Cultured H9c2 Cardiomyocytes Research Articles

Ablation of CXCR4 expression in cardiomyocytes exacerbates isoproterenol‑induced cell death and heart failure.

CXCR4 is a seven‑transmembrane‑spanning Gi‑coupled receptor for the SDF‑1 chemokine and plays a critical role in cardiovascular development and post‑injury repair. However, the specific role of CXCR4 in cardiomyocytes is incompletely understood. It was hypothesized that CXCR4 activation in cardiomyocytes antagonizes β‑adrenoceptor/Gs signaling‑induced cardiac dysfunction. Cardiomyocyte‑specific CXCR4 knockout (CXCR4‑CMKO) mice were generated by crossing CXCR4fl/fl and MHC‑Cre+/‑ mice. Their cardiac structure and function in the basal state are equivalent to that of the control MHC‑Cre+/‑ littermates until at least 4months old. However, following continuous subcutaneous administration of isoproterenol (Iso) via an osmotic mini‑pump, the ventricular myocardial contractility, dilation, cardiomyocyte apoptosis, and interstitial fibrosis are worse in CXCR4‑CMKO mice than in MHC‑Cre+/‑ littermates. In the cultured H9C2 cardiomyocytes, SDF‑1 treatment markedly attenuated Iso‑induced apoptosis and reduction in phospho‑Akt, and this protective effect was lost by knockdown of CXCR4 or by co‑treatment with Gi inhibitors. In conclusion, CXCR4 promotes cardiomyocyte survival and heart function during β‑adrenergic stress.

Salidroside attenuates myocardial ischemia/reperfusion injury via AMPK-induced suppression of endoplasmic reticulum stress and mitochondrial fission

Ischemic heart disease (IHD) is the primary cause of death worldwide. Salidroside (Sal), the major active compound derived from Rhodiola rosea, is believed to have cardioprotective effects. AMP-activated protein kinase (AMPK), is a pivotal AMP-activated protein kinase in energy metabolism. Whether Sal plays an anti-endoplasmic reticulum stress/mitochondrial fission role through AMPK remains elusive. In this study, we established a myocardial ischemia/reperfusion (I/R) rat model. Rat hearts exposed to Sal with or without compound C were then subjected to I/R. Further, H9c2 cardiomyocytes were subjected to simulated ischemia/reperfusion (SIR) by hypoxia-reoxygenation. The rats and cardiomyocytes were pretreated with Sal, followed by Compound C and AMPK-siRNA to block AMPK activity. We found that Sal significantly ameliorated cardiac function, mitigated infarct size and serum content of lactate dehydrogenase and creatine kinase, improved mitochondrial function, and reduced mitochondrial fission and apoptosis. Furthermore, in cultured H9c2 cardiomyocytes, Sal increased the cell viability and inhibited SIR-induced myocardial apoptosis and mitochondrial fission. Furthermore, the translocation of Drp1 from the cytoplasm to mitochondria induced by salidroside was confirmed both in vivo and in vitro. However, the use of Compound C or AMPK siRNA to block AMPK activity leads to blockade of the protective effects of Sal. In summary, protects against myocardial I/R by activating the AMPK signaling pathway, inhibiting ER stress, and reducing mitochondrial fission and apoptosis.

Mass Spectrometry-Based Quantitative Proteomics Analysis for Better Understanding of Telomeric Zinc Finger-Associated Protein-Induced Pathogenesis in Cardiomyocytes.

Telomere length is highly related to cardiovascular diseases. Telomeric zinc finger-associated protein (TZAP) directly binds to telomeric TTAGGG repeats via zinc finger domains and triggers the initiation of the telomere trimming process. However, proteomics analysis of TZAP in cardiomyocytes is slightly unknown. In our study, TZAP was overexpressed by adenovirus transfection in cultured H9c2 cardiomyocytes, and then mass spectrometry-based quantitative proteomics research strategies, including Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, subcellular localizations, predicted functional domains, and protein-protein interaction (PPI) analysis, were performed to explore TZAP-induced potential pathogenesis in cardiomyocytes. A total of 184 upregulated and 228 downregulated differentially expressed proteins (DEPs) were identified among identified 5693 quantifiable proteins in TZAP-overexpressed cardiomyocytes. These DEPs were mainly distributed in the nucleus, cytoplasm, and plasma membrane. DEPs were enriched in biological processes including cardiac muscle cell contraction, acute inflammatory response, cell-cell junction assembly, and macromolecule biosynthetic process. They were enriched in 9 KEGG pathways, including Hippo signaling pathway, protein digestion and absorption, and cytokine receptor interaction, and enriched in 17 protein domains, including translation initiation factor 1A/IF-1, class I histocompatibility antigen, and zinc finger. PPI analysis indicated that TZAP interacted with NDUFC2, Gja1, and HDAC2. Further, as proteins closely related to cardiovascular function, the mRNA levels of BRD4, Gja1, HDAC2, MAP2K3, Plakophilin 4, and Syndecan 1 significantly decreased, while Trpm7, clusterin, and NDUFC2 remarkably increased in TZAP-overexpressed cardiomyocytes by RT-PCR assay, which were consistent with the proteomics analysis. Collectively, we provided candidate proteins and enrichment pathways in TZAP-overexpressed cardiomyocytes, which need further investigation.

