Apoptosis In H9c2 Cells Research Articles

Silencing of miR-223 expression inhibits the apoptosis of H2O2-induced cardiomyocytes by increasing the activity of the IGF-1R/PI3K/AKT pathway

To study the: (1) function of micro (mi)R-223 on H2O2-induced H9C2 cells; (2) relationship between miR-223 and insulin-like growth factor 1 receptor (IGF-1R); and (3) role of miR-223 on the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway. H9C2 cells were selected to establish the H2O2-injury model. Overexpression/low expression of miR-223 in H9C2 cells was constructed, respectively. Flow cytometry and western blotting were applied to measure the apoptosis, cell activity, and expression of related proteins. Dual-luciferase reporter gene assays (DLRGAs) were applied to test if miR-223 targeted IGF-1R. Overexpression/low expression of IGF-1R was constructed to test if miR-223 regulated IGF-1R expression negatively. Increases in miR-223 expression were observed in H2O2-induced H9C2 cells. miR-223 absence improved H2O2-induced H9C2-cell apoptosis accompanied by an increase in B-cell lymphoma (Bcl)-2 expression and decrease in expression of Bax and cleaved caspase-3 ( P < 0.05). miR-223 silencing increased expression of IGF-1R, p-PI3K, and p-AKT in H2O2-induced H9C2 cells ( P < 0.05). miR-223 overexpression aggravated H2O2-induced H9C2-cell apoptosis and reduced expression of the proteins of IGF-1R, p-PI3K, and p-AKT. DLRGAs showed IGF-1R to be a downstream gene of miR-223. IGF-1R silencing significantly inhibited expression of p-PI3K and p-AKT proteins ( P < 0.05). miR-223 negatively regulated IGF-1R expression for H9C2-cell apoptosis and the PI3K/AKT pathway. miR-223 absence can ameliorate H2O2-induced cardiomyocyte apoptosis by targeting IGF-1R to regulate PI3K/AKT activity.

Long non-coding RNA FTX alleviates hypoxia/reoxygenation-induced cardiomyocyte injury via miR-410-3p/Fmr1 axis.

Long non-coding RNAs (lncRNAs) are involved in the development of myocardial ischemia/reperfusion (I/R) injury. In this study, we aimed to investigate the roles and underlying mechanisms of five prime to Xist (FTX) in myocardial I/R injury using cardiomyocyte hypoxia/reoxygenation (H/R) model. Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to determine the expression of FTX, microRNA-410-3p (miR-410-3p) and fragile X mental retardation 1 (Fmr1) mRNA. Cell Counting Kit-8 (CCK-8) assay and flow cytometry analysis were employed to evaluate cell proliferation and apoptosis, respectively. Western blot assay was conducted to examine the protein levels of apoptosis-associated factors and Fmr1. Specific kits were used to detect the levels of oxidative stress-associated factors. Dual-luciferase reporter assay was performed to verify the association between miR-410-3p and FTX or Fmr1. FTX was reduced in myocardial I/R injury patients' serum and H/R-stimulated H9c2 cells. FTX overexpression relieved cell damage caused by H/R treatment through inducing cell proliferation and repressing cell apoptosis and oxidative stress in H9c2 cells. FTX was a sponge for miR-410-3p and the impact of FTX overexpression on H/R-induced cell injury was abolished by miR-410-3p elevation in H9c2 cells. Fmr1 was identified as a target of miR-410-3p and Fmr1 knockdown reversed the effect on H/R-induced cell damage mediated by miR-410-3p inhibition in H9c2 cells. Moreover, FTX positively regulated Fmr1 expression through sponging miR-410-3p in H9c2 cells. FTX regulated H/R-induced cardiomyocyte damage by upregulating Fmr1 via sponging miR-410-3p.

LncRNA XIST attenuates hypoxia-induced H9c2 cardiomyocyte injury by targeting the miR-122-5p/FOXP2 axis

