H9c2 Cardiac Myocytes Research Articles

Abstract P320: Novel Alpha-calcitonin Gene Related Peptide Agonist Analogs In The Treatment Of Cardiovascular Disease

Alpha-calcitonin gene related peptide (α-CGRP) is a potent vasodilator and protects against heart failure and hypertension in various animal models; however, rapid clearance of the peptide from the circulation makes it difficult to use as a therapeutic agent. Recently, we have synthesized two α-CGRP agonist analogs linked with two n-methoxyethylglycine (NMEG) peptoid molecules at either end: 1)- α-CGRP with NMEG molecules at the N-terminal end (N-ter NMEG), and 2)- α-CGRP with NMEG molecules at the C-terminal end (C-ter NMEG). Bioactivity and toxicity of modified peptides were evaluated in in vitro and in vivo conditions. Peptide-peptoid hybrids, N-ter NMEG and C-ter NMEG, were synthesized using a solid-phase peptide synthesis method with a >98% purity. To test the in vitro cell viability of the analogs, rat H9C2 cardiac myocyte cells were treated with 1 μM, 3 μM, and 10 μM concentrations of N-ter NMEG and C-ter NMEG peptides for 4 days followed by an MTT assay. Our in vitro results demonstrated that neither α-CGRP analogs were toxic to H9C2 cells. To test the bioactivity of these peptides, blood pressure (BP) was measured in wild-type C57BL6 mice using a tail-cuff BP analysis system. A bolus dose of 1.2, 3.6, and 12 (in mg/kg b.wt./mouse) of either peptide was injected subcutaneously (n= 4 mice/dose) followed by BP measurement at increasing time points. Our results demonstrate that there is a variance in the bioactivity of the two peptides. Subcutaneous delivery of N-ter NMEG reduced BP similar to the native peptide. After ten minutes of N-ter NMEG delivery, the BP (in mmHg ±SD) at a dose of 1.2 mg/kg was 76 ±5 (baseline BP= 118 ±15), at a dose of 3.6 mg/kg was 79 ±7 (baseline BP= 113 ±9), and at a dose of 12 mg/mg was 83 ±12 (baseline BP= 104 ±6). In contrast, C-ter NMEG administration did not reduce BP at any peptide concentration tested. In summary, in this study we determined that the α-CGRP analog N-ter NMEG, but not C-ter NMEG, is bioactive. Furthermore, N-ter NMEG showed no toxic effects on H9C2 cardiac myocytes in an in vitro viability assay. These results demonstrate that the analog N-ter NMEG is an effective α-CGRP agonist and a promising candidate molecule to treat cardiac diseases.

Effects of Δ9-Tetrahydrocannabinol and the Aminoalkylindole K2/Spice Constituent JWH-073 on Cardiac Tissue and Mesenteric Vascular Reactivity.

Background: Although use of Cannabis sativa is not associated with serious adverse effects, recreational use of aminoalkylindole (AAI) cannabinoid receptor agonists found in K2/Spice herbal blends has been reported to cause adverse cardiovascular events, including angina, arrhythmia, changes in blood pressure, ischemic stroke, and myocardial infarction. Δ9-Tetrahydrocannabinol (Δ9-THC) is the primary CB1 agonist found in cannabis and JWH-073 is one of the AAI CB1 agonists found in K2/Spice brands sold to the public. Methods: This study used in vitro, in vivo, and ex vivo approaches to investigate potential differences on cardiac tissue and vascular effects betweenJWH-073 and Δ9-THC. Male C57BL/6 mice were treated with JWH-073 or Δ9-THC and cardiac injury was assessed by histology. Effects of JWH-073 and Δ9-THC on H9C2 cell viability and ex vivo mesenteric vascular reactivity were also determined. Results: JWH-073 or Δ9-THC induced typical cannabinoid effects of antinociception and hypothermia but did not promote death of cardiac myocytes. No differences in cell viability were observed in cultured H9C2 cardiac myocytes after 24 h of treatment. In isolated mesenteric arteries from drug-naive animals, JWH-073 produced significantly greater maximal relaxation (96%±2% vs. 73%±5%, p<0.05) and significantly greater inhibition of phenylephrine-mediated maximal contraction (Control 174%±11%KMAX) compared with Δ9-THC (50%±17% vs. 119%±16%KMAX, p<0.05). Discussion: These findings suggest that neither cannabinoid at the concentrations/dose studied caused cardiac cell death, but JWH-073 has the potential for greater vascular adverse events than Δ9-THC through an increased vasodilatory effect.

Yang-xin-xue keli exerts therapeutic effects via regulating mitochondrial homeostasis and function in doxorubicin-induced rat heart failure.