Resveratrol Inhibits High Glucose-Induced H9c2 Cardiomyocyte Hypertrophy and Damage via RAGE-Dependent Inhibition of the NF-κB and TGF-β1/Smad3 Pathways.

Hyperglycaemia is associated with the development of cardiac vascular disease. Resveratrol (RES) is a naturally occurring polyphenolic compound that possesses many biological properties, including anti-inflammatory properties and antioxidation functions. Our study aimed to explore the RES's protective roles on high glucose (HG)-induced H9c2 cells and the underlying mechanisms. Small-molecule inhibitors, western blotting (WB), as well as reverse-transcription PCR (RT-PCR) were employed to investigate the mechanisms underlying HG-induced damage in H9c2 cells. RES (40 μg/mL) treatment significantly alleviated HG-induced cardiac hypertrophy and cardiac dysfunction. RES abated the HG‐induced increase in the levels of extracellular matrix (ECM) components and inflammatory cytokines, reducing ECM accumulation and inflammatory responses. Additionally, RES administration prevented HG‐induced mitochondrion‐mediated cardiac apoptosis of myocardial cells. In terms of mechanisms, we demonstrated that RES ameliorated the HG‐induced overexpression of receptor for advanced glycation endproducts (RAGE) and downregulation of NF-κB signalling. Moreover, RES inhibited HG‐induced cardiac fibrosis by inhibiting transforming growth factor beta 1 (TGF‐β1)/Smad3‐mediated ECM synthesis in cultured H9c2 cardiomyocytes. Further studies revealed that the effects of RES against HG‐induced upregulation of NF-κB and TGF‐β1/Smad3 pathways were similar to those of FPS-ZM1, a RAGE inhibitor. Collectively, the results implied that RES might help alleviate HG‐induced cardiotoxicity via RAGE‐dependent downregulation of the NF-κB and TGF‐β/Smad3 pathways. This study provided evidence that RES can be developed as a promising cardioprotective drug.

Pinocembrin mediates antiarrhythmic effects in rats with isoproterenol-induced cardiac remodeling

BackgroundHigh levels of circulating catecholamines are related to raise risk of cardiac arrhythmias. In addition, our recent studies have suggested that pinocembrin could decrease the susceptibility to arrhythmias in several rat models, including chronic ischemic heart failure, myocardial infarction and depression. In this research, the effects of pinocembrin on ventricular fibrillation (VF) susceptibility were investigated in rats treated with isoproterenol (ISO) and further explored the possible mechanism. MethodsCardiac remodeling was induced by intraperitoneally injection ISO (5 mg/kg) 7 days. Simultaneously, Rats were received pinocembrin (5 mg/kg) or saline by tail vein injection. The effects of pinocembrin were evaluated by electrocardiogram parameters, ventricular electrophysiological parameters, echocardiographic, western blot, ventricular histology, biochemical examinations. In vitro, we cultured H9C2 cardiomyocytes to further define the mechanisms. ResultsCompared with ISO group, pinocembrin remarkably decreased VF inducibility rate, attenuated the shortening of QT and corrected QT (QTc) interval, action potential duration (APD), ventricular effective refractory period (ERP), and increased the protein levels of Kv4.2 and Kv4.3 and Cav1.2 and decreased phosphorylated Ca2+ calmodulin-dependent kinase Ⅱ (p-CaMK Ⅱ). Pinocembrin also alleviated ventricular fibrosis, hypertrophy and increased expression of connexin protein 43 (Cx43). In addition, pinocembrin markedly downregulated levels of malondialdehyde (MDA), hydrogen peroxide (H2O2), oxidized glutathione (GSSG) and increased the activity of superoxide dismutase (SOD) and glutathione (GSH) levels in circulation and cardiac tissue. Pinocembrin reduced the reactive oxygen species (ROS) levels. Furthermore, after treatment of pinocembrin the content of NADPH Oxidase-4 (NOX4) and NADPH Oxidase-2 (NOX2) was significantly lower and the level of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was significantly higher. In vitro, we found that Nrf2 inhibitor remarkably reduced the antioxidant effects of pinocembrin, which further demonstrated that the effect of pinocembrin was related to activation of Nrf2. ConclusionOur data demonstrate that pinocembrin decreases ventricular electrical remodeling, ion remodeling, ventricular fibrosis, hypertrophy and suppresses isoproterenol-induced oxidative stress. The findings shown that pinocembrin mediates antiarrhythmic effects in rats with isoproterenol-induced cardiac remodeling related to Nrf2/HO-1 pathway.