ObjectiveTo investigate the effect of lncRNA XIST on apoptosis induced by hypoxia. MethodsWe analyzed the expression levels of lncRNA XIST and miR-122-5p using RT-qPCR in hypoxia-induced cardiomyocytes. The mechanism by which lncRNA XIST affects myocardial ischemia was investigated using the cell transfection, CCK-8, and dual-luciferase reporter assays, as well as by flowcytometry, western blotting, and RNA immunoprecipitation. ResultsHypoxic H9c2 cells demonstrated a decrease in their migration and invasion abilities and XIST expression and an increase in the extent of their apoptosis and expression of microRNA-122-5p. Overexpression of XIST significantly increased the H9c2 cell viability, enhanced cell migration and invasion, and decreased cell apoptosis in a hypoxic environment. The luciferase activity of XIST-WT in H9c2 cells co-transfected with XIST-WT and microRNA-122-5p mimics had decreased. The results of RNA immunoprecipitation showed that XIST interacted directly with miRNA-122-5p. Overexpression of XIST decreased the level of miRNA-122-5p significantly. mi-122-5p mimics increased H9c2 cell apoptosis and downregulated FOXP2 expression. Overexpression of FOXP2 upregulated the expression of the Bcl-2 protein in H9c2 cells transfected with microRNA-122-5p mimics and inhibited the expression of HIF-alpha, Bax, and the cleaved-caspase 9 protein. ConclusionlncRNA XIST could regulate the miR-122-5p/FOXP2 axis to attenuate hypoxia-induced H9c2 cardiomyocyte injury.

Swainsonine protects H9c2 cells against lipopolysaccharide-induced apoptosis and inflammatory injury via down-regulating miR-429

ABSTRACT Pediatric myocarditis (PM) is usually related to myocardial dysfunction. Generally, 30% of PM patients will die or undergo heart transplantation. Swainsonine (SW) is a natural alkaloid and an anti-cancer substance. Our goal was to determine the roles of SW in PM in current study. H9c2 cells were pre-treated by lipopolysaccharide (LPS). Viability and apoptosis were evaluated utilizing CCK-8 assay and flow cytometry. Inflammatory cytokines’ mRNA expression and production were assessed by western blot and ELISA. Western blot was utilized to distinguish apoptosis and immune-related factors expression. Sequentially, the abovementioned parameters were reassessed when miR-429 was overexpressed. LPS declined viability as well as raised apoptosis and inflammatory injury in H9c2 cells. SW alleviated apoptosis and inflammatory injury induced by LPS. MiR-429 expression was elevated by LPS and suppressed by SW. SW-induced the increasing of viability and the reduction of inflammatory injury were reversed by overexpression of miR-429. Eventually, SW inhibited p38MAPK/NF-κB pathway which activated by LPS via overexpressing miR-429. SW exerted its anti-apoptosis and anti-inflammatory function in LPS-treated H9c2 cells through p38MAPK/NF-κB pathway and down-regulation of miR-429.

Teneligliptin prevents doxorubicin-induced inflammation and apoptosis in H9c2 cells

Doxorubicin is a common chemotherapy treatment with numerous negative ramifications of use such as nephropathy and radiation-induced cardiotoxicity. Doxorubicin has been shown to cause overexpression of proinflammatory cytokines including MCP-1 and IL-1β via activation of the NF-κB pathway. Furthermore, apoptosis marked by dysregulation of the Bax/Bcl-2 ratio and oxidative stress and the production of reactive oxygen species (ROS) are also exacerbated by doxorubicin administration. Teneligliptin is part of the wider dipeptidyl peptidase-4 (DPP-4) inhibitor family which has until recently been almost exclusively used to treat type 2 diabetes mellitus. DPP-4 inhibitors such as teneligliptin control the overexpression of glucagon-like peptidase 1 (GLP-1) which has the downstream effects of general insulin resistance and high blood sugar levels. Our findings indicate a significant protective effect of teneligliptin against the aftereffects of doxorubicin as a chemotherapy treatment. This protective effect includes but is not limited to the reduction of inflammation and the mitigation of dysregulated apoptosis, as evidenced by reduced expression of IL-1β and MCP-1, inhibition of NF-κB activation, and improvement of the Bax/Bcl-2 ratio. The aim of the present study was to establish teneligliptin as a potentially useful agent for the treatment of radiation-induced cardiotoxicity, and our findings support this notion.

LncRNA nuclear-enriched abundant transcript 1 regulates hypoxia-evoked apoptosis and autophagy via mediation of microRNA-181b.

Nuclear-enriched abundant transcript 1 (NEAT1), a vital long noncoding RNA (lncRNA), exhibits the functions in disparate cancers. Nevertheless, the influences of NEAT1 in congenital heart disease (CHD) remain unreported. The research delves into whether NEAT1 affects H9c2 cells apoptosis and autophagy under the hypoxia condition. Overexpressed NEAT1 vector was transfected into H9c2 cells; then, functions of NEAT1 in cell viability, apoptosis, autophagy, PI3K/AKT/mTOR and JAK1/STAT3 pathways were detected in H9c2 cells under hypoxia condition. Expression of NEAT1 and miR-181b in hypoxia and blood samples from CHD was evaluated. After miR-181b inhibitor transfection, functions of miR-181b repression in the above-mentioned cell behavior and PI3K/AKT/mTOR and JAK1/STAT3 pathways were reassessed. Overexpressed NEAT1 clearly allayed hypoxia-triggered H9c2 cells apoptosis and autophagy. The decreased NEAT1 and miR-181b were showcased in hypoxia and blood samples from CHD; meanwhile, elevated miR-181b evoked by overexpressed NEAT1 was observed in hypoxia-managed H9c2 cells. More importantly, miR-181b inhibition obviously overturned the influences of NEAT1 in hypoxia-affected H9c2 cells apoptosis and autophagy. Besides, overexpressed NEAT1 facilitated PI3K/AKT/mTOR and JAK1/STAT3 activations via enhancing miR-181b. The research exposed that NEAT1 eased hypoxia-triggered H9c2 cells apoptosis and autophagy by expediting PI3K/AKT/mTOR and JAK1/STAT3 pathways via elevating miR-181b.