Background: Heart failure, especially chronic heart failure, is generally induced by the accumulation of reactive oxygen species (ROS), as well as the subsequent loss of mitochondrial permeability transition pore (mPTP) openings and pathological mitochondrial dysfunction. Herein, we explored the therapeutic effects of the Chinese medicine Yangxin Keli (YXXKL) on chronic heart failure and its underlying working mechanism. Methods: To mimic oxidative stress-induced chronic heart failure, a rat heart failure model was induced by the administration of DOX. Transthoracic echocardiography was performed to confirm the successful establishment of the heart failure model by observing significantly decreased cardiac function in the rats. Mitochondrial membrane potential, function, and ATP synthesis activity were measured after YXXKL was employed. Results The administration of YXXKL not only significantly improved cardiac function but also reversed the myocardium loss and fibrosis induced via DOX. Moreover, the administration of YXXKL also increased ATP synthesis and mitochondrial DNA mass in left ventricular tissues, which indicated that mitochondria may be a key target of YXXKL. Thus, we employed rat cardiomyocyte H9c2 and primary rat cardiac myocytes (RCMs) to induce oxidative stress-induced myocardial injury via DOX treatment. YXXKL-medicated serum promoted cell proliferation, which was inhibited by the addition of IC30 DOX, and the serum also inhibited cell apoptosis, which was promoted by the addition of IC50 DOX. YXKL-medicated serum was able to scavenge ROS and maintain the mitochondrial membrane potential as well as promote mitochondrial function, including the promotion of ATP synthesis, mitochondrial DNA mass, and transcriptional activity. Furthermore, we also observed that YXXKL-medicated serum inhibited DOX-induced autophagy/mitophagy by scavenging ROS. Conclusion: Taken together, we conclude that YXXKLI may exert therapeutic effects on oxidative stress-related heart failure via the regulation of mitochondria.

Sirt6 activation blocks LPS induced expression of inflammatory cytokines in cardiac cells

Sepsis induced myocardial dysfunction is a common complication that has been linked to an increased mortality. The activation of NFkB pathway and excessive production of pro‐inflammatory cytokines is considered to be one of the most important signaling pathway involved in the pathogenesis of sepsis. Earlier studies have shown that Sirt6, a member of class III of histone deacetylases, attenuates hyper‐activation of NFkB signaling that contributes to inflammation and the aging process. In the present study we tested the hypothesis whether pharmacological activation of Sirt6 can prevent LPS induced myocardial inflammation. Primary cultures of adult rat cardiac fibroblasts were treated with UBCS039, an allosteric activator of Sirt6, for 48 hours. The results showed a dose‐dependent increase in steady‐state levels of Sirt6 as well as deacetylation of its target H3K9 by Western analysis. A similar effect of UBCS039 treatment was observed in H9C2 cardiac myocytes. We also tested effects of UBCS039 on LPS induced NFkB activation and cytokine production in these cells. Our data showed that pretreatment of cardiac fibroblasts with UBCS039 for 48 hrs significantly reduced LPS (100ng/ml)‐induced phosphorylated levels of NFkB (pNFkB). By real‐time PCR analysis we found that LPS led to increased expression (4‐9 fold) of mRNA of IL‐6, IL‐1b, TLR4 and CD‐38 in cardiac fibroblasts within 6h of treatment. This effect of LPS in cardiac fibroblasts was completely blocked when cells were pretreated with UBCS039. These data thus indicated that pharmacological activation of Sirt6 is capable of blocking LPS induced inflammatory response in cardiac cells, and has the potential to alleviate sepsis induced myocardial dysfunction and related health consequences.

Epoxyeicosatrienoic acids prevent cardiomyocytes against sepsis by A2AR-induced activation of PI3K and PPARγ

Although epoxyeicosatrienoic acids (EETs) have multiple protective effects against different diseases, whether they can improve the pathogenesis of lipopolysaccharide (LPS)-induced septic cardiac dysfunction remains unknown. We investigated the effects of EETs on the LPS-induced inflammatory response in myocardial dysfunction mice and H9c2 cardiac myocytes. Cardiac-specific CYP2J2 transgenic mice (Tr) showed improved cardiac function and reduced inflammation response after administration with LPS, while the protective effects were not observed in A2A adenosine receptor (A2AR/ADORA2A)–deficient mice (knockout/KO). In vitro, EETs prevented LPS-induced inflammation and apoptosis in the cardiomyocytes via A2AR activation. Moreover, ZM241385 (A2AR inhibitor) attenuated the cardioprotective properties of EETs. Further investigation demonstrated that A2AR signal pathway activation partly regulated phosphatidylinositol 3-kinase (PI3K) and peroxisome proliferator-activated receptor-γ (PPARγ) expression. This is the first report on EETs exerting cardioprotective effects against LPS-induced cardiomyocyte injury via A2AR activation.