Pinocembrin ameliorates post-infarct heart failure through activation of Nrf2/HO-1 signaling pathway

BackgroundOxidative stress is an important factor involved in the progress of heart failure. The current study was performed to investigate whether pinocembrin was able to ameliorate post-infarct heart failure (PIHF) and the underlying mechanisms.MethodsRats were carried out left anterior descending artery ligation to induce myocardial infarction and subsequently raised for 6 weeks to produce chronic heart failure. Then pinocembrin was administrated every other day for 2 weeks. The effects were evaluated by echocardiography, western blot, Masson’s staining, biochemical examinations, immunohistochemistry, and fluorescence. In vitro we also cultured H9c2 cardiomyocytes and cardiac myofibroblasts to further testify the mechanisms.ResultsWe found that PIHF-induced deteriorations of cardiac functions were significantly ameliorated by administrating pinocembrin. In addition, the pinocembrin treatment also attenuated collagen deposition and augmented vascular endothelial growth factor receptor 2 in infarct border zone along with an attenuated apoptosis, which were related to an amelioration of oxidative stress evidenced by reduction of reactive oxygen species (ROS) in heart tissue and malondialdehyde (MDA) in serum, and increase of superoxide dismutase (SOD). This were accompanied by upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2)/ heme oxygenase-1 (HO-1) pathway. In vitro experiments we found that specific Nrf2 inhibitor significantly reversed the effects resulted from pinocembrin including antioxidant, anti-apoptosis, anti-fibrosis and neovascularization, which further indicated the amelioration of PIHF by pinocembrin was in a Nrf2/HO-1 pathway-dependent manner.ConclusionPinocembrin ameliorated cardiac functions and remodeling resulted from PIHF by ROS scavenging and Nrf2/HO-1 pathway activation which further attenuated collagen fibers deposition and apoptosis, and facilitated angiogenesis.

Effects of the Wenyang Zhenshuai Granule on the Expression of LncRNA-MiR143HG/miR-143 Regulating ERK5 in H9C2 Cardiomyocytes.

Chronic heart failure (CHF) is a complex clinical syndrome caused by a variety of heart problems, with a high incidence. The 5-year survival rate of patients with clinical symptoms is similar to that of malignant tumors. Wenyang Zhenshuai granules are a safe and effective granule of traditional Chinese medicine components, including aconite, dried ginger, licorice, and red ginseng. In contemporary clinical applications, it is widely used in acute and chronic heart insufficiency, coronary heart disease, and arrhythmia. This research cultured H9C2 cardiomyocytes and divided them into the normal control group, LncRNA-MiR143HG overexpression group, LncRNA-MiR143HG silence group, Adriamycin (ADR) group, ADR + medicated serum group, ADR + LncRNA-MiR143HG overexpression + medicated serogroup, and ADR + LncRNA-MiR143HG silence + medicated serogroup. The cells of each group were treated differently, and the survival rate of each group of cells and the expression levels of LncRNA-MiR143HG/miR-143 and ERK5 were detected at the end of the experiment, and the expression of LncRNA-MiR143HG/miR-143 in H9C2 cardiomyocytes was regulated by Wenyang Zhenshuai granules' impact. The results of this study showed that, in the doxorubicin-induced H9C2 cardiomyocyte injury model, the expression of miR-143 was upregulated, and the expression of LncRNA-MiR143HG and ERK5 was significantly downregulated. Wenyang Zhenshuai granules can downregulate the expression of miR-143 to promote ERK5 protein expression and phosphorylation. The process is regulated by LncRNA-MiR143HG/miR-143, which may be one of its important mechanisms for the treatment of chronic heart failure.

Liquiritigenin attenuates isoprenaline‑induced myocardial fibrosis in mice through the TGF‑β1/Smad2 and AKT/ERK signaling pathways.