Prostaglandin E1 protects cardiomyocytes against hypoxia-reperfusion induced injury via the miR-21-5p/FASLG axis.

Background: Prostaglandin-E1 (PGE1) is a potent vasodilator with anti-inflammatory and antiplatelet effects. However, the mechanism by which PGE1 contributes to the amelioration of cardiac injury remains unclear.Methods: The present study was designed to investigate how PGE1 protects against hypoxia/reoxygenation (H/R)-induced injuries by regulating microRNA-21-5p (miR-21-5p) and fas ligand (FASLG). Rat H9C2 cells and isolated primary cardiomyocytes were cultured under hypoxic conditions for 6 h (6H, hypoxia for 6 h), and reoxygenated for periods of 6 (6R, reoxygenation for 6 h), 12, and 24 h, respectively. Cells from the 6H/6R group were treated with various doses of PGE1; after which, their levels of viability and apoptosis were detected.Results: The 6H/6R treatment regimen induced the maximum level of H9C2 cell apoptosis, which was accompanied by the highest levels of Bcl-2-associated X protein (Bax) and cleaved-caspase-3 expression and the lowest level of B-cell lymphoma 2 (Bcl-2) expression. Treatment with PGE1 significantly diminished the cell cytotoxicity and apoptosis induced by the 6H/6R regimen, and also decreased expression of IL-2, IL-6, P-p65, TNF-α, and cleaved-caspase-3. In addition, we proved that PGE1 up-regulated miR-21-5p expression in rat cardiomyocytes exposed to conditions that produce H/R injury. FASLG was a direct target of miR-21-5p, and PGE1 reduced the ability of H/R-injured rat cardiomyocytes to undergo apoptosis by affecting the miR-21-5p/FASLG axis. In addition, we proved that PGE1 could protect primary cardiomyocytes against H/R-induced injuries.Conclusions: These results indicate that PGE1 exerts cardioprotective effects in H9C2 cells during H/R by regulating the miR-21-5p/FASLG axis.

Long Noncoding RNA NEAT1 Promotes Myocardiocyte Apoptosis and Suppresses Proliferation Through Regulation of miR-129-5p.

Recent studies have revealed the important role of long noncoding RNAs (lncRNAs) in heart development and pathogenesis. This study was aimed to investigate the role of NEAT1 in hypoxia-induced cardiac injury and explore its possible molecular mechanism. Real-time PCR (RT-PCR) was used to determine the relative RNA expression of NEAT1 and its potential target microRNA, miR-129-5p, in the plasma of patients with acute myocardial infarction, heart failure, and angina, as well as in H2O2-treated H9c2 cells. The role of NEAT1 overexpression or inhibition in H9c2 cell migration and proliferation was assessed by transwell assay and Edu staining, respectively. Collagen deposition and apoptosis were evaluated by Western blot detection of collagen and apoptotic proteins, including Capase3, Bax, and Bcl2. We showed that H2O2 treatment significantly decreased H9c2 cell migration and proliferation while increasing H9c2 cell apoptosis. Inhibition of NEAT1 attenuated the cell apoptosis and alleviated proliferation inhibition induced by hypoxia. Bioinformatics analysis showed that miR-129-5p was the direct target of NEAT1, which was confirmed by luciferase assay. NEAT1 upregulation aggravated apoptosis by downregulating miR-129-5p. In conclusion, we uncovered a novel NEAT1-miR-129 axis and its implication in H2O2-induced heart failure.

Rapamycin regulates the balance betweencardiomyocyte apoptosis and autophagy in chronic heart failure by inhibiting mTOR signaling.