Inhibition of mitochondrial reactive oxygen species reduces high glucose-induced pyroptosis and ferroptosis in H9C2 cardiac myocytes

To observe the effect of inhibiting mitochondrial oxidative stress and NLRP3 inflammasomes on high glucose (HG)-induced pyroptosis and ferroptosis in H9C2 cardiac muscle cells and explore the possible interactions between mitochondrial reactive oxygen species (ROS) and inflammasomes. H9C2 cells exposed to high glucose (35 mmol/L) were treated with the mitochondrial antioxidant mitoquinone (MitoQ), the NLRP3 antagonist MCC950, or both MCC950 and rotenone (a mitochondrial electron transport antagonist), and the cell viability was measured with CCK-8 assay. The cellular and mitochondrial ROS levels were measured using CellRox and Mitosox fluorescent probes, respectively. The cellular NLRP3 inflammasome level was detected with immunofluorescence assay, and the expressions of the key proteins related with pyroptosis and ferroptosis were determined with Western blotting. HG exposure significantly lowered the viability of H9C2 cells (P < 0.01), reduced the expression of GPX4 protein (a key protein related with ferroptosis) (P < 0.01), and increased the fluorescence intensities of NLRP3 (P < 0.01) and ROS (at both the cellular and mitochondrial levels, P < 0.01) and the protein expressions of NLRP3 and GSDMD-NT (P < 0.01). Treatment with either MitoQ or MCC950 significantly increased the viability of HG-exposed cells (P < 0.01), increased GPX4 expression (P < 0.01), and reduced the fluorescence intensities of NLRP3 (P < 0.01) and cellular and mitochondrial ROS (P < 0.01) and the protein expressions of NLRP3 and GSDMD-NT (P < 0.05). Compared with MCC950 treatment, treatment with both MCC950 and rotenone significantly reduced the viability of HG-exposed cells (P < 0.01), lowered GPX4 expression (P < 0.01), and increased the fluorescence intensities of ROS and NLRP3 (P < 0.01) and the protein levels of NLRP3 and GSDMD-NT (P < 0.05). MitoQ inhibits mitochondrial ROS production to reduce HGinduced NLRP3 inflammasome activation and thus suppress pyroptosis and ferroptosis of cardiac muscle cells. There may be an interaction between mitochondrial ROS and NLRP3 inflammasomes.

RETRACTED ARTICLE: Role of aminolevulinic acid synthase 1 in doxorubicin-induced oxidative stress to the ardiomyocyte.

Aminolevulinic acid synthase 1(ALAS 1) is the rate-limiting enzyme for heme synthesis, which is the essential source of intracellular oxidative stress under specific conditions. This study aimed to identify the role of ALAS1 in the oxidative injury of cardiomyocyte induced by doxorubicin. H9c2 cardiac myocytes were treated with a 5-μM concentration of doxorubicin for 24 h. ALAS1 small interfering RNA (siRNA) or rALAS1 plasmid was transfected into H9c2 cardiac myocytes to establish ALAS1 knockdown or over-expressing H9c2 cells. Apoptotic cells were detected using flow cytometric analysis. ALAS1 expression was detected by real-time PCR and western blot assay. The levels of heme were detected by ELISA. MitoSOX Red was introduced for detection of mitochondrial superoxide in H9c2 cells and was validated with confocal fluorescence microscopy. Doxorubicin increase the expression of ALAS1, and heme promotes the oxidative stress and apoptosis in H9c2 cardiac myocytes. Over-expression of ALAS1 can produce similar apoptotic effect on H9c2 cells with doxorubicin, although at a relatively low level. Inhibition of ALAS1 by ALAS1 siRNA decreased heme and mitochondrial superoxide levels, results in inhibition of doxorubicin-induced apoptosis. ALAS1 plays a pivotal role in the doxorubicin-induced oxidative stress and apoptosis in H9c2 cardiac myocytes.