Myocardial fibrosis is a pathological process characterized by excessive accumulation of extracellular matrix in myocardial interstitial spaces. Myocardial fibrosis is a fundamental process in ventricular remodeling and a primary contributor to the progression of heart failure. Liquiritigenin (LQ) is a flavanone compound with anti-oxidative, anti-carcinogenic, anti-inflammatory and estrogenic properties. The present study aimed to investigate the regulatory potential of LQ treatment in a mouse model of isoprenaline (ISO)-induced cardiac fibrosis and in cultured H9C2 cardiomyocytes stimulated with angiotensin II (Ang II). The treatment of ISO-induced mice with LQ significantly decreased the levels of cardiac injury-related proteins in the serum and ECM accumulation in mouse heart tissues. LQ treatment also effectively alleviated cardiac dysfunction in ISO-treated mice. Further analyses revealed that LQ inhibited ISO-induced collagen formation and activation of the transforming growth factor-β1 (TGF-β1)/Smad2 and protein kinase B (AKT)/extracellular signal-regulated kinase (ERK) signaling pathways. As a major pathological event in myocardial fibrosis, the apoptosis of cardiomyocytes has been considered a key mechanism contributing to impaired left ventricle performance. The pretreatment of rat cardiomyocytes with LQ significantly reduced the apoptosis of H9C2 cells, and inhibited Ang II-induced activation of the TGF-β1/Smad2 and AKT/ERK pathways. In conclusion, the present study revealed that LQ ameliorated ISO-induced myocardial fibrosis in mice and inhibited the apoptosis of cardiomyocytes in vitro by inhibiting the TGF-β1/Smad2 and AKT/ERK signaling pathways. These results suggested the anti-fibrotic and cardioprotective potential of LQ in fibrosis, thus supporting the use of LQ for the management of cardiomyocyte injury and myocardial fibrosis in patients with cardiac diseases.

Effects of protein disulfide isomerase on hyperglycemia and hypoxia/reoxygenation injury in H9c2 cardiomyocytes

Objective: To explore the effect of protein disulfide isomerase (PDI) in diabetic ischemic heart disease. Methods: We established an in vitro model of high glucose and hypoxia/reoxygenation in H9c2 rat myocardial cells. Cultured cells were divided into four groups: Control, high glucose (HG), hypoxia/reoxygenation (H/R) and HG+H/R. Changes in PDI expression mediated by PDI adenovirus(Ad-PDI) infection and siRNA(PDI-siRNA) transfection in myocardial cells were observed by inverted fluorescence microscopy. We also measured lactate dehydrogenase(LDH) activity and malondialdehyde(MDA) and high molecular weight(HMW)-APN concentrations. PDI, APN, cleaved caspase-3, and glucose regulated protein 78 (Grp78) protein expression were detected. Results: PDI expression was significantly decreased in the HG, H/R and HG+H/R groups compared to the Control group; however, LDH activity[(179.7±10.4) U/L、(218.4±18.4) U/L、(328.2±5.3) U/L vs (91.0±11.0) U/L], MDA concentration[(7.0±0.4) μmol/L、(10.0±1.0) μmol/L、(11.7±1.0) μmol/L vs (4.2±1.8) μmol/L], cleaved caspase-3, and Grp78 expression were increased. Interestingly, APN and HMW-APN expression were decreased [(2.01±0.21) μg/L、(1.64±0.27) μg/L、(1.20±0.14) μg/L vs (2.62±0.12) μg/L, all P<0.05]. Over expression of PDI attenuated high glucose and hypoxia/reoxygenation induced apoptosis and oxidative stress in H9c2 cardiomyocytes(all P<0.05), and simultaneously increased APN and HMW-APN expression [(2.86±0.03) μg/L vs (3.03±0.10) μg/L、(2.06±0.05) μg/L vs (2.31±0.06) μg/L、(1.83±0.07) μg/L vs (1.96±0.11) μg/L、(1.20±0.06) μg/L vs (1.39±0.09) μg/L]. PDI-siRNA transfection increased LDH activity, MDA concentration, and cleaved caspase-3 and Grp78 expression, and decreased APN and HMW-APN expression [(0.75±0.09) μg/L vs (0.59±0.09) μg/L、(0.62±0.04) μg/L vs (0.53±0.05) μg/L、(0.55±0.14) μg/L vs (0.51±0.12) μg/L、(0.48±0.12) μg/L vs (0.35±0.08) μg/L] in response to different treatments in cultured H9c2 cardiomyocytes (all P<0.05). Conclusion: PDI may regulate the expression of APN and HMW-APN, and play an important role in the function of diabetic ischemia-reperfusion cardiomyocytes.

Redd1 knockdown prevents doxorubicin-induced cardiac senescence.