The progressive loss of cardiomyocytes caused by cell death leads to cardiac dysfunction and heart failure (HF). Rapamycin has been shown to be cardioprotective in pressure-overloaded and ischemic heart diseases by regulating the mechanistic target of rapamycin (mTOR) signaling network. However, the impact of rapamycin on cardiomyocyte death in chronic HF remains undetermined. Therefore, in the current study we addressed this issue using a rat myocardial infarction (MI)-induced chronic HF model induced by ligating the coronary artery. Following surgery, rats were randomly divided into six groups, including the sham-, vehicle- and rapamycin-operated groups, at 8 or 12 weeks post-MI. A period of 4 weeks after MI induction, the rats were treated with rapamycin (1.4 mg-kg-day) or vehicle for 4 weeks. Cardiac function was determined using echocardiography, the rats were subsequently euthanized and myocardial tissues were harvested for histological and biochemical analyses. In the cell culture experiments with H9c2 rat cardiomyocytes, apoptosis was induced using angiotensin II (100 nM; 24 h). Cardiomyocyte apoptosis and autophagy were assessed via measuring apoptosis- and autophagy-associated proteins. The activities of mTOR complex 1 (mTORC1) and mTORC2 were evaluated using the phosphorylation states of ribosomal S6 protein and Akt, respectively. The activity of the endoplasmic reticulum (ER) stress pathway was determined using the levels of GRP78, caspase-12, phospho-JNK and DDIT3. Echocardiographic and histological measurements indicated that rapamycin treatment improved cardiac function and inhibited cardiac remodeling at 8 weeks post-MI. Additionally, rapamycin prevented cardiomyocyte apoptosis and promoted autophagy at 8 weeks post-MI. Rapamycin treatment for 4 weeks inhibited the mTOR and ER stress pathways. Furthermore, rapamycin prevented angiotensin II-induced H9c2 cell apoptosis and promoted autophagy by inhibiting the mTORC1 and ER stress pathways. These results demonstrated that rapamycin reduced cardiomyocyte apoptosis and promoted cardiomyocyte autophagy, by regulating the crosstalk between the mTOR and ER stress pathways in chronic HF.

Sheng Mai San protects H9C2 cells against hyperglycemia-induced apoptosis

BackgroundSheng Mai San (SMS) has been proven to exhibit cardio-protective effects. This study aimed to explore the molecular mechanisms of SMS on hyperglycaemia (HG)-induced apoptosis in H9C2 cells.MethodsHG-induced H9C2 cells were established as the experimental model, and then treated with SMS at 25, 50, and 100 μg/mL. H9C2 cell viability and apoptosis were quantified using MTT and Annexin V-FITC assays, respectively. Furthermore, Bcl-2/Bax signalling pathway protein expression and Fas and FasL gene expression levels were quantified using western blotting and RT-PCR, respectively.ResultsSMS treatments at 25, 50, 100 μg/mL significantly improved H9C2 cell viability and inhibited H9C2 cell apoptosis (p < 0.05). Compared to the HG group, SMS treatment at 25, 50, and 100 μg/mL significantly downregulated p53 and Bax expression and upregulated Bcl-2 expression (p < 0.05). Moreover, SMS treatment at 100 μg/mL significantly downregulated Fas and FasL expression level (p < 0.05) when compared to the HG group.ConclusionSMS protects H9C2 cells from HG-induced apoptosis probably by downregulating p53 expression and upregulating the Bcl-2/Bax ratio. It may also be associated with the inhibition of the Fas/FasL signalling pathway.

MicroRNA-133b Alleviates Hypoxia Injury by Direct Targeting on NOD-Like Receptor Protein 3 in Rat H9c2 Cardiomyocyte.

Objective MiR-133b was dysregulated in myocardial infarction. However, the role and mechanism of miR-133b in myocardial infarction remains unclear. This study was aimed to explore the role of miR-133b in H9c2 cell injury induced by hypoxia and to investigate the underlying molecular mechanism. Methods Cell injury was assessed by cell viability, migration, invasion, and apoptosis assays. The expression of miR-133b and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) mRNA was determined by qRT-PCR. The levels of apoptosis-related proteins and NLRP3 were detected by western blotting. Results Results showed that hypoxia significantly reduced cell viability, migration, and invasion, but increased apoptosis of H9c2 cells. Downregulation of miR-133b aggravated the cell injury induced by hypoxia. MiR-133b was directly targeted on NLRP3. Overexpression of NLRP3 significantly inhibited cell viability, migration, and invasion but induced cell apoptosis in H9c2 treated with hypoxia. Conclusions Thus, miR-133b protects H9c2 against hypoxia injury via downregulation of NLRP3.

The inhibition of miR‑101a‑3p alleviates H/R injury in H9C2 cells by regulating the JAK2/STAT3 pathway.