GRK5‐mediated inhibitory phosphorylation is essential for inverse agonism at the cardiac mineralocorticoid receptor

BackgroundIn the heart, aldosterone binds the mineralocorticoid receptor (MR) to exert damaging effects, e.g. fibrosis, apoptosis, oxidative stress, etc. GRK2 and GRK5 are the most abundant cardiac G protein‐coupled receptor (GPCR)‐kinases (GRKs). Both phosphorylate GPCRs but also non‐GPCR substrates. We recently showed that GRK5 blocks the cardio‐toxic MR‐dependent effects of aldosterone in the heart by directly phosphorylating the cardiac MR and inhibiting its transcriptional activity. MR antagonist (MRA) drugs are beneifical in human CHF by blocking the cardiac MR. Novel non‐steroidal MRAs, such as finerenone, may provide better cardio‐protection against aldosterone's cardio‐toxic actions than the classic steroidal MRAs, such as sprironolactone and eplerenone. Indeed, finerenone was very recently shown to be a more potent and efficacious inverse agonist at the MR than eplerenone in terms of cardiac fibrosis/adverse remodeling attenuation. Thus, in the present study, we sought to investigate potential differences between finerenone and eplerenone at engaging GRK5‐dependent cardiac MR phosphorylation and subsequent blockade.MethodsWe used the cardiomyocyte cell line H9c2 and neonatal rat ventricular myocytes (NRVMs). We measured MR phosphorylation via phospho‐serine immunoblotting &amp; transcriptional activity via the luciferase reporter assay.ResultsGRK5 phosphorylates the MR in H9c2 cardiomyocytes in response to finerenone but not to eplerenone. Contrary to eplerenone, finerenone alone potently and efficiently suppresses MR transcriptional activity in H9c2 cardiac myocytes. GRK5‐dependent phosphorylation is necessary for the robust inverse agonism displayed by finerenone at the cardiac MR, since CRISPR‐mediated GRK5 gene deletion renders finerenone incapable of blocking cardiac MR transcriptional activity. Conversely, this non‐steroidal MRA fully suppresses cardiac MR transcriptional activity upon GRK5 overexpression in H9c2 cardiomyocytes. Eplerenone alone cannot suppress cardiac MR basal activity upon GRK5 overexpression or knockout. Finally, finerenone blocks aldosterone's effects more efficiently than eplerenone in control and GRK5‐overexpressing NRVMs.ConclusionsFinerenone appears a more efficacious cardiac MR blocker than eplerenone, partly via GRK5‐dependent cardiac MR inhibition, which eplerenone does not induce. This non‐canonical effect of GRK5 on the MR is essential for the efficient blockade of aldosterone's deleterious actions in the heart.Support or Funding Information1) American Heart Association 2) NSU President's Faculty Research &amp; Development GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Melanocortin MC5R as a New Target for Treatment of High Glucose-Induced Hypertrophy of the Cardiac H9c2 Cells

The study explored the anti-hypertrophic effect of the melanocortin MC5R stimulation in H9c2 cardiac myocytes exposed to high glucose. This has been done by using α-MSH and selective MC5R agonists and assessing the expression of GLUT4 and GLUT1 transporters, miR-133 and urotensin receptor levels as a marker of cardiac hypertrophy. The study shows for the first time an up-regulation of MC5R expression levels in H9c2 cardiomyocytes exposed to high glucose medium (33 mM D-glucose) for 48 h, compared to cells grown in normal glucose medium (5.5 mM D-glucose). Moreover, H9c2 cells exposed to high glucose showed a significant reduction in cell viability (-40%), a significant increase in total protein per cell number (+109%), and an increase of the urotensin receptor expression levels as an evidence of cells hypertrophy. The pharmacological stimulation of MC5R with α-MSH (90 pM)of the high glucose exposed H9c2 cells increased the cell survival (+50,8%) and reduced the total protein per cell number (-28,2%) with respect to high glucose alone, confirming a reduction of the hypertrophic state as per cell area measurement. Similarly, PG-901 (selective agonist, 10-10 M) significantly increased cell viability (+61,0 %) and reduced total protein per cell number (-40,2%), compared to cells exposed to high glucose alone. Interestingly, the MC5R agonist reduced the GLUT1/GLUT4 glucose transporters ratio on the cell membranes exhibited by the hypertrophic H9c2 cells and increased the intracellular PI3K activity, mediated by a decrease of the levels of the miRNA miR-133a. The beneficial effects of MC5R agonism on the cardiac hypertrophy caused by high glucose was also observed also by echocardiographic evaluations of rats made diabetics with streptozotocin (65 mg/kg i.p.). Therefore, the melanocortin MC5R could be a new target for the treatment of high glucose-induced hypertrophy of the cardiac H9c2 cells.

MiR-34a-5p aggravates hypoxia-induced apoptosis by targeting ZEB1 in cardiomyocytes.