Regulated in development and DNA damage response-1 (Redd1) is a stress-response gene that is transcriptionally induced by diverse stressful stimuli to influence cellular growth and survival. Although evidence suggests that aging may drive Redd1 expression in skeletal muscles, the expression patterns and functions of Redd1 in senescent cardiomyocytes remain unspecified. To address this issue, in vitro and in vivo models of cardiomyocyte senescence were established by administration of doxorubicin (Dox). Redd1 overexpression and knockdown was achieved in cultured H9c2 cardiomyocytes and mouse tissues using, respectively, lentivirals and adeno-associated virus 9 (AAV9) vectors. In the hearts of both aged (24 months old) and Dox-treated mice, as well as in Dox-exposed H9c2 cardiomyocytes, high Redd1 expression accompanied the increase in both cellular senescence markers (p16INK4a and p21) and pro-inflammatory cytokine expression indicative of a stress-associated secretory phenotype (SASP). Notably, Redd1 overexpression accentuated, whereas Redd1 silencing markedly attenuated, Dox-induced cardiomyocyte senescence features both in vitro and in vivo. Notably, AAV9-shRNA-mediated Redd1 silencing significantly alleviated Dox-induced cardiac dysfunction. Moreover, through pharmacological inhibition, immunofluorescence, and western blotting, signaling pathway analyses indicated that Redd1 promotes cardiomyocyte senescence as a downstream effector of p38 MAPK to promote NF-kB signaling via p65 phosphorylation and nuclear translocation.

Improving antibacterial, biocompatible, and reusable properties of polyvinyl chloride via the addition of aluminum alkoxides

AbstractGlobal bacterial infections associated with conventional polyvinyl chloride (PVC) medical devices place a heavy burden on healthcare systems and thus it will be desirable if medical devices are made from antimicrobial PVC. There are numerous studies focusing on polymer surface modifications to either leach antimicrobial agents or kill pathogenic microbes upon direct contact. In this work, mannitol fumarate ester‐based aluminum metal alkoxide (MFE‐Al) additive was developed to confer simultaneously improved antibacterial property and enhanced high temperature sterilization resistance of the resultant PVC. Data obtained confirm that the MFE‐Al stabilized PVC sheets significantly inhibit 98% bacterial growth. They also show biocompatibility with cultured H9C2 cardiomyocytes and hemocompatibility in vitro. Dry heat sterilization is generally not suitable for PVC medical wares due to their poor thermal compatibility. Surprisingly, our antimicrobial‐biocompatible PVC can maintain stability at 180°C for 90 min. Such a high thermal stability indicates the MFE‐Al stabilized PVC can endure 90 cycles of dry‐heat sterilization without significant damage. This study may provide a solution to reduce PVC medical waste for a maximum benefit without compromising human health or the environment.

Regulation of cardiomyocyte DNA damage and cell death by the type 2A protein phosphatase regulatory protein alpha4

The type 2A protein phosphatase regulatory protein alpha4 (α4) constitutes an anti-apoptotic protein in non-cardiac tissue, however it’s anti-apoptotic properties in the heart are poorly defined. To this end, we knocked down α4 protein expression (α4 KD) using siRNA in cultured H9c2 cardiomyocytes and confirmed the lack of DNA damage/cell death by TUNEL staining and MTT assay. However, α4 KD did increase the phosphorylation of p53 and ATM/ATR substrates, decreased the expression of poly ADP-ribose polymerase and associated fragments. Expression of anti-apoptotic proteins Bcl-2 and Bcl-xL was reduced, whereas expression of pro-apoptotic BAX protein did not change. Alpha4 KD reduced basal H2AX Ser139 phosphorylation, whereas adenoviral-mediated re-expression of α4 protein following α4 KD, restored basal H2AX phosphorylation at Ser139. The sensitivity of H9c2 cardiomyocytes to doxorubicin-induced DNA damage and cytotoxicity was augmented by α4 KD. Adenoviral-mediated overexpression of α4 protein in ARVM increased PP2AC expression and augmented H2AX Ser139 phosphorylation in response to doxorubicin. Furthermore, pressure overload-induced heart failure was associated with reduced α4 protein expression, increased ATM/ATR protein kinase activity, increased H2AX expression and Ser139 phosphorylation. Hence, this study describes the significance of altered α4 protein expression in the regulation of DNA damage, cardiomyocyte cell death and heart failure.

Apocynin prevents reduced myocardial nerve growth factor, contributing to amelioration of myocardial apoptosis and failure.