Hypoxia/reoxygenation (H/R) is used as an in vivo model of ischemia/reperfusion injury, and myocardial ischemia can lead to heart disease. Therefore, it is necessary to prevent myocardial H/R injury to avoid the risk of heart disease. The aim of the present study was to investigate whether inhibiting microRNA (miR)-101a-3p attenuated H9C2 cell H/R injury, apoptosis mechanisms and key target proteins. Cell viability and apoptosis were determined by Cell Counting Kit-8 assays and flow cytometry using a cell apoptosis kit, respectively. The contents of creatine kinase (CK) and lactate dehydrogenase (LDH) were detected using colorimetric assays. Dual luciferase assays were carried out to determine if miR-101a-3p inhibited Janus kinase (JAK)2. Western blot analysis and reverse transcription-quantitative PCR were used to determine proteins levels and mRNAs expression. It was found that the inhibition of miR-101a-3p increased the growth of H9C2 cells and decreased H9C2 cell apoptosis during H/R injury. The inhibition of miR-101a-3p reduced the amounts of CK and LDH in H/R model H9C2 cells. The inhibition of miR-101a-3p lowered the levels of Bax, interleukin-6 and tumor necrosis factor-α, but raised the levels of phosphorylated (p)-STAT3 and p-JAK2 in H9C2 cells subjected to H/R injury treatment. miR-101a-3p mimic was found to inhibit H9C2 cell viability, raise p-JAK2 level and slightly increase p-STAT3 during H/R injury. AG490 induced H9C2 cell apoptosis, and decreased the levels of p-JAK2 and p-STAT3 during H/R injury. The data indicated that inhibiting miR-101a-3p reduced H/R damage in H9C2 cells and decreased apoptosis via Bax/Bcl-2 signaling during H/R injury. In addition, it was suggested that the inhibition of miR-101a-3p decreased H/R injury in H9C2 cell by regulating the JAK2/STAT3 signaling pathway.

Four new flavonol glycosides from the leaves of Ginkgo biloba

Four new flavonol glycosides, 5, 7, 5′-trihydroxy-3′, 4′-dimethoxyflavonol-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (1), quercetin 3-O-(6-trans-feruloyl)-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranoside (2), kaempferol 3-O-(6-trans-caffeoyl)-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranoside (3), myricetin 3-O-(6-trans-p-coumaroyl)-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranoside (4), together with nine known flavonoids and two known lignans, were isolated from the leaves of Ginkgo biloba. Their structures were determined by extensive spectroscopic analyses. Their cardioprotective effects against H2O2-induced apoptosis in H9c2 cells were also evaluated. The flavonol glycosides had stronger activity than the acylated flavonol glycosides at the concentration of 50 µM.

CCN1 triggers adaptive autophagy in cardiomyocytes to curb its apoptotic activities.

Autophagy occurs at basal levels for cellular homeostasis under normal conditions and is increased in response to nutrient starvation or stress to ensure cell survival. However, excessive autophagy can be deleterious to cardiomyocytes. CCN1/Cyr61, a matricellular protein, is expressed in the stressed heart to induce cardiomyopathy. The role of autophagy in CCN1-associated cardiotoxicity was not clear. Here, we found that autophagy was induced in the myocardium of the isoproterenol (ISO; 100mg/kg/day for 5days; s.c.) treated mice, where CCN1 expression is colocalized. The knock-in mice carrying an integrin α6β1-binding-defective mutant allele Ccn1-dm were resistant to the ISO-induced cardiac injury and autophagy. Our in vitro studies demonstrated that CCN1 dose- and time-dependently induced GFP-LC3-labeled autophagosome formation in rat cardiomyoblast H9c2 cells. The formation of autolysosomes in response to CCN1 (5μg/ml; 3h) treatment was identified by the acridine orange staining. The autophagy induction was confirmed by the elevated protein levels of Beclin 1, Atg5, and LC3-II, and the decrease of p62. Inhibition of autophagy by 3-methyladenine or by silencing Atg5 gene enabled CCN1-induced apoptosis in H9c2 cells, suggesting a protective role of autophagy. CCN1 binds to integrin α6β1 to induce autophagy through reactive oxygen species, and the activation of ERK and JNK. Furthermore, mitophagy was observed after CCN1 treatment for the clearance of depolarized mitochondria. Together, these results demonstrated that autophagy is induced in response to CCN1/α6β1 signaling in cardiomyocytes to alleviate CCN1-associated cardiotoxicity.

Deletion of Neuropeptide Y Attenuates Cardiac Dysfunction and Apoptosis During Acute Myocardial Infarction.

