H9c2 Rat Cardiomyocytes Research Articles

Regulation of Cx43 and its role in trichloroethylene-induced cardiac toxicity in H9C2 rat cardiomyocytes

Trichloroethylene (TCE), a widespread environmental contaminant, has been linked to congenital heart defects. Abnormal regulation of Connexin 43 is closely associated with various cardiac diseases. However, it is yet to be established how Cx43 responds to environmental pollutants. Here, we aim to explore the role of Cx43 in TCE-induced cardiac toxicity using H9C2 cardiomyocytes. EdU incorporation assay and cell cycle analysis revealed that increased number of TCE-treated cells entered into the S stage, indicating that TCE exposure provoked cell proliferation. Additionally, compromised mitochondrial function was observed in TCE-treated cells, and inhibition of mitochondrial permeability transition pore (mPTP) with Cyclosporin A or eliminating mitochondrial ROS by MitoQ alleviated the TCE-induced cardiac toxicity. Importantly, TCE exposure increased the protein expression levels of Cx43 and stimulated the recruitment of Cx43 to the mitochondria. TCE exposure disrupted canonical Wnt signal pathway, resulting in downregulation of antioxidant genes and β-catenin. The adverse effects of TCE on Wnt signal pathway activation, mitochondrial function and cell proliferation were efficiently counteracted by either Cx43 knockdown or pharmaceutical activator of Wnt signaling, CHIR-99021. Taken together, our results for the first time revealed that dysregulation of Cx43 mediates TCE-induced heart defects via mitochondrial dysfunction and Wnt signaling inhibition, suggesting that Cx43 can be a potential molecular marker or therapeutic target for cardiac diseases caused by environmental pollutants.

Parecoxib attenuates inflammation injury in septic H9c2 cells by regulating the MAPK signaling pathway.

Parecoxib, a non-steroidal anti-inflammatory drug, has been reported to possess protective effects against sepsis. However, its detailed role and underlying mechanisms in septic cardiomyopathy remain unclear. Therefore, the goal of the present study was to clarify the function and to investigate the mechanisms of parecoxib in lipopolysaccharide (LPS)-treated H9c2 rat cardiomyocytes. TNF-α, IL-1β and IL-6 expression levels in parecoxib-treated H9c2 cells stimulated with LPS were assessed using ELISA. Parecoxib-treated H9c2 cells stimulated with LPS were tested for viability using the Cell Counting Kit-8 assay. Western blotting analysis and 5-ethynyl-2'-deoxyuridine were used to evaluate cell proliferation. Apoptosis was assessed using TUNEL and western blotting. To assess the protein expression of the MAPK signaling pathway, western blotting was performed. The data showed that parecoxib significantly and dose-dependently reduced the inflammatory responses of LPS-treated H9c2 cells. Parecoxib also significantly and dose-dependently increased the proliferation and inhibited the apoptosis of LPS-treated H9c2 cells. In addition, parecoxib significantly suppressed the activation of the MAPK (p38, JNK and ERK) signaling pathway. The current study indicated that parecoxib could be a viable therapeutic option for septic cardiomyopathy.

Chronic high‑salt intake induces cardiomyocyte autophagic vacuolization during left ventricular maladaptive remodeling in spontaneously hypertensive rats.

The role of autophagy in high-salt (HS) intake associated hypertensive left ventricular (LV) remodeling remains unclear. The present study investigated the LV autophagic change and its association with the hypertensive LV remodeling induced by chronic HS intake in spontaneously hypertensive rats (SHR). Wistar Kyoto (WKY) rats and SHR were fed low-salt (LS; 0.5% NaCl) and HS (8.0% NaCl) diets and were subjected to invasive LV hemodynamic analysis after 8, 12 and 16 weeks of dietary intervention. Reverse transcription-quantitative PCR and western blot analysis were performed to investigate the expression of autophagy-associated key components. The LV morphologic staining was performed at the end of the study. The rat H9c2 ventricular myoblast cell-associated experiments were performed to explore the mechanism of HS induced autophagic change. A global autophagy-associated key component, as well as increased cardiomyocyte autophagic vacuolization, was observed after 12 weeks of HS intake. During this period, the heart from HS-diet-fed SHR exhibited a transition from compensated LV hypertrophy to decompensation, as shown by progressive impairment of LV function and interstitial fibrosis. Myocardial extracellular [Na+] and the expression of tonicity-responsive enhancer binding protein (TonEBP) was significantly increased in HS-fed rats, indicating myocardial interstitial hypertonicity by chronic HS intake. The global autophagic change and overt deterioration of LV function were not observed in LS-fed SHR and HS-fed WKY rats. The study of rat H9c2 cardiomyocytes demonstrated a cytosolic [Na+] elevation-mediated, reactive oxygen species-dependent the autophagic change occurred when exposed to an increased extracellular [Na+]. The present findings demonstrated that a myocardial autophagic change participates in the maladaptive LV remodeling induced by chronic HS intake in SHR, which provides a possible target for future intervention studies on HS-induced hypertensive LV remodeling.

