Rat Cardiomyocyte H9c2 Cells Research Articles

Downregulation of the Cardiotrophin-1 Gene Expression by Valsartan and Spironolactone in Hypertrophied Heart Rats In Vivo and Rat Cardiomyocyte H9c2 Cell Line In Vitro

The incidence, prevalence, and hospitalization rates associated with heart failure (HF) are projected to increase substantially in the world. Among all medications used clinically to treat HF, valsartan (VAL) and spironolactone (SPL) have been shown to reduce morbidity and mortality. Recently, a novel cardiac gene cardiotrophin-1 (CT-1) has been shown to play a crucial role in the pathogenesis of HF. However, the ability of VAL and SPL to modulate the expression of CT-1 has not been investigated yet. Therefore, healthy and isoproterenol (ISO)-induced hypertrophy adult male Wistar albino rats were treated with either VAL or SPL for 14 days. Thereafter, cardiac markers of cardiotoxicity and hypertrophy, creatine kinase, heart weight/body weight ratio, and atrial natriuretic peptide mRNA levels were measured. In addition, CT-1 mRNA and protein levels were determined by real-time polymerase chain reaction and Western blot analysis. Our results showed that the increases in all HF markers, creatine kinase, heart weight/body weight ratio, and atrial natriuretic peptide mRNA levels in ISO-treated rats were significantly restored to their normal levels by VAL and SPL. In addition, induction of cardiac hypertrophy by ISO caused remarkable induction in CT-1 mRNA and protein expression levels by approximately 3.5- and 3-fold, respectively. Importantly, VAL and SPL significantly decreased the induction of CT-1 gene at the mRNA and protein levels in heart hypertrophied rats. On the other hand, treatment of cardiac-derived rat myoblast H9c2 cells with VAL and SPL significantly decreased angiotensin II-induced CT-1 mRNA levels through transcriptional mechanism, as demonstrated by the effect of transcription inhibitor, actinomycin D. In conclusion, VAL and SPL exhibited their cardioprotective effect through inhibiting the expression of CT-1 gene in cardiac hypertrophied rats.

The role of aryl hydrocarbon receptor signaling pathway in cardiotoxicity of acute lead intoxication in vivo and in vitro rat model

Lead (Pb2+) is a naturally occurring systemic toxicant heavy metal that affects several organs in the body including the kidneys, liver, and central nervous system. However, Pb2+-induced cardiotoxicity has never been investigated yet and the exact mechanism of Pb2+ associated cardiotoxicity has not been studied. The current study was designed to investigate the potential effect of Pb2+ to induce cardiotoxicity in vivo and in vitro rat model and to explore the molecular mechanisms and the role of aryl hydrocarbon receptor (AhR) and regulated gene, cytochrome P4501A1 (CYP1A1), in Pb2+-mediated cardiotoxicity. For these purposes, Wistar albino rats were treated with Pb2+ (25, 50 and 100mg/kg, i.p.) for three days and the effects on physiological and histopathological parameters of cardiotoxicity were determined. At the in vitro level, rat cardiomyocyte H9c2 cell lines were incubated with increasing concentration of Pb2+ (25, 50, and 100μM) and the expression of hypertrophic genes, α- and β-myosin heavy chain (α-MHC and β-MHC), brain Natriuretic Peptide (BNP), and CYP1A1 were determined at the mRNA and protein levels using real-time PCR and Western blot analysis, respectively. The results showed that Pb2+ significantly induced cardiotoxicity and heart failure as evidenced by increase cardiac enzymes, lactate dehydrogenase and creatine kinase and changes in histopathology in vivo. In addition, Pb2+ treatment induced β-MHC and BNP whereas inhibited α-MHC mRNA and protein levels in vivo in a dose-dependent manner. In contrast, at the in vitro level, Pb2+ treatment induced both β-MHC and α-MHC mRNA levels in time- and dose-dependent manner. Importantly, these changes were accompanied with a proportional increase in the expression of CYP1A1 mRNA and protein expression levels, suggesting a role for the CYP1A1 in cardiotoxicity. The direct evidence for the involvement of CYP1A1 in the induction of cardiotoxicity by Pb2+ was evidenced by the ability of AhR antagonist, resveratrol, to significantly inhibit the Pb2+-modulated effect on β-MHC and α-MHC mRNAs. It was concluded that acute lead exposure induced cardiotoxicity through AhR/CYP1A1-mediated mechanism.