Myocardial infarction (MI) is an unsolved health problem which seriously affects human health around the world. miR-34a-5p acting as a tumor-suppressor is associated with left ventricular remodeling. We aimed to explore the functional roles of miR-34a-5p in cardiomyocytes. Hypoxia-induced cell injury in H9c2, HL-1 and human cardiac myocytes was analyzed according to the decrease of cell viability and increase of apoptosis. Expression of miR-34a-5p was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) when the concentration of O2 was decreased. Then, the effects of aberrantly expressed miR-34a-5p on proliferation and apoptosis of cardiomyocytes incubated under hypoxia were assessed. Finally, the downstream protein and signaling pathways of miR-34a-5p were explored. The hypoxic model was successfully constructed after incubation under hypoxia for 48 h. When the concentration of O2 decreased, the miR-34a-5p level was increased significantly. Then, we found miR-34a-5p aggravated hypoxia-induced alterations of proliferation and apoptosis in cardiomyocytes. Zinc finger E-box binding homeobox 1 (ZEB1) was identified as a target of miR-34a-5p, and miR-34a-5p conferred its function via targeting ZEB1. Finally, miR-34a-5p inhibition reversed hypoxia-induced decreases of phosphorylated kinases in the JAK/STAT and PI3K/AKT pathways through up-regulating ZEB1. Our study revealed that miR-34a-5p inhibition protected cardiomyocytes against hypoxia-induced cell injury through activating the JAK/STAT and PI3K/AKT pathways by targeting ZEB1.

Inhibition of protein kinase R protects against palmitic acid-induced inflammation, oxidative stress, and apoptosis through the JNK/NF-kB/NLRP3 pathway in cultured H9C2 cardiomyocytes.

Double-stranded RNA-dependent protein kinase (PKR) is a critical regulator of apoptosis, oxidative stress, and inflammation under hyperlipidemic and insulin resistance conditions. Saturated free fatty acids, such as palmitic acid (PA), are known inducers of apoptosis in numerous cell types. However, the underlying molecular mechanism is not fully understood. The aim of the present study was to examine the effect of PA on cultured rat H9C2 cardiac myocytes cells and to investigate the PKR mediated harmful effects of PA in vitro in cultured cardiomyocytes. PKR expression was determined by immunofluorescence and immunoblotting. Oxidative stress and apoptosis were determined by flow cytometry and assay kits. The expression of different gene markers of apoptosis, oxidative stress, and inflammation were measured by Western blot analysis and reverse transcription polymerase chain reaction. PKR expression, reactive oxygen species levels as well as apoptosis were increased in PA-treated cultured H9C2 cardiomyocytes. The harmful effects of PA were attenuated by a selective PKR inhibitor, C16. Moreover, we observed that upregulation of c-Jun N-terminal kinase (JNK), nuclear factor-kB (NF-kB) and NACHT, LRR and PYD domains-containing protein 3 (NLRP3) pathways is associated with increased expression of interleukin 6 and tumor necrosis factor-α in PA-treated cardiomyocytes and attenuation by a selective PKR inhibitor. Our study reports, for the first time, that PKR-mediated harmful effects of PA in cultured cardiomyocytes via activation of JNK, NF-kB, and NLRP3 pathways. Inhibition of PKR is one of the possible mechanistic approaches to inhibit inflammation, oxidative stress, and apoptosis in lipotoxicity-induced cardiomyocyte damage.

Berberine Inhibits Oxygen Consumption Rate Independent of Alteration in Cardiolipin Levels in H9c2 Cells.

Small clinical studies have shown that oral treatment with the plant alkaloid berberine (BBR) reduces blood glucose levels similar to that of metformin and have promoted its use as a novel anti-diabetic therapy. However, in vitro studies have shown that high concentrations of BBR potently inhibit cell proliferation through inhibition of mitochondrial function. Cardiolipin (Ptd2Gro) is a key phospholipid required for regulating mitochondrial bioenergetic function. We examined if BBR inhibited oxygen consumption rate in H9c2 cardiac myocytes through alteration in Ptd2Gro metabolism. Treatment of H9c2 cells with BBR resulted in a rapid (within minutes) concentration-dependent decrease in the oxygen consumption rate (OCR) as determined using a Seahorse XF24 analyzer. Concentrations of BBR as low as 1µM were effective in inhibiting OCR. In addition, all concentrations of BBR inhibited the fatty acid-mediated increase in OCR that was observed in untreated cells. Treatment of H9c2 cells with up to 25µM BBR for 24h markedly reduced [3H]thymidine incorporation into cells but did not alter the pool size of Ptd2Gro. In contrast, 12.5µM BBR increased [1-14C]palmitate incorporation into Ptd2Gro and 12.5µM and 25µM BBR reduced [1-14C]oleate incorporation into Ptd2Gro. Protein kinase C delta (PKCδ) activation through its increased membrane association is known to alter Ptd2Gro distribution within mitochondria. BBR treatment resulted in a decrease in membrane-associated PKCδ and attenuated the palmitate-mediated increase in PKCδ membrane-association. Thus, BBR treatment of H9c2 cardiac myocytes inhibits cellular OCR independent of alteration in Ptd2Gro levels.