A four-compound remedy AGILe protected H9c2 cardiomyocytes against oxygen glucose deprivation via targeting the TNF-α/NF-κB pathway: Implications for the therapy of myocardial infarction.

Myocardial infarction (MI) is a highly prevalent and lethal disease worldwide. Prevention and timely recovery are critical for the control of the recurrence and heart failure in MI survivors. The present study was designed to investigate the cardioprotective activity of the herbal medicine formula Baoyuan Decoction (BYD) and identify the active compounds and molecular targets. The ethanolic BYD extract (BYDE) was prepared by water extraction and ethanol precipitation of four herbal medicines, Astragali Radix, Ginseng Radix et Rhizoma, Cinnamomi Cortex, and Glycyrrhizae Radix et Rhizoma. Initially, BYDE was validated for the cardioprotective effectiveness in a mouse model of ischemia injury and rat cardiomyocyte H9C2 cells. As results, BYDE effectively reduced infarct size from 56% to 37% and preserved cardiac functions in mouse MI model while protected H9C2 cells against oxygen glucose deprivation. Subsequent network pharmacology analysis revealed that 122 bioactive ingredients, including flavonoids and saponins from the UPLC-MS/MS profile of BYDE, might target 37 MI-related proteins, including inflammatory and apoptotic mediators (e.g., TNF, NFKB1, CASPs, TNFRSF1A, CXCL12, BCL2A1). Pathway enrichment analysis suggested that BYDE might control the cardiac inflammation via targeting the tumor necrosis factor-alpha (TNF-α)/nuclear factor-κB (NF-κB) pathway while the selected targets were also implicated in IL-17 signaling pathway, lipid and atherosclerosis. Consequently, adenosine, ginsenoside Rh2, isoliquiritigenin, and licochalcone A were selected to generate the four-compound mixture AGILe and validated for the inhibitory effects on the TNF-α/NF-κB pathway. The results of the present study suggested that the mixture AGILe might be a potential cardioprotective remedy against MI.

Momordicine I alleviates isoproterenol-induced cardiomyocyte hypertrophy through suppression of PLA2G6 and DGK-ζ.

This study aimed to observe the protective effect of momordicine I, a triterpenoid compound extracted from momordica charantia L., on isoproterenol (ISO)-induced hypertrophy in rat H9c2 cardiomyocytes and investigate its potential mechanism. Treatment with 10 μM ISO induced cardiomyocyte hypertrophy as evidenced by increased cell surface area and protein content as well as pronounced upregulation of fetal genes including atrial natriuretic peptide, β-myosin heavy chain, and α-skeletal actin; however, those responses were markedly attenuated by treatment with 12.5 μg/ml momordicine I. Transcriptome experiment results showed that there were 381 and 447 differentially expressed genes expressed in comparisons of model/control and momordicine I intervention/model, respectively. GO enrichment analysis suggested that the anti-cardiomyocyte hypertrophic effect of momordicine I may be mainly associated with the regulation of metabolic processes. Based on our transcriptome experiment results as well as literature reports, we selected glycerophospholipid metabolizing enzymes group VI phospholipase A2 (PLA2G6) and diacylglycerol kinase ζ (DGK-ζ) as targets to further explore the potential mechanism through which momordicine I inhibited ISO-induced cardiomyocyte hypertrophy. Our results demonstrated that momordicine I inhibited ISO-induced upregulations of mRNA levels and protein expressions of PLA2G6 and DGK-ζ. Collectively, momordicine I alleviated ISO-induced cardiomyocyte hypertrophy, which may be related to its inhibition of the expression of glycerophospholipid metabolizing enzymes PLA2G6 and DGK-ζ.