Reduced nerve growth factor (NGF) is associated with cardiac sympathetic nerve denervation in heart failure (HF) which is characterized by increased oxidative stress. Apocynin is considered an antioxidant agent which inhibits NADPH oxidase activity and improves reactive oxygen species scavenging. However, it is unclear whether apocynin prevents reduced myocardial NGF, leading to improvement of cardiac function in HF. In this study, we tested the hypothesis that apocynin prevents reduced myocardial NGF, contributing to amelioration of myocardial apoptosis and failure. Rabbits with myocardial infarction (MI) or sham operation were randomly assigned to receive apocynin or placebo for 4weeks. MI rabbits exhibited left ventricular (LV) dysfunction, and elevation in oxidative stress, as evidenced by a decreased reduced-to-oxidized glutathione ratio and an increased 4-hydroxynonenal expression, and reduction in NGF and NGF receptor tyrosine kinase A (TrKA) expression in the remote non-infarcted myocardium. Apocynin treatment ameliorated LV dysfunction, reduced oxidative stress, prevented decreases in NGF and TrKA expression and reduced cardiomyocyte apoptosis after MI. In cultured H9C2 cardiomyocytes, hypoxia or hydrogen peroxide decreased NGF expression, and apocynin normalized hypoxia-induced reduction of NGF. Recombinant NGF attenuated hypoxia-induced apoptosis. Apocynin prevented hypoxia-induced apoptosis, and the suppressive effect of apocynin on apoptosis was abolished by NGF receptor TrKA inhibitor K252a. We concluded that apocynin prevented reduced myocardial NGF, leading to attenuation of cardiomyocyte apoptosis and LV remodelling and dysfunction in HF after MI. These findings suggest that strategies to prevent NGF reduction by inhibition of oxidative stress may be of value in amelioration of LV dysfunction in HF.

Upregulation of PKR pathway mediates glucolipotoxicity induced diabetic cardiomyopathy in vivo in wistar rats and in vitro in cultured cardiomyocytes

AimsProtein Kinase R (PKR) plays a key role in inflammation and insulin resistance. Cytokines, high fat diet, infection and various stress signals can activate PKR. However, the functional significance of PKR in diabetic cardiomyopathy (DCM) is not explored so far. Thus the aim of the present study was to investigate the role of PKR in DCM in vivo in a rat model of DCM and underlying molecular mechanism. Methods and resultsDCM was induced in Wistar rats by recipe of high fat diet and single injection of streptozotocin. Vital parameters were measured by non-invasive BP apparatus. Morphology, fibrosis and protein expression in heart was done by haematoxylin & eosin staining, masson’s trichome/sirius red staining and western blotting respectively. For molecular mechanism studies, PKR gene silencing was done in cultured H9C2 cardiomyocytes and effect was observed in the presence of high glucose and high fat. Significant upregulation of PKR along with increase in cardiac biomarkers, decreased systolic and diastolic cardiac functions, oxidative stress, inflammatory markers, markers of fibrosis, enhanced cell death and AGEs’ was observed in DCM disease model. Moreover, selective inhibition of PKR alleviated cardiac dysfunction, fibrosis, oxidative stress, inflammation and cell death. Additionally knockdown of PKR attenuated glucolipotoxicty-induced markers of inflammation, oxidative stress and apoptosis in cultured H9C2 cardiomyocytes. ConclusionOur present study reports for the first time that inhibition of PKR may have great therapeutic potential in the treatment of DCM by attenuating inflammation, oxidative stress, apoptosis and fibrosis.

Effects of hypoxia/reoxygenation on apoptosis, autophagy and pyroptosis in H9C2 cardiomyocytes

Objective To investigate the effects of hypoxia/reoxygenation (HR) on apoptosis, autophagy and pyroptosis in H9C2 cardiomyocytes. Methods The cultured H9C2 cardiomyocytes were divided into two groups according to the random number table method: a normal control (Ctrl) group and an HR group. The H9C2 cardiomyocytes in the HR group were deprived of oxygen and glucose for 8 h in a hypoxic incubator, followed by reoxygenation for 12 h. Cell damage was assessed through detection of lactate dehy-drogenase (LDH) content in culture medium and determination of cell viability by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) method (n=6). Apoptosis related proteins [cysteinyl aspartate-specific proteinase (caspase)-3, B-cell lymphoma/leuke-mia 2 (Bcl-2) and Bcl-associate x protein (Bax)], autophagy related proteins [(light chain 3 (LC3) Ⅱ/Ⅰ , p62, Beclin-1, and phosphory-lated mammalian target of rapamycin (p-mTOR)]and pyroptosis related proteins [(nod-like receptor pyrin domain 3 (NLRP3), apoptosis associated speck-like protein (ASC), caspase-1p20, interleukin (IL)-1β, and IL-18]were detected by Western blot (n=9). Cell apopto-sis, autophagy and pyroptosis were further assessed by immunofluorescence staining (n=6) Results Compared with the Ctrl group, the HR group presented reduced viability of H9C2 cardiomyocytes (P<0.05), increased LDH release (P<0.05), and up-regulated expres-sion of activated caspase-3 and Bax (P<0.05), as well as decreased expression of Bcl-2 (P<0.05). TUNEL staining showed that apoptosis significantly was enhanced in the cells after HR (P<0.05). These results indicated that apoptosis and cell damage were enhanced after HR. In contrast, the expression of p-mTOR and p62 increased (P<0.05), but the expression of LC3 Ⅱ/Ⅰ and Beclin-1 protein decreased(P<0.05). The immunofluorescence staining of LC3 Ⅱ/Ⅰ showed that the number of autophagosome within the cells increased after HR(P<0.05), indicating that autophagy in H9C2 myocardial cells was significantly inhibited. Meanwhile, the expression of NLRP3 inflamma-some, ASC and caspase-1p20 protein was remarkably up-regulated (P<0.05), and the expression of activated inflammatory cellular factors IL-1β and IL-18 increased (P<0.05), suggesting that NLRP3 inflammasomes were activated and pyroptosis enhanced after HR. Conclusions Autophagy is inhibited in H9C2 cardiomyocytes during HR injury, but pyroptosis and apoptosis are enhanced. Key words: Autophagy; Apoptosis; Pyroptosis; Cardiomyocytes; Hypoxia-reoxygenation injury