Insulin upregulates GRIM-19 and protects cardiac mitochondrial morphology in type 1 diabetic rats partly through PI3K/AKT signaling pathway

Insulin is involved in the development of diabetic heart disease and is important in the activities of mitochondrial complex I. However, the effect of insulin on cardiac mitochondrial nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) 1 subunit of retinoic-interferon-induced mortality 19 (GRIM-19) has not been characterized. The aim of this study was to investigate the effect of insulin on the mitochondrial GRIM-19 in the hearts of rats with streptozotocin (STZ)-induced type 1 diabetes. Protein changes of GRIM-19 were evaluated by western blotting and reverse transcription-quantitative polymerase chain reaction. Furthermore, the effects of insulin on mitochondrial complex I were detected in HeLa cells and H9C2 cardiac myocytes. During the development of diabetic heart disease, the cardiac function did not change within the 8 weeks, but the mitochondrial morphology was altered. The hearts from the rats with STZ-induced diabetes exhibited reduced expression of GRIM-19. Prior to the overt cardiac dilatation, mitochondrial alterations were already present. Following subcutaneous insulin injection, it was demonstrated that GRIM-19 protein was altered, as well as the mitochondrial morphology. The phosphoinositide 3-kinase inhibitor LY294002 had an effect on insulin signaling in H9C2 cardiacmyocytes, and decreased the level of GRIM-19 by half compared with that in the insulin group. The results indicate that insulin is essential for the control of cardiac mitochondrial morphology and the GRIM-19 expression partly via PI3K/AKT signaling pathways.

FoxO1 regulates myocardial glucose oxidation rates via transcriptional control of pyruvate dehydrogenase kinase 4 expression.

Pyruvate dehydrogenase (PDH) is the rate-limiting enzyme for glucose oxidation and a critical regulator of metabolic flexibility during the fasting to feeding transition. PDH is regulated via both PDH kinases (PDHK) and PDH phosphatases, which phosphorylate/inactivate and dephosphorylate/activate PDH, respectively. Our goal was to determine whether the transcription factor forkhead box O1 (FoxO1) regulates PDH activity and glucose oxidation in the heart via increasing the expression of Pdk4, the gene encoding PDHK4. To address this question, we differentiated H9c2 myoblasts into cardiac myocytes and modulated FoxO1 activity, after which Pdk4/PDHK4 expression and PDH phosphorylation/activity were assessed. We assessed binding of FoxO1 to the Pdk4 promoter in cardiac myocytes in conjunction with measuring the role of FoxO1 on glucose oxidation in the isolated working heart. Both pharmacological (1 µM AS1842856) and genetic (siRNA mediated) inhibition of FoxO1 decreased Pdk4/PDHK4 expression and subsequent PDH phosphorylation in H9c2 cardiac myocytes, whereas 10 µM dexamethasone-induced Pdk4/PDHK4 expression was abolished via pretreatment with 1 µM AS1842856. Furthermore, transfection of H9c2 cardiac myocytes with a vector expressing FoxO1 increased luciferase activity driven by a Pdk4 promoter construct containing the FoxO1 DNA-binding element region, but not in a Pdk4 promoter construct lacking this region. Finally, AS1842856 treatment in fasted mice enhanced glucose oxidation rates during aerobic isolated working heart perfusions. Taken together, FoxO1 directly regulates Pdk4 transcription in the heart, thereby controlling PDH activity and subsequent glucose oxidation rates.NEW & NOTEWORTHY Although studies have shown an association between FoxO1 activity and pyruvate dehydrogenase kinase 4 expression, our study demonstrated that pyruvate dehydrogenase kinase 4 is a direct transcriptional target of FoxO1 (but not FoxO3/FoxO4) in the heart. Furthermore, we report here, for the first time, that FoxO1 inhibition increases glucose oxidation in the isolated working mouse heart.

JS-K, a GST-activated nitric oxide donor prodrug, enhances chemo-sensitivity in renal carcinoma cells and prevents cardiac myocytes toxicity induced by Doxorubicin.