Protective effect of Guanxin Danshen formula on myocardial ischemiareperfusion injury in rats

Myocardial ischemia/reperfusion injury (MIRI) leads to myocardial tissue necrosis, which will increase the size of myocardial infarction. The study examined the protective effect and mechanism of the Guanxin Danshen formula (GXDSF) on MIRI in rats. MIRI model was performed in rats; rat H9C2 cardiomyocytes were hypoxia-reoxygenated to establish a cell injury model. The GXDSF significantly reduced myocardial ischemia area, reduced myocardial structural injury, decreased the levels of interleukin (IL-1β, IL-6) in serum, decreased the activity of myocardial enzymes, increased the activity of superoxide dismutase (SOD), and reduced glutathione in rats with MIRI. The GXDSF can reduce the expression of nucleotide- binding oligomerization domain, leucine-rich repeat and pyrin domain containing nod-like receptor family protein 3 (NLRP3), IL-1β, caspase-1, and gasdermin D (GSDMD) in myocardial tissue cells. Salvianolic acid B and notoginsenoside R1 protected H9C2 cardiomyocytes from hypoxia and reoxygenation injury and reduced the levels of tumor necrosis factor α (TNF-α) and IL-6 in the cell supernatant, decreasing the NLRP3, IL-18, IL-1β, caspase-1, and GSDMD expression in H9C2 cardiomyocytes. GXDSF can reduce the myocardial infarction area and alleviate the damage to myocardial structure in rats with MIRI, which may be related to the regulation of the NLRP3. GXDSF reduces MIRI in rat myocardial infarction injury, improves structural damage in myocardial ischemia injury, and reduces myocardial tissue inflammation and oxidative stress by lowering inflammatory factors and controlling focal cell death signaling pathways.

Mitochondria-targeting EGCG derivatives protect H9c2 cardiomyocytes from H2O2-induced apoptosis: design, synthesis and biological evaluation.

Pathologies related to cardiovascular diseases mostly emerge as a result of oxidative stress buildup in cardiomyocytes. The heavy load of mitochondrial oxidative phosphorylation in cardiac tissues corresponds to a surge in oxidative stress leading to mitochondrial dysfunction and cellular apoptosis. Thus, scavenging the reactive oxygen species (ROS) linked to mitochondria can significantly improve cardio-protection. Epigallocatechin-3-gallate (EGCG), the major polyphenol found in green tea has been extensively studied for its profound health-beneficial activities. Herein, we designed and synthesized a series of mitochondrial-targeting EGCG derivatives, namely MitoEGCGn (n = 4, 6, 8) by incorporating triphenylphosphonium ion onto it using different linkers. MitoEGCGn were found to be non-toxic to H9c2 rat cardiomyocyte cells even at higher doses in comparison to its parent molecule EGCG. Interestingly, MitoEGCG4 and MitoEGCG6 protected the H9c2 cardiomyocyte cells from the oxidative damage induced by H2O2 whereas EGCG was found to be toxic and ineffective in protecting the cells from H2O2 damage. MitoEGCG4 and MitoEGCG6 also protected the cells from the H2O2-induced disruption of mitochondrial membrane potential as well as activation of apoptosis as revealed by pro-caspase 3 expression profile, DNA fragmentation assay, and AO/EtBr staining. Taken together, our study shows that the mitochondria targeting EGCG derivatives were able to effectively combat the H2O2-induced oxidative stress in H9c2 cardiomyocytes. They eventually augmented the mitochondrial health of cardiomyocytes by maintaining the mitochondrial function and attenuating apoptosis. Overall, MitoEGCG4 and MitoEGCG6 could provision a cardioprotective role to H9c2 cardiomyocytes at the time of oxidative insults related to mitochondrial dysfunction-associated injuries.

Soluble tumor endothelial marker 1 in heart failure with reduced ejection fraction: A pilot study.

Tumor endothelial marker 1 (TEM1/CD248) is a transmembrane protein that expresses in mesenchymal lineage derived cells during embryogenesis and becomes undetectable in normal adults after birth. Re-expression of TEM1 is found in organ fibrosis, wound healing and cardiac remodeling indicating its potential role in heart failure (HF). The purpose of this study is to explore the role of soluble TEM1 (sTEM1) in patients with HF with reduced ejection fraction. We examined endomyocardial biopsy specimens from three HF patients and blood samples from 48 patients admitted for acute decompensated HF (age 72 years, men 61.7%). The expression of TEM1 in cardiac tissue and concentrations of sTEM1 in plasma were evaluated. Cultured rat cardiomyocytes (H9c2) and human cardiac fibroblasts (HCF) were stimulated with hypoxia or transforming growth factor beta (TGF-β) to observe the release of sTEM1 into culture media. The conditioned media of hypoxia-stimulated H9c2 cells was harvested and added into cultured cardiac fibroblast to evaluate its biological effect. Immunofluorescence study of biopsy specimens from three HF patients showed TEM1 expression in cardiomyocytes and cardiac fibroblasts. The plasma level of sTEM1 was significantly higher in patients (0.90 ± 0.23 vs. 0.33 ± 0.10 ng/mL, p = 0.032) with LVEF ≤ 35% compared with those with LVEF 36-49%. The sTEM1 levels had correlations with HF biomarkers of cardiac fibrosis, including growth differentiation factor-15 (GDF-15) and galectin-3. There was a significant increase in sTEM1 levels in the cultured media of H9c2 and HCF after being stressed with hypoxia or TGF-β. The conditioned media derived from hypoxia-stimulated H9c2 cells significantly increased cell proliferation of cardiac fibroblasts. This effect was partially reversed by anti-TEM1 antibody. This pilot study demonstrated that cardiac TEM1 expression was upregulated in HF. The levels of sTEM1 were significantly higher in HF patients with LVEF ≤ 35% and correlated with other biomarkers of cardiac fibrosis. In vitro study proved that functional sTEM1 was released into cultured media after stressing cardiomyocytes and HCF.