Activation of CaMKII via ER-stress mediates coxsackievirus B3-induced cardiomyocyte apoptosis.

Cardiomyocyte apoptosis contributes to the development of coxsackievirus B3 (CVB3)-induced myocarditis, but the mechanism for the apoptosis by CVB3 infection remains unclear. Here, we showed that CVB3-induced endoplasmic reticulum (ER) stress response and apoptosis in cultured H9c2 cardiomyocytes. We found that Ca2+ -calmodulin-dependent kinase II (CaMKII) was activated by ER stress-dependent intracellular Ca2+ overload in the CVB3-infected H9c2 cardiomyocytes. Treatment with an inhibitor of ER stress, 4-phenylbutyric acid (4-PBA), attenuated intracellular Ca2+ accumulation indirectly and reduced CaMKII activity. Inhibition of CaMKII with pharmacological inhibitor (KN-93) or short hairpin RNA reduced CVB3-induced H9c2 apoptosis and repressed cytochrome c release from mitochondria to cytoplasm; whereas overexpression of the activated mutant of CaMKII (CaMKII-T287D) enhanced CVB3-induced H9c2 apoptosis and mitochondrial cytochrome c release, which could be alleviated by blocking of mitochondrial Ca2+ uniporter or mitochondrial permeability transition pore. Further in vivo investigation revealed that blocking of CaMKII with KN-93 prevented cardiomyocytes apoptosis and improved cardiac contractile function in CVB3-infected mouse heart. Collectively, these findings provide a novel evidence that CaMKII plays a vital role in the promotion of CVB3-induced cardiomyocyte apoptosis, which links ER stress and mitochondrial Ca2+ uptake.

Isorhynchophylline enhances Nrf2 and inhibits MAPK pathway in cardiac hypertrophy.

Isorhynchophylline (IRN) is one of the major tetracyclic oxindole alkaloids found in Uncaria rhynchophylla. Studies have found that IRN has diverse biological activities including antioxidant, anti-apoptosis, and neuroprotection. However, little is known about the effect of IRN on the development of cardiac hypertrophy. In this study, we investigated the change of the cell surface area and nascent protein synthesis of cultured H9c2 cardiomyocytes on exposure to phenylephrine (PE) plus IRN, and thus confirmed that IRN ameliorated cardiomyocyte hypertrophy induced by PE in vitro. Meanwhile, it turns out that IRN is also effective in neonatal rat ventricular myocytes (NRVMs) stimulated with angiotensin II (AngII). We also showed that IRN prevented cardiac dysfunction in mice with pressure overload due to transverse aortic constriction (TAC) and attenuated cardiac hypertrophy and fibrosis. IRN treatment improved the cardiac function assessed by echocardiographic parameters fractional shortening (FS) as well as suppressed the cardiac hypertrophy phenotypes, such as the increasing of ventricular mass/body weight and myocyte cross-sectional area. RT-PCR analysis showed that IRN treatment also alleviated the expression of fetal genes of ANP, BNP, Myh7, and the correlated fibrosis genes including TGF-β1, collagen I, collagen III, and CTGF in vivo. Meanwhile, IRN had anti-oxidative effects on cardiac remodeling with suppressed 4-HNE and MDA. Western blot analysis showed that the Nrf2 nuclear translocation and MAPK pathway were involved in the potential mechanisms of IRN on cardiac hypertrophy inhibition. The results of our study provide further evidence that IRN is a promising drug for the treatment of cardiac hypertrophy.

SIRT1 Mediates Septic Cardiomyopathy in a Murine Model of Polymicrobial Sepsis.