Impact of N-Acetylcysteine on Neonatal Cardiomyocyte Ischemia-Reperfusion Injury

Reactive oxygen species (ROS) are hypothesized to play a key role in myocardial ischemia-reperfusion (IR) injury after cardiopulmonary bypass in children. Clinical studies in adults and several animal models suggest that myocardial IR injury involves cardiomyocyte apoptosis and necrosis. This study investigated a potential relationship between IR-induced ROS production and neonatal cardiomyocyte apoptosis using both in vitro and ex vivo techniques. For in vitro experiments, embryonic rat cardiomyocytes (H9c2 cells) exposed to hypoxia-reoxygenation (HR) showed a time-dependent increase in gp91 phox (a marker for ROS production by NADPH oxidases), caspase-3 (a key mediator of apoptosis) expression, and a decrease in the glutathione redox ratio. N-acetylcysteine (NAC; 0.25-2 mM), a potent antioxidant, decreased gp91 phox and caspase-3 expression, inhibited apoptosis and restored the glutathione redox ratio. For ex vivo study, IR injury significantly reduced left ventricular (LV) function and increased the expression of gp91 phox and caspase-3 in Langendorff-perfused neonatal (7-14 d) rabbit hearts. NAC (0.4 mM) treatment completely attenuated LV dysfunction after IR. In summary, neonatal myocardial IR injury is associated with an increase in cardiomyocyte oxidative stress and apoptosis. NAC attenuates apoptosis in an in vitro embryonic rat cardiomyocyte model of HR, and myocardial dysfunction in an ex vivo neonatal rabbit model of myocardial IR injury.

Sodium thiosulfate exposure disrupts in vitro and in vivo heart development

It is well-known that the majority of malformations found in the human population is based on complex gene-environment interactions. As an industrial chemical sodium thiosulfate (STS) is used heavily in many industries. Nevertheless, there is little known about the effects of STS on embryo development. In the present study, we have investigated the effects of STS on cardiac development in rat cardiomyocyte H9C2 cell line and chick embryos. As determined by MTT assays, the proliferation of H9C2 cells was inhibited by STS in a dose-dependent manner. Fertilized eggs injected via the yolk sac with STS at Hamburger-Hamilton (HH) stages 6, 9 and 12 showed significantly increased cardiotoxicity at HH stage 18, including cardiomyocyte apoptosis and animal mortality. Western blot analysis showed that STS significantly affected the expression of the apoptosis-related genes bcl-2, bax, and caspase-3 in a dose-dependent manner in the H9C2 cell line and in chick embryos. Dysregulation of apoptosis was correlated with embryonic heart malformations. Thus, STS may be a potent cardiac teratogen during embryo development.

Lagerstroemia speciosa extract inhibit TNF-induced activation of nuclear factor-κB in rat cardiomyocyte H9c2 cells

Aim of the study Lagerstroemia speciosa has been used as a folk medicine among people with diabetes in the Philippines. It is known to exhibit antidiabetic, antiobesity, and glucose transport activities through mechanisms not well defined. Diabetes leads to cardiomyocyte hypertrophy in association with an upregulation of vasoactive factors and activation of nuclear factor (NF)-κB and activating protein-1. We therefore investigated the effect of Lagerstroemia speciosa on the activation of NF-κB as a key mediator of cardiomyocyte hypertrophy, in rat cardiomyocyte H9c2 cells. Materials and methods Water extract of Lagerstroemia speciosa (Lythraceae family) was prepared. H9c2 cells were used for treatment of Lagerstroemia speciosa extract with/without tumor necrosis factor (TNF). To examine NF-κB's activation, we performed an electrophoretic mobility shift assay (EMSA). Results The activation of NF-κB by TNF was completely blocked by a Lagerstroemia speciosa extract in a dose- and time-dependent manner in H9c2 cells. Conclusion Overall, our results indicate that Lagerstroemia speciosa can inhibit DNA-binding of NF-κB. This may explain its possible inhibition of diabetes-induced caridomyocyte hypertrophy.

Induction of cellular glutathione-linked enzymes and catalase by the unique chemoprotective agent, 3H-1,2-dithiole-3-thione in rat cardiomyocytes affords protection against oxidative cell injury

Considerable evidence suggests that reactive oxygen species (ROS) are crucially involved in the pathogenesis of cardiovascular diseases, such as myocardial ischemia–reperfusion injury. Consistent with this notion, administration of exogenous antioxidative compounds has been shown to provide protection against oxidative cardiac injury. However, whether induction of endogenous cellular antioxidants by chemicals (drugs) also offers protection against oxidative cardiac injury has not been extensively investigated. In the present study, with rat cardiomyocyte H9C2 cells as an in vitro model, we have investigated the induction of cellular antioxidants by the unique chemoprotective agent, 3 H -1,2-dithiole-3-thione (D3T) and the protective effects of the D3T-induced cellular antioxidants against ROS-mediated injury in cardiac cells. Incubation of H9C2 cells with micromolar concentrations of D3T for 24 h resulted in a significant induction of a battery of cellular antioxidants, including reduced glutathione (GSH), GSH peroxidase, GSSG reductase, GSH S-transferase and catalase. To further examine the protective effects of the induced endogenous antioxidants against oxidative cell injury, H9C2 cells were pre-treated with D3T and then incubated with xanthine oxidase (XO) plus xanthine, a system that generates ROS. We observed that D3T pre-treatment of H9C2 cells led to significant protection against XO/xanthine-induced cytotoxicity as determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction and morphological changes. Taken together, this study demonstrates for the first time that a number of endogenous antioxidants in cardiomyocytes can be induced by exposure to D3T, and that this chemical (drug) induction of cellular antioxidants is accompanied by markedly increased resistance to ROS-mediated cardiac cell injury.