Coxsackievirus B3 infection induces glycolysis to facilitate viral replication.

Coxsackievirus B3 (CVB3) is a leading cause of viral myocarditis, but no effective treatment strategy against CVB3 is available. Viruses lack an inherent metabolic system and thus depend on host cellular metabolism for their benefit. In this study, we observed that CVB3 enhanced glycolysis in H9c2 rat cardiomyocytes and HL-1 mouse cardiomyocytes. Therefore, three key glycolytic enzymes, namely, hexokinase 2 (HK2), muscle phosphofructokinase (PFKM), and pyruvate kinase M2 (PKM2), were measured in CVB3-infected H9c2 and HL-1 cells. Expression levels of HK2 and PFKM, but not PKM2, were increased in CVB3-infected H9c2 cells. All three key glycolytic enzymes showed elevated expression in CVB3-infected HL-1 cells. To further investigate this, we used 2 deoxyglucose, sodium citrate, and shikonin as glycolysis inhibitors for HK2, PFKM, and PKM2, respectively. Glycolysis inhibitors significantly reduced CVB3 replication, while the glycolysis enhancer dramatically promoted it. In addition, glycolysis inhibitors decreased autophagy and accelerated autophagosome degradation. The autophagy inducer eliminated partial inhibition effects of glycolysis inhibitors on CVB3 replication. These results demonstrate that CVB3 infection enhances glycolysis and thus benefits viral replication.

Gender Differences in the Correlations Between Immune Cells and Organ Damage Indexes of Acute Myocardial Infarction Patients.

There are clear gender differences in the pathological process and outcome in acute myocardial infarction (AMI) patients but inflammatory responses remain clarified. Here, we aimed to analyse the correlations between inflammatory cells and organ injury parameters in AMI patients and compared between male and female groups. A total of 603 AMI patients who underwent percutaneous coronary intervention (PCI) within 24 hours of the onset were analysed retrospectively. Basic information and hematological parameters detected 6 hours before the PCI were collected, neutrophil-to-lymphocyte ratio (NLR) and monocyte-to-lymphocyte ratio (MLR) were calculated. Renal, liver function indicators, and myocardial enzymes were measured. Left ventricular ejection fraction (EF) and fractional shortening (FS) on days 5-7 after PCI were obtained. Western blot was performed to detect iNOS, eNOS and nNOS expression in H9C2 rat cardiomyocytes treated with IL-6 with and without estrogen and testosterone. WBC, NEU, MON, MLR, CK, ALT and CREA of male patients were significantly higher than females, but FS was lower in females. NEU, MON and MLR were positively correlated with CK, CK-MB, AST, and ALT in males, whereas LYM were correlated with these parameters in female. NEU and NLR were inversely correlated with EF or FS only in female. Estrogen and testosterone reduced IL-6-induced iNOS protein expression in H9C2 cardiomyocytes, estrogen enhanced IL-6-induced nNOS protein expression. NEU, MON, MLR in male AMI patients, and LYM in female patients were associated with organ injury parameters. Estrogen regulation of nitric oxide pathway may mediate the protective effects in female.