Cardiac dysfunction, a common complication from severe sepsis, is associated with increased morbidity and mortality. However, the molecular mechanisms of septic cardiac dysfunction are poorly understood. SIRT1, a member of the sirtuin family of NAD+-dependent protein deacetylases, is an important immunometabolic regulator of sepsis, and sustained SIRT1 elevation is associated with worse outcomes and organ dysfunction in severe sepsis. Herein, we explore the role of SIRT1 in septic cardiac dysfunction using a murine model of sepsis. An in vitro model of inflammation in isolated H9c2 cardiomyocytes was used to confirm SIRT1 response to stimulation with lipopolysaccharide (LPS), followed by a murine model of cecal ligation and puncture (CLP) to investigate the molecular and echocardiographic response to sepsis. A selective SIRT1 inhibitor, EX-527, was employed to test for SIRT1 participation in septic cardiac dysfunction. SIRT1 mRNA and protein levels in cultured H9c2 cardiomyocytes were significantly elevated at later time points after stimulation with LPS. Similarly, cardiac tissue harvested from C57BL/6 mice 36 h after CLP demonstrated increased expression of SIRT1 mRNA and protein compared with sham controls. Administration of EX-527 18 h after CLP reduced SIRT1 protein expression in cardiac tissue at 36 h. Moreover, treatment with EX-527 improved cardiac performance with increased global longitudinal strain and longitudinal strain rate. Our findings reveal that SIRT1 expression increases in isolated cardiomyocytes and cardiac tissue after sepsis inflammation. Moreover, rebalancing SIRT1 excess in late sepsis improves cardiac performance, suggesting that SIRT1 may serve as a therapeutic target for septic cardiomyopathy.

Cinnamaldehyde Ameliorates High-Glucose-Induced Oxidative Stress and Cardiomyocyte Injury Through Transient Receptor Potential Ankyrin 1.

Oxidative stress plays a critical role in diabetic cardiomyopathy. Transient receptor potential ankyrin subtype 1 (TRPA1) has antioxidative property. In this study, we tested whether activation of TRPA1 with cinnamaldehyde protects against high-glucose-induced cardiomyocyte injury. Cinnamaldehyde remarkably decreased high-glucose-induced mitochondrial superoxide overproduction, upregulation of nitrotyrosine, P22, and P47, and apoptosis in cultured H9C2 cardiomyocytes (P < 0.01), which were abolished by a TRPA1 antagonist HC030031 (P < 0.01). Nrf2 and its induced genes heme oxygenase-1 (HO-1), glutathione peroxidase-1 (GPx-1), and quinone oxidoreductase-1 (NQO-1) were slightly increased by high glucose (P < 0.01) and further upregulated by cinnamaldehyde (P < 0.05 or P < 0.01). Feeding with cinnamaldehyde (0.02%)-containing diet for 12 weeks significantly decreased cardiac nitrotyrosine levels (P < 0.01), fibrosis, and cardiomyocyte hypertrophy (P < 0.05), while increased expression of antioxidative enzymes (HO-1, GPx-1, NQO-1, and catalase) (P < 0.01) in the myocardial tissue of db/db diabetic mice. These results suggest that cinnamaldehyde protects against high-glucose-induced oxidative damage of cardiomyocytes likely through the TRPA1/Nrf2 pathway.

Yellow Wine Polyphenolic Compounds prevents Doxorubicin-induced cardiotoxicity through activation of the Nrf2 signalling pathway.

Doxorubicin (DOX) is considered as the major culprit in chemotherapy‐induced cardiotoxicity. Yellow wine polyphenolic compounds (YWPC), which are full of polyphenols, have beneficial effects on cardiovascular disease. However, their role in DOX‐induced cardiotoxicity is poorly understood. Due to their antioxidant property, we have been suggested that YWPC could prevent DOX‐induced cardiotoxicity. In this study, we found that YWPC treatment (30 mg/kg/day) significantly improved DOX‐induced cardiac hypertrophy and cardiac dysfunction. YWPC alleviated DOX‐induced increase in oxidative stress levels, reduction in endogenous antioxidant enzyme activities and inflammatory response. Besides, administration of YWPC could prevent DOX‐induced mitochondria‐mediated cardiac apoptosis. Mechanistically, we found that YWPC attenuated DOX‐induced reactive oxygen species (ROS) and down‐regulation of transforming growth factor beta 1 (TGF‐β1)/smad3 pathway by promoting nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) nucleus translocation in cultured H9C2 cardiomyocytes. Additionally, YWPC against DOX‐induced TGF‐β1 up‐regulation were abolished by Nrf2 knockdown. Further studies revealed that YWPC could inhibit DOX‐induced cardiac fibrosis through inhibiting TGF‐β/smad3‐mediated ECM synthesis. Collectively, our results revealed that YWPC might be effective in mitigating DOX‐induced cardiotoxicity by Nrf2‐dependent down‐regulation of the TGF‐β/smad3 pathway.