Increasing neuropeptide Y (NPY) has been shown to be a risk factor for cardiovascular diseases. However, its role and mechanism in myocardial infarction (MI) have not yet been fully understood. H9c2 cells and neonatal rat ventricular myocytes with loss of function of NPY and rats with global knockout were used in this study. MI model of rats was induced by the ligation of left coronary artery, and the extent of MI was analyzed through echocardiographic, pathological, and molecular analyses. Our data demonstrated that NPY expression was significantly increased in MI rats and hypoxia/hydrogen peroxide (H2O2)-treated cardiomyocytes. At the same time, NPY-knockout rats exhibited a remarkable decrease in infarct size, serum lactate dehydrogenase activity, cardiomyocyte apoptosis, and caspase-3 expression and activity and a strong improvement in heart contractile function compared with MI rats. Meanwhile, NPY small interfering RNA (siRNA) inhibited the cell apoptosis in H2O2-treated H9c2 cells and hypoxia-treated neonatal rat ventricular myocytes. NPY deletion increased miR-499 expression and decreased FoxO4 expression in MI in vivo and in vitro. Moreover, NPY type 1 receptor antagonist BIBO3304 can reverse miR-499 decrease and FoxO4 increase in H2O2-induced cardiomyocytes. NPY siRNA inhibited cell apoptosis in H2O2-treated H9c2 cells that were reversed by miR-499 inhibitor. Additionally, FoxO4 was validated as the direct target of miR-499. Moreover, BIBO3304 and FoxO4 siRNA significantly increased the cell activity, inhibited the cell apoptosis, and decreased caspase-3 expression and activity in H2O2-treated cardiomyocytes that NPY presented the opposite effect. Collectively, deletion of NPY reduced myocardial ischemia, improved cardiac function, and inhibited cardiomyocyte apoptosis by NPY type 1 receptor–miR-499–FoxO4 axis, which provides a new treatment for MI.

GFI-1 Protects Against Lipopolysaccharide-Induced Inflammatory Responses and Apoptosis by Inhibition of the NF-κB/TNF-α Pathway in H9c2 Cells.

Growth factor independence 1 (Gfi-1) has been widely studied for its anti-inflammatory and anti-apoptotic effects. However, whether Gfi-1 has similar effects on H9c2 cardiomyocytes has not yet been reported. In this study, we explored the effect of Gfi-1 on lipopolysaccharide (LPS)-induced inflammatory responses and apoptosis in H9c2 cells. We found that LPS induced the increased expression of TNF-α and IL-6 in the LPS group. After transfection of the Gfi-1 overexpression plasmid, the expression of TNF-α and IL-6 decreased significantly in the LPS + Gfi-1 group. Gfi-1 clearly blocked LPS-induced NF-κB, TNF-α, TNFR1, cleaved-caspase-3 and cleaved-caspase-8 expression and increased Gfi-1 and Bcl-xL expression in H9c2 cells. Similarly, compared with the LPS group, Gfi-1 significantly decreased the expression of cleaved-caspase3/8 and increased the expression of Bcl-xL in the LPS + Gfi-1 group, as verified by immunocytochemical analysis. Furthermore, Gfi-1 markedly inhibited LPS-induced H9c2 cardiomyocyte apoptosis in the LPS + Gfi-1 group, as determined by TEM, TUNEL and flow cytometry. Taken together, these results demonstrate that Gfi-1 may have protective effects against LPS-induced inflammatory responses and apoptosis in H9c2 cells. Gfi-1 may be a novel molecule for treating septic cardiomyopathy.

P4463The pineal hormone melatonin inhibits doxorubicin-induced mitochondrial dysfunction and apoptosis in cardiomyocytes