Abstract 12308: Presenilin-2 Regulates Mitophagy via Promoting Intracellular Translocation of Parkin to Mitochondria

Introduction: Presenilin-2 (PS2) is a component of the γ-secretase complex, intracellular protease. Variant in PSEN2 , the causative genes of Alzheimer's disease, are also implicated in the development of dilated cardiomyopathy. Hypoxia increases PSEN2 expression 10-fold over normal, and PSEN2 knockout (PS2KO) cells exhibit significantly lower ATP production. However, the mechanism of the mitochondrial dysfunction has not been fully elucidated. Hypothesis: PS2 regulates mitochondrial selective autophagy (mitophagy) by promoting the translocation of Parkin to mitochondria. Methods: Expression of autophagy and mitophagy markers was examined using cultured rat cardiomyocytes (H9c2) silenced for PSEN2 and PSEN1 , exposed to 10 μM carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler used to induce mitophagy. Results: Mitochondrial respiration of PS2KO-H9c2 was showed reduced (maximal respiration capacity, 68.4 ± 27.0 pmol/min/cells) compared to PS1KO-H9c2 (147.6 ± 27.2 pmol/min/cells) or normal cells (276.6 ± 11.9 pmol/min/cells) (P<0.001). When mitophagy-dye modified with perylene-3,4-dicarboxylic anhydride was used, mitophagy detection rates were significantly increased by the addition of FCCP to normal H9c2 (34% increased, P=0.041) and PS1KO-H9c2 (46% increased, P=0.035), but there was no change with PS2KO-H9c2 (25% decreased, P=0.232). When FCCP was added to PS2KO-H9c2, Parkin was not only enhanced in whole cell lysate of PS2KO-H9c2, but also increased even at mRNA level; however, in the isolated mitochondrial fraction, Parkin was significantly reduced, and PINK1 and phosphorylated ubiquitin (serine 65) were also attenuated. In Immunofluorescent staining confirmed that Parkin was not co-localized with mitochondria in PS2KO-H9c2, implicating PS2 in promoting the transfer of Parkin from the cytoplasm to mitochondria. As mitochondrial depolarization causes PINK1 stabilization needed for Parkin recruitment, we interrogated mitochondrial membrane potential measurement using Rhodamine-123 and examined expression of OPA1 isoforms; no significant results were obtained. Conclusions: PS2 contributes to the intracellular transport of Parkin to regulate mitophagy.

Cytoprotective Polyketides from Sponge-Derived Fungus Lopadostoma pouzarii.

The new polyketides lopouzanones A and B, as well as the new 1-O-acetyl and 2-O-acetyl derivatives of dendrodochol B, were isolated from the sponge-derived marine fungus Lopadostoma pouzarii strain 168CLC-57.3. Moreover, six known polyketides, gliorosein, balticolid, dendrodolide G, dihydroisocoumarine, (-)-5-methylmellein, and dendrodochol B, were identified. The structures of the isolated compounds were determined by a combination of NMR and ESIMS techniques. The absolute configurations of the lopouzanones A and B were determined using the Mosher's method. The cytotoxicity of the isolated compounds against human prostate cancer cells PC-3 and normal rat cardiomyocytes H9c2 was investigated. Gliorosein showed weak DPPH radical-scavenging activity and in vitro cardioprotective effects toward rotenone toxicity and CoCl2-mimic hypoxia.

Integrated strategy of derivatization and separation for sensitive analysis of salvianolic acids using capillary electrophoresis with laser-induced fluorescence detection

Salvianolic acids (SAs) are a class of natural active substances that have been widely used in clinical treatment and food adjuvant therapy. In this work, we found that SAs could form the ternary complex with borax and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), thereby sensitizing their weak intrinsic fluorescence to maximized 92-fold for salvianolic acid B (SAB). The formation of ternary complex was dynamic and could complete once mixed, and the fluorescence intensity remained stable within 3 h. On this basis, an integrated strategy of derivatization and separation was proposed for the sensitive analysis of SAs using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. After the systematic investigation, a solution consisting of 20 mM borax and 20 mM HP-β-CD (pH 8.5) was selected as the running buffer. By the direct injection of SAs, the dynamic derivatization was realized in the capillary, and the baseline separation was achieved within 6 min, with the detection limits of 1.2-21.2 nM for four SAs (S/N=3). Then, the developed CE-LIF method was successfully applied to the quantitative analysis of SAs in four traditional ginsengs, including Salvia miltiorrhiza, Codonopsis pilosula, Panax quinquefolius and Panax ginseng with the recoveries ranging from 95.2% to 110.7%. Except for four target SAs, a large number of unknown electrophoretic peaks had also been observed in four ginsengs, and then were utilized for the identification of ginseng species via principal component analysis. Furthermore, a hypoxia/reoxygenation model was constructed using Rat cardiomyocyte H9c2 cells, and subsequently, the developed method was applied to continuously monitor the consumption of SAB in cell culture medium after its intervention.