Doxorubicin, a highly effective and widely used anthracycline antibiotic in multiple chemotherapy regimens, has been limited by its cardiotoxicity. The aim of this study is to investigate the effect of nitric oxide donor prodrug JS-K on proliferation and apoptosis in renal carcinoma cells and cardiac myocytes toxicity induced by Doxorubicin and to explore possible p53-related mechanism in renal carcinoma cells. The effect of JS-K on anti-cancer activity of Doxorubicin was investigated in renal carcinoma cells via detecting cell proliferation, cytotoxicity, cell death and apoptosis and expressions of apoptotic-related proteins. Effect of p53 on the combination of JS-K and Doxorubicin was determined using p53 inhibitor Pifithrin-α and p53 activator III. Furthermore, the effect of JS-K on cardiac myocytes toxicity of Doxorubicin was investigated in H9c2 (2-1) cardiac myocytes via measuring cell growth, cell death and apoptosis, expressions of proteins involved in apoptosis and intracellular reactive oxygen species. We demonstrated that JS-K could increase Doxorubicin-induced renal carcinoma cell growth suppression and apoptosis and could increase expressions of proteins that are involved in apoptosis. Additionally, Pifithrin-α reversed the promoting effect of JS-K on Doxorubicin-induced renal carcinoma cell apoptosis; conversely, the p53 activator III exacerbated the promoting effect of JS-K on Doxorubicin-induced renal carcinoma cell apoptosis. Furthermore, JS-K protected H9c2 (2-1) cardiac myocytes against Doxorubicin-induced toxicity and decreased Doxorubicin-induced reactive oxygen species production. JS-K enhances the anti-cancer activity of Doxorubicin in renal carcinoma cells by upregulating p53 expression and prevents cardiac myocytes toxicity of Doxorubicin by decreasing oxidative stress.

The role of ZFP580, a novel zinc finger protein, in TGF-mediated cytoprotection against chemical hypoxia‑induced apoptosis in H9c2 cardiac myocytes.

Zing finger protein 580 (ZFP580) is a novel Cys2-His2 zinc-finger transcription factor that has an anti-apoptotic role in myocardial cells. It is involved in the endothelial transforming growth factor-β1 (TGF-β1) signal transduction pathway as a mothers against decapentaplegic homolog (Smad)2 binding partner. The aim of the present study was to determine the involvement of ZFP580 in TGF-β1-mediated cytoprotection against chemical hypoxia-induced apoptosis, using H9c2 cardiac myocytes. Hypoxia was chemically induced in H9c2 myocardial cells by exposure to cobalt chloride (CoCl2). In response to hypoxia, cell viability was decreased, whereas the expression levels of hypoxia inducible factor-1α and ZFP580 were increased. Pretreatment with TGF-β1 attenuated CoCl2-induced cell apoptosis and upregulated ZFP580 protein expression; however, these effects could be suppressed by SB431542, an inhibitor of TGF-β type I receptor and Smad2/3 phosphorylation. Furthermore, suppression of ZFP580 expression by RNA interference reduced the anti-apoptotic effects of TGF-β1 and thus increased CoCl2-induced apoptosis. B-cell lymphoma (Bcl)-2-associated X protein/Bcl-2 ratio, reactive oxygen species generation and caspase-3 activation were also increased following ZFP580 inactivation. In conclusion, these results indicate that ZFP580 is a component of the TGF-β1/Smad signaling pathway, and is involved in the protective effects of TGF-β1 against chemical hypoxia-induced cell apoptosis, through inhibition of the mitochondrial apoptotic pathway.

Icariin attenuated oxidative stress induced-cardiac apoptosis by mitochondria protection and ERK activation.

Our previous study showed that Icariin (ICA) has anti-cardiac hypertrophy effect in rats with an unknown mechanism. In the present study, we aimed to clarify the cardiac protective effect and mechanism of ICA in vitro. H9C2 cardiac myocytes were incubated with H2O2 to build up the oxidative stress injury model. The results showed that pre-treatment of ICA protected cells against the toxicity induced by H2O2. H2O2 treatment significantly reduced H2O2-induced apoptosis, evidenced by lower Annexin V/PI stained cells and less PARP and caspase-3/9 activation. Mitochondria membrane potential (MMP) dissipation occurred following the exposure of H2O2, which could be prevented by ICA treatment. Moreover, Ca2+ homeostasis was preserved by ICA and ROS generation was significantly suppressed by ICA incubation. Interestingly, ICA treatment increased the phosphorylation of upstream ERK mitogen-activated protein kinase (MAPK) while ERK inhibitor U1026 could reverse the protective effect of ICA. Overall, ICA seems to protect the cardiac cells from oxidative stress injury through ROS scavenge and stimulation of ERK pathway which may explain its effects in vivo.