Abstract Background Heart failure (HF) is a major end-point of cardiovascular diseases (CVD). The pathogenesis of HF is mostly unresolved but involves metabolic alterations. Treatment of animals and cardiomyocytes with β-adrenergic receptor agonists induces HF. Mitochondrial dysfunction and HF are common complications of anticancer drugs such as doxorubicin (DOX). Melatonin synthesis dramatically decreases with age and in patients with CVD. Purpose The aim of this study was to investigate whether DOX-induced cardiac dysfunction can be attenuated by melatonin. Methods The Seahorse XF analyser was utilised (with the XFp Cell Energy Phenotype kit) to measure oxygen consumption rate [OCR; oxidative phosphorylation (OXPHOS)] and extracellular acidification rate (ECAR; glycolysis) in living rat cardiomyocyte-derived H9c2 cell line. Mono-layers of cells were treated with cardiotoxic drugs [isoproterenol (ISO, 100μM) or DOX (0.1μM)] for 24hr with and without melatonin co-treatment (MEL, 1μM). Cyan ADP flow cytometry was used to examine the anti-apoptotic properties of MEL (1μM) on DOX-treatment (0.5μM, 24hr). Data are given as mean±SEM (n=separate experiments) and analysis was performed using ANOVA and two-tail unpaired Student's T-test, as applicable. Results Isoproterenol-treatment increased peak OCR of H9c2 cells by ∼30% which was inhibited by MEL [CON, 384±17; ISO, 496±33; ISO+MEL, 412±31pmol/min; n=3 (six replicates); CON vs. ISO, p&lt;0.05; ISO vs. ISO+MEL, p&lt;0.05; CON vs. ISO+MEL, p&gt;0.05]. Doxorubicin-treatment decreased OCR by ∼40% which was reversed by MEL [CON, 934±69; DOX, 554±52; DOX+MEL, 858±97pmol/min; n=3 (six replicates); CON vs. DOX, p&lt;0.05; DOX vs. DOX+MEL, p&lt;0.05; CON vs. DOX+MEL, p&gt;0.05]. ISO and DOX significantly increased (∼30%) and decreased (∼25%) ECAR respectively (n=3, p&lt;0.05) which was not inhibited by MEL. Melatonin alone had no significant effect on OCR and ECAR. Melatonin inhibited DOX-induced apoptosis in H9c2 cells [CON, 6.3±0.8%; DOX, 22±1.8%; DOX+MEL, 11±1.7%, n=4 (two replicates); CON vs. DOX, p&lt;0.001; DOX vs. DOX+MEL, p&lt;0.004; CON vs. DOX+MEL, p&gt;0.05]. Conclusions ISO and DOX-treatment induced mitochondrial dysfunction in H9c2 cells by alteration of OXPHOS and glycolysis; changes in OXPHOS were prevented by MEL. These data indicate that DOX-induced apoptosis in cardiac cells may be mediated, at least in part, by OXPHOS dysfunction which was attenuated by MEL treatment.

&lt;p&gt;Micelles Loaded With Puerarin And Modified With Triphenylphosphonium Cation Possess Mitochondrial Targeting And Demonstrate Enhanced Protective Effect Against Isoprenaline-Induced H9c2 Cells Apoptosis&lt;/p&gt;

BackgroundThe protective role of puerarin (PUE) against myocardial infarction is closely related to its regulation on mitochondria. However, free PUE can hardly reach the mitochondria of ischemic cardiomyocytes due to the lack of mitochondrial targeting of PUE. Here PUE was loaded into mitochondria-targeted micelles (PUE@TPP/PEG-PE) for precisely delivering PUE into mitochondria with the aim of enhancing the anti-apoptosis effect.MethodsThe mitochondriotropic polymer TPP-PEG-PE was synthesized for the preparation of PUE@TPP/PEG-PE micelles modified with triphenylphosphonium (TPP) cation. The physicochemical properties and anti-apoptosis effect of PUE@TPP/PEG-PE micelles were investigated. The coumarin 6 (C6)-labeled TPP/PEG-PE (C6@TPP/PEG-PE) micelles were used to observe the enhanced cellular uptake, mitochondrial targeting and lysosomes escape. Moreover, in vivo and ex vivo biodistribution of lipophilic near-infrared dye 1,1ʹ-dioctadecyl-3,3,3′,3ʹ-tetramethylindotricarbocyanine iodide (DiR)-labeled PUE@TPP/PEG-PE (DiR@TPP/PEG-PE) micelles were detected through fluorescence imaging.ResultsThe successful synthesis of TPP-PEG-PE conjugate was confirmed. PUE@TPP/PEG-PE micelles had a particle size of 17.1 nm, a zeta potential of −6.2 mV, and a sustained-release behavior. The in vitro results showed that the intracellular uptake of C6@TPP/PEG-PE micelles was significantly enhanced in H9c2 cells. C6@TPP/PEG-PE micelles could deliver C6 to mitochondria and reduce the capture of lysosomes. In addition, compared with the PUE@PEG-PE micelles and free PUE, the PUE@TPP/PEG-PE micelles exerted an enhanced protective effect against isoprenaline-induced H9c2 cell apoptosis, as evident by the decreased percentage of apoptotic cells, Caspase-3 activity, ROS level, Bax expression, and increased Bcl-2 expression. The in vivo detecting results of the targeting effect using DiR probe also indicated that TPP/PEG-PE micelles could accumulate and retain in the ischemic myocardium.ConclusionThe results of this study demonstrate the promising potential of applying PUE@TPP/PEG-PE micelles in mitochondria-targeted drug delivery to achieve maximum therapeutic effects of PUE.