Targeted UHPLC-MS Analysis Reveals Disparate Polyphenol Composition and Concentration in Muscadine Grape Supplements with Proportional Antioxidant Activity

Muscadine grape supplements (MGS) with high polyphenol content are a potential therapeutic option to combat oxidative stress; however, the precise identity and concentration of individual phenolics in commercially processed MGSs is not well defined. We probed for 17 phenolic compounds by ultra-high pressure liquid chromatography and mass spectroscopy from distinct lots of four commercially processed MGSs composed of MG seed and/or skin waste products. The total phenolic content (TPC) and antioxidant capacity were highest in a dried water-extract MGS as compared to three ground seed and/or skin products. The TPC was not different between MGS lots from individual companies and remained stable for 3 years without microbial contamination. The extract MGS had the highest concentration of epicatechin, ellagic acid, gallic acid, procyanidin B2, catechin and catechin gallate compared to the other supplements. Only ellagic acid and gallic acid were detected in all four MGSs, while catechin and catechin gallate were below detection in two supplements. Based on gram weight, only the extract MGS prevented the angiotensin II-induced increase in malondialdehyde and 4-hydroxynonenol in rat H9c2 cardiomyocytes as well as upregulated superoxide dismutase and catalase. This study demonstrates that commercial MGSs differ in phenolic composition and concentration, resulting in disparate antioxidant activity.

Potential cardiotoxicity induced by Euodiae Fructus: In vivo and in vitro experiments and untargeted metabolomics research.

Background: Euodiae Fructus, a well-known herbal medicine, is widely used in Asia and has also gained in popularity in Western countries over the last decades. It has known side effects, which have been observed in clinical settings, but few studies have reported on its cardiotoxicity. Methods: In the present study, experiments using techniques of untargeted metabolomics clarify the hazardous effects of Euodiae Fructus on cardiac function and metabolism in rats in situations of overdosage and unsuitable syndrome differentiation. In vitro assays are conducted to observe the toxic effects of evodiamine and rutaecarpine, two main chemical constituents of Euodiae Fructus, in H9c2 and neonatal rat cardiomyocytes (NRCMs), with their signaling mechanisms analyzed accordingly. Results: The cardiac cytotoxicity of evodiamine and rutaecarpine in in vivo experiments is associated with remarkable alterations in lactate dehydrogenase, creatine kinase, and mitochondrial membrane potential; also with increased intensity of calcium fluorescence, decreased protein expression of the cGMP-PKG pathway in H9c2 cells, and frequency of spontaneous beat in NRCMs. Additionally, the results in rats with Yin deficiency receiving a high-dosage of Euodiae Fructus suggest obvious cardiac physiological dysfunction, abnormal electrocardiogram, pathological injuries, and decreased expression of PKG protein. At the level of endogenous metabolites, the cardiac side effects of overdose and irrational usage of Euodiae Fructus relate to 34 differential metabolites and 10 metabolic pathways involving among others, the purine metabolism, the glycerophospholipid metabolism, the glycerolipid metabolism, and the sphingolipid metabolism. Conclusion: These findings shed new light on the cardiotoxicity induced by Euodiae Fructus, which might be associated with overdose and unsuitable syndrome differentiation, that comes from modulating the cGMP-PKG pathway and disturbing the metabolic pathways of purine, lipid, and amino acid. Continuing research is needed to ensure pharmacovigilance for the safe administration of Chinese herbs in the future.

TMEM43 Protects against Sepsis-Induced Cardiac Injury via Inhibiting Ferroptosis in Mice

A previous study found that transmembrane protein 43 (TMEM43) was highly associated with arrhythmogenic right ventricular dysplasia/cardiomyopathy. However, as a transmembrane protein, TMEM43 may be involved in ferroptosis in cardiovascular disease. In this study, we aimed to explore the role of TMEM43 in lipopolysaccharide (LPS)-induced cardiac injury and the underlying mechanism. Mice were injected with LPS (10 mg/kg) for 12 h to generate experimental sepsis. Mice were also subjected to AAV9-shTMEM43 to knock down TMEM43 or AAV9-TMEM43 to overexpress TMEM43 in hearts. H9c2 rat cardiomyocytes were also transfected with Ad-TMEM43 or TMEM43 siRNA to overexpress/knock down TMEM43. As a result, TMEM43 knockdown in hearts deteriorated LPS-induced mouse cardiac injury and dysfunction. LPS increased cardiac ferroptosis as assessed by malonaldehyde (MDA) and cardiac iron density, which were aggravated by TMEM43 knockdown. Moreover, TMEM43 overexpression alleviated LPS-induced cardiac injury, dysfunction, and ferroptosis. In vitro experiments showed that TMEM43 overexpression inhibited LPS-induced lipid peroxidation and cardiomyocyte injury while TMEM43 knockdown aggravated LPS-induced ferroptosis and injury in cardiomyocytes. Mechanistically, LPS increased the expression of P53 and ferritin but decreased the level of Gpx4 and SLC7A11. TMEM43 could inhibit the level of P53 and ferritin enhanced the level of Gpx4 and SLC7A11. Furthermore, ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis, could protect against LPS-induced cardiac injury and also counteracted the deteriorating effects of TMEM43 silencing in the heart. Based on these findings, we concluded that TMEM43 protects against sepsis-induced cardiac injury via inhibiting ferroptosis in mice. By targeting ferroptosis in cardiomyocytes, TMEM43 may be a therapeutic strategy for preventing sepsis in the future.