Pharmacological evaluation of novel alagebrium analogs as methylglyoxal scavengers in vitro in cardiac myocytes and in vivo in SD rats

BackgroundMethylglyoxal (MG) is a byproduct of glucose metabolism and an inducer of advanced glycation end products (AGEs). AGEs are implicated in the pathogenesis of diabetes as well as hypertension. Most of the currently available MG scavengers are non-specific and have other effects as well. Alagebrium (ALA), developed by Alteon Corporation is a MG scavenger. Thus the aim of the present study was to investigate the potential of novel ALA analogs as possible MG scavengers and whether they could prevent any deleterious effects of MG. Methods and resultsMG levels were measured by HPLC. The different biochemical and molecular parameters were measured by assay kits, RT-PCR and immunocytochemistry. Out of the 15 ALA analogs tested in vitro, compound no. 13 was found to be an effective inhibitor of MG in a concentration and time dependent manner. Compound no. 13 significantly attenuated the MG levels in vitro in MG treated cultured H9C2 cardiomyocytes as well as in vivo in MG treated SD rats. MG induced oxidative stress and apoptosis were attenuated by pretreatment of H9C2 cardiac myocytes with compound no. 13. MG induced cardiac hypertrophy and apoptosis were also attenuated by treating MG treated SD rats with compound no. 13. ConclusionOur results indicate compound 13 as an effective inhibitor of MG in vitro in cultured cardiomyocytes and in vivo in SD rats and thus it may prove very useful in blocking the multiple deleterious effects of MG, including AGEs and vascular complications of diabetes.

Cardiac mitochondrial energy metabolism in heart failure: Role of cardiolipin and sirtuins

Mitochondrial oxidation of fatty acids accounts for the majority of cardiac ATP production in the heart. Fatty acid utilization by cardiac mitochondria is controlled at the level of fatty acid uptake, lipid synthesis, mobilization and mitochondrial import and oxidation. Consequently defective mitochondrial function appears to be central to the development of heart failure. Cardiolipin is a key mitochondrial phospholipid required for the activity of the electron transport chain. In heart failure, loss of cardiolipin and tetralinoleoylcardiolipin helps to fuel the generation of excessive reactive oxygen species that are a by-product of inefficient mitochondrial electron transport chain complexes I and III. In this vicious cycle, reactive oxygen species generate lipid peroxides and may, in turn, cause oxidation of cardiolipin catalyzed by cytochrome c leading to cardiomyocyte apoptosis. Hence, preservation of cardiolipin and mitochondrial function may be keys to the prevention of heart failure development. In this review, we summarize cardiac energy metabolism and the important role that fatty acid uptake and metabolism play in this process and how defects in these result in heart failure. We highlight the key role that cardiolipin and sirtuins play in cardiac mitochondrial β-oxidation. In addition, we review the potential of pharmacological modulation of cardiolipin through the polyphenolic molecule resveratrol as a sirtuin-activator in attenuating mitochondrial dysfunction. Finally, we provide novel experimental evidence that resveratrol treatment increases cardiolipin in isolated H9c2 cardiac myocytes and tetralinoleoylcardiolipin in the heart of the spontaneously hypertensive rat and hypothesize that this leads to improvement in mitochondrial function. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.

Trichostatin A induces a unique set of microRNAs including miR-129-5p that blocks cyclin-dependent kinase 6 expression and proliferation in H9c2 cardiac myocytes.

The pan-histone deacetylase inhibitor (HDACI), trichostatin A (TSA), was shown to normalize interleukin-18-induced cardiac hypertrophy in vivo and in vitro; evidently, this occurred via epigenetic mechanisms that profoundly altered cardiac gene expression (Majumdar et al. in, Physiol Genom, 43: 1392, 2011; BMC Genom, 13: 709, 2012). Here, we tested the hypothesis that TSA-induced changes in chromatin architecture also led to altered expression of microRNAs that in turn, contributed to the unique transcriptome of cardiac myocytes exposed to the HDACI. Using miRCURY LNA™ Universal microRNA PCR system, we demonstrate that H9c2 cells exposed to TSA for 6 and 24h elicited differential expression of 19 and 16 microRNAs, respectively. H9c2 cells incubated in medium-containing 100nM of TSA elicited a rapid and robust induction of miR-129-5p. Enhanced expression of miR-129-5p was also observed in the hearts of TSA-treated mice. Induction of miR-129-5p in H9c2 cells was accompanied by reduced expression of its direct target, cyclin-dependent kinase 6 (CDK6) that is a key regulator of cell cycle. Using cell division-dependent dilution of Cell Trace™ violet measurements we showed that concomitant induction of miR-129-5p and reduced CDK6 expression were mechanistically involved in TSA-induced inhibition of proliferation of H9c2 cells. Consistent with this scenario, cells expressing an antagomiR of miR-129-5p were resistant to the anti-proliferative actions of TSA. These data indicate that although TSA treatment led to altered expression of several microRNAs, the overarching action of TSA (i.e., inhibition of cell division) in H9c2 cells was achieved via miR-129-5p.