the ERK1/2 and PI3K-Akt signaling pathways.

The active ingredient (DSH-20) of Salvia miltiorrhiza flower reduces oxidative damage and apoptosis in cardiomyocytes by regulating miR-1.

DSH-20, the active ingredient of Salvia miltiorrhiza flower extract, is used to treat cardiovascular diseases. However, its mechanism of action remains unclear. Herein, we investigated the intervention of DSH-20 in H2O2-induced oxidative damage and apoptosis in cardiomyocytes. METHODS ANDRESULTS: H2O2 was used to induce oxidative damage and apoptosis in H9c2 cardiomyocytes. Based on concentration gradient studies, we found that 62.5µg/mL DSH-20 significantly reduced reactive oxygen species and lactate dehydrogenase levels and increased superoxide dismutase levels. DSH-20 also alleviated the apoptosis rate, the changes in mRNA of apoptosis-related genes (Bcl-2, BAX, and Caspase-3) and miR-1 expression. Moreover, transfection of miR-1 mimics aggravated oxidative damage and apoptosis, whereas DSH-20 alleviated these effects. DSH-20 reduced H2O2-induced oxidative damage and apoptosis in H9c2 cardiomyocytes likely by downregulating miR-1 expression.

A-kinase anchoring protein 5 anchors protein kinase A to mediate PLN/SERCA to reduce cardiomyocyte apoptosis induced by hypoxia and reoxygenation

The A-kinase anchoring protein 5 (AKAP5) has a variety of biological activities. This study explored whether AKAP5 was involved in cardiomyocyte apoptosis induced by hypoxia and reoxygenation (H/R) and its possible mechanism. H9C2 cells were used to construct an H/R model in vitro, followed by AKAP5 overexpression. Flow cytometry was performed to determine the rate of cardiomyocyte apoptosis. Phosphorylation of phospholamban (PLN), sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA2a), and apoptosis-related proteins was determined by western blotting. Immunofluorescence staining and immunoprecipitation were performed to detect the distribution and interaction between AKAP5, protein kinase A (PKA), and PLN. After H/R induction, H9C2 cells exhibited significantly reduced AKAP5 protein expression. Upregulation of AKAP5 promotes cell survival and significantly reduces lactate dehydrogenase (LDH) levels and apoptosis rates in H9C2 cells. In addition, the overexpression of AKAP5 was accompanied by the activation of the PLN/SERCA2a signaling pathway and a reduction in apoptosis. Immunofluorescence staining and immunoprecipitation revealed that AKAP5 co-localized and interacted with PLN and PKA. Interestingly, St-Ht31, an inhibitory peptide that disrupts AKAP interactions with regulatory subunits, inhibits the effect of AKAP5 overexpression on H/R-induced apoptosis in H9C2 cardiomyocytes. AKAP5 overexpression alleviated H/R-induced cardiomyocyte apoptosis possibly by anchoring PKA to mediate the PLN/SERCA pathway, suggesting that AKAP5 is a potential therapeutic target for the prevention and treatment of ischemia-reperfusion injury.

Quercetin Mitigates Cisplatin-Induced Oxidative Damage and Apoptosis in Cardiomyocytes through Nrf2/HO-1 Signaling Pathway

Cisplatin is massively used to treat solid tumors. However, several severe adverse effects, such as cardiotoxicity, are obstacles to its clinical application. Cardiotoxicity may lead to congestive heart failure and even sudden cardiac death in patients receiving cisplatin. Therefore, finding a novel therapeutic strategy for the prevention of cisplatin-induced cardiotoxicity is urgent. Quercetin is a flavonol compound that can be found in dietary fruits and vegetables. The antioxidant function and anti-inflammatory capacity of quercetin have been reported. However, whether quercetin could protect against cisplatin-caused apoptosis and cellular damage in cardiomyocytes is still unclear. H9c2 cardiomyocytes were treated with cisplatin (40 μM) for 24 h to induce cellular damage with or without quercetin pretreatment. We found that quercetin activates Nrf2 and HO-1 expression, thereby mitigating cisplatin-caused cytotoxicity in H9c2 cells. Quercetin also increases SOD levels, maintains mitochondrial function, and reduces oxidative stress under cisplatin stimulation. Quercetin attenuates cisplatin-induced apoptosis and inflammation in H9c2 cardiomyocytes; however, these cytoprotective effects were diminished by silencing Nrf2 and HO-1. In conclusion, this study reports that quercetin has the potential to antagonize cisplatin-caused cardiotoxicity by reducing ROS-mediated mitochondrial damage and inflammation via the Nrf2/HO-1 and p38MAPK/NF-[Formula: see text]Bp65/IL-8 signaling pathway. This study provided the theoretical basis and experimental proof for the clinical application of quercetin as a new effective strategy to relieve chemotherapy-induced cardiotoxicity.