Protective activity of Malus doumeri leaf extract on H2O2-induced oxidative injury in H9C2 rat cardiomyocytes.

In this study, Malus doumeri leaf extract (MDLE) was used to test its anti-oxidation capacity in vitro, it has been preliminarily analyzed for H2O2-induced oxidative damage in H9C2 cells and its main active components. The antioxidant capacity through DPPH (1, 1-Diphenyl-2-Picrylhydrazyl), ABTS+• [2,2,2'-azino-BIS-(3-ethylbenzo-thiazoline-6-sulfonic acid)] radical ion, •OH (hydroxyl radical), and (superoxide anion) were determined in vitro. The proliferation of H9C2 cells was examined by MTT [3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-Tetrazolium bromide]. MDA (malondialdehyde), SOD (superoxide dismutase), CAT (catalase), GSH (glutathione), and GSH-Px (glutathione peroxidase) were determined by colorimetry. Apoptosis induced by oxidative damage was detected by flow cytometry. The mRNA expression of antioxidant related genes of SOD, CAT, GSH, and GSH-Px were checked by qRT-PCR (quantitative real-time polymerase chain reaction). The MDLE main active components were analyzed by HPLC (high-performance liquid chromatography). MDLE had significant scavenging effects on DPPH, ABTS+•, •OH, and superoxide anion radicals in a concentration-dependent manner. H2O2 treatment could significantly lead to oxidative stress injury of H9C2 cells, and MDLE treatment significantly improved the degree of H9C2 cell damage, and showed a positive correlation with concentration. MDLE can also reduce apoptosis caused by oxidative damage. MDLE treatment could significantly reduce MDA content and increase CAT, SOD, GSH, and GSH-Px contents and expression. In addition, by HPLC analysis, the following six bioactive components were detected from MDLE: chlorogenic acid, isoquercitrin, quercetin, baicalin, and phloretin. Therefore, MDLE has a good protective effect on myocardial cells.

H2S regulation of ferroptosis attenuates sepsis‑induced cardiomyopathy.

Ferroptosis is a non-apoptotic form of cell death mediated by reactive oxygen species (ROS). Iron metabolism disorders play a key role in sepsis-induced cardiomyopathy (SIC). While hydrogen sulfide (H2S) inhibits SIC, it is unknown if it does so by controlling ferroptosis. The present study evaluated whether sodium hydrosulfide (NaHS), an H2S donor, alleviates SIC by decreasing ferroptosis. Lipopolysaccharide (LPS) was employed to induce an in vitro model of septic myocardial injury in rat H9c2 cardiomyocytes. The myocardial injury model of septic rats was established by cecal ligation and puncture (CLP). Cardiomyocyte injury was evaluated using Cell Counting Kit-8 and myocardial enzyme assay and hematoxylin and eosin (H&E) staining. The cardiac function of rats was assessed using echocardiography and changes in myocardial fibers and mitochondria were evaluated using H&E staining and transmission electron microscopy, respectively. Fe2+, glutathione and malondialdehyde levels in cardiomyocytes were detected using assay kits, ROS and mitochondrial membrane potential changes were detected using fluorescent probes and ferroptosis and Beclin 1 (BECN1) signaling pathway-associated protein expression levels were semi-quantified using western blotting. NaHS decreased ferroptosis of H9c2 cells induced by LPS and decreased injury of myocardial cells by improving iron metabolism disorder and oxidative stress levels. Furthermore, in vivo results demonstrated that NaHS attenuated CLP-induced septic myocardial ferroptosis and significantly improved cardiac dysfunction in septic rats compared with the CLP group. NaHS was demonstrated to attenuate sepsis-induced myocardial cell and tissue injury by significantly inhibiting the phosphorylation of BECN1 and significantly increasing expression levels of the ferroptosis regulatory proteins solute carrier family 7 member 11 and glutathione peroxidase 4. The results of the present study suggested that by regulating the BECN1 signaling pathway, NaHS may decrease the incidence of myocardial ferroptosis, thereby improving SIC.