Exosomes derived from cardiac progenitor cells attenuate CVB3-induced apoptosis via abrogating the proliferation of CVB3 and modulating the mTOR signaling pathways

Viral myocarditis is potentially fatal and lacking a specific treatment. Exosomes secreted by cardiac progenitor cells (CPCs) have emerged as a promising tool for cardioprotection and repair. In this study, we investigated whether CPCs-derived exosomes (CPCs-Ex) could utilize the mTOR signal pathway to reduce the apoptosis in viral myocarditis. In vitro, exosomes were, respectively, added to H9C2 cells after CVB3 infection to detect the anti-apoptosis effect of CPCs-Ex. Compared with the controls, the apoptosis rate was reduced, accompanied with the depressed expression of viral capsid protein 1 (VP1) and pro-apoptosis factors of Bim/caspase families. Meanwhile, the phosphorylation of Akt, mTOR, and p70S6K were promoted, but that of 4EBP1 was suppressed. In vivo, the results of apoptosis, expression of CVB3 and pro-apoptosis factors, and phosphorylation of Akt/mTOR factors of CVB3-infected cardiomyocytes were consistent with that of vitro. Following that, we use Rapamycin and MK-2206 to inhibit the Akt/mTOR signaling pathway, meanwhile, Rattus 4EBP1, p70S6K, Akt1 and Akt2 were transfected to H9C2 cells to establish the stably transfected cell lines. In the group with Rapamycin or MK-2206 pretreatment, CPCs-Ex also could decrease the apoptosis of H9C2 cells and expression of CVB3 mRNA, followed by decreased expression of apoptosis factors. In Akt2, p70S6K and 4EBP1 overexpression groups, CPCs-Ex promoted CVB3-induced apoptosis, VP1 expression and cleavage of caspase-3. Our results therefore identify CPCs-Ex exerts an anti-apoptosis effect in CVB3-infected cells by abrogating the proliferation of CVB3 and modulating the mTOR signaling pathways as well as the expression of Bcl-2 and caspase families. Viral myocarditis, mainly caused by CVB3 infection, is lacking a specific treatment. Our study identified an anti-apoptosis role of CPCs-Ex in CVB3-infected cells and rats, which shown that CPCs-Ex may be an effective tool to treat viral myocarditis. We believe that with more in-depth research on the functionality of CPCs-Ex, there will be a breakthrough in the treatment of viral myocarditis.

Baicalin relieves hypoxia-aroused H9c2 cell apoptosis by activating Nrf2/HO-1-mediated HIF1α/BNIP3 pathway

Background Myocardial ischemia is the main reason for ischemic heart disease. Baicalin is a plant-derived flavonoid with cardio-protective activity. Herein, we tested the influences of baicalin on cardiomyocytes H9c2 apoptosis aroused by hypoxia stimulation. Methods Firstly, H9c2 cells were subjected to hypoxia and/or baicalin exposure. Cell viability and apoptosis, along with hypoxia-inducible factor 1α (HIF1α) and Bcl-2/adenovirus E1B 19-KDa interacting protein 3 (BNIP3) expressions were tested respectively. Then, si-HIF1α was transfected into H9c2 cells to probe whether up-regulation of HIF1α attended to the influences of baicalin on hypoxia-stimulated H9c2 cells. Finally, the regulatory effect of nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) pathway on HIF1α expression was analyzed. Results Hypoxia exposure aroused H9c2 cell viability reduction and apoptosis. Baicalin mitigated H9c2 cell viability reduction and apoptosis aroused by hypoxia. Moreover, HIF1α/BNIP3 pathway was further activated by baicalin in hypoxia-exposed H9c2 cells. Silencing HIF1α lowered the functions of baicalin on hypoxia-exposed H9c2 cells. Besides, baicalin enhanced hypoxia-caused activation of Nrf2/HO-1 pathway. Activation of Nrf2/HO-1 pathway was associated with the up-regulation of HIF1α and protective functions of baicalin on hypoxia-exposed H9c2 cells. Conclusion Baicalin relieved cardiomyocytes H9c2 apoptosis aroused by hypoxia might be achieved through activating Nrf2/HO-1-mediated HIF1α/BNIP3 pathway. Highlights Baicalin mitigates H9c2 cell viability loss and apoptosis aroused by hypoxia; Baicalin activates HIF1a/BNIP3 pathway in hypoxia-exposed H9c2 cells; Silencing HIF1α weakens the influences of baicalin on hypoxia-exposed H9c2 cells; Baicalin promotes Nrf2/HO-1 pathway in hypoxia-exposed H9c2 cells; Promotion of Nrf2/HO-1 pathway is related to the up-regulation of HIF1α.