Cobinamide is a strong and versatile antioxidant that overcomes oxidative stress in cells, flies, and diabetic mice.

Increased oxidative stress underlies a variety of diseases, including diabetes. Here, we show that the cobalamin/vitamin B12 analog cobinamide is a strong and multifaceted antioxidant, neutralizing superoxide, hydrogen peroxide, and peroxynitrite, with apparent rate constants of 1.9 × 108, 3.7 × 104, and 6.3 × 106 M−1 s−1, respectively, for cobinamide with the cobalt in the +2 oxidation state. Cobinamide with the cobalt in the +3 oxidation state yielded apparent rate constants of 1.1 × 108 and 8.0 × 102 M−1 s−1 for superoxide and hydrogen peroxide, respectively. In mammalian cells and Drosophila melanogaster, cobinamide outperformed cobalamin and two well-known antioxidants, imisopasem manganese and manganese(III)tetrakis(4-benzoic acid)porphyrin, in reducing oxidative stress as evidenced by: (i) decreased mitochondrial superoxide and return of the mitochondrial membrane potential in rotenone- and antimycin A-exposed H9c2 rat cardiomyocytes; (ii) reduced JNK phosphorylation in hydrogen-peroxide-treated H9c2 cells; (iii) increased growth in paraquat-exposed COS-7 fibroblasts; and (iv) improved survival in paraquat-treated flies. In diabetic mice, cobinamide administered in the animals’ drinking water completely prevented an increase in lipid and protein oxidation, DNA damage, and fibrosis in the heart. Cobinamide is a promising new antioxidant that has potential use in diseases with heightened oxidative stress.

Abstract P095: Arginine Vasopressin Infusion Is Sufficient To Cause Pregnancy-specific Cardiac Hypertrophy

Preeclampsia is a prevalent gestational disorder characterized by hypertension, proteinuria, and renal damage. Mothers with a history of preeclampsia (PE) are at increased risk of future cardiovascular disease (CVD). Yet, the mechanism by which preeclampsia predisposes patients to future CVD is unknown. Previously published data demonstrated that arginine vasopressin (AVP) infusion in mice recapitulates clinical features of PE including pregnancy-specific hypertension. We hypothesized that chronic AVP infusion in pregnant mice would cause cardiac hypertrophy. C57Bl6/J mice were implanted with an osmotic minipump to deliver 24ng/hr of AVP throughout gestation until necropsy on gestational day 18. Blood pressures were monitored by tail cuff plethysmography and showed no increase in mean arterial pressure (MAP) in nonpregnant mice. However in pregnant mice, an increase of 18.8 ± 7.4 mmHg (82.1 vs 100.8, p=0.05) in MAP was observed in AVP-infused mice compared to saline-infused pregnant mice. Similarly, there was pregnancy-specific 13.4 ± 0.9 mg (93.6 vs 106.9, p=0.0001) increase in the heart weight of AVP-infused mice. Plasma levels of NT-ProBNP were measured by ELISA and showed a significant 163.6 ± 61.00 pg/ml (426.0 vs 589.6, p=0.01) increase in pregnant AVP-infused mice. Human 3rd trimester maternal plasma samples were obtained from the Iowa Maternal Fetal Tissue Bank (IRB# 200910784) for a case control study. In plasma samples from preeclamptic patients there was a significant 264.6 ± 79.72 pg/ml (185.1 vs 449.8, p=0.001) and a 27.9 ± 11.5 pg/ml (64.6, n=32 vs 92.5, n=66, p=0.02) increase in NT-ProBNP and MR-proANP respectively compared to normal pregnancy plasma. H9C2 rat cardiomyocytes treated with pooled plasma from preeclamptic patients showed a 6.6 and 1.8 fold increase in rat ANP and BNP expression respectively. These data suggest that AVP infusion in mice specifically during pregnancy is sufficient to cause cardiac hypertrophy beyond typical hypertrophy seen in a normal pregnancy and that unknown circulating factors in preeclamptics may contribute to the development of hypertrophy. Importantly, these data support using AVP-infusion as a novel mouse model of pregnancy-specific cardiac hypertrophy to study postpartum cardiomyopathies.