H9c2 Cardiac Myoblasts Research Articles

PI3K p85α/HIF-1α accelerates the development of pulmonary arterial hypertension by regulating fatty acid uptake and mitophagy.

Pulmonary arterial hypertension (PAH) is characterized by lipid accumulation and mitochondrial dysfunction. This study was designed to investigate the effects of hypoxia-inducible factor-1α (HIF-1α) on fatty acid uptake and mitophagy in PAH. Peripheral blood samples were obtained from PAH patients. Human pulmonary arterial smooth muscle cells and rat cardiac myoblasts H9c2 were subjected to hypoxia treatment. Male Sprague-Dawley rats were treated with monocrotaline (MCT). Right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), pulmonary artery remodeling, and lipid accumulation were measured. Cell proliferation and ROS accumulation were assessed. Mitochondrial damage and autophagosome formation were observed. Co-immunoprecipitation was performed to verify the interaction between HIF-1α and CD36/PI3K p85α. HIF-1α, CD36, Parkin, and PINK1 were upregulated in PAH samples. HIF-1α knockdown or PI3K p85α knockdown restricted the expression of HIF-1α, PI3K p85α, Parkin, PINK1, and CD36, inhibited hPASMC proliferation, promoted H9c2 cell proliferation, reduced ROS accumulation, and suppressed mitophagy. CD36 knockdown showed opposite effects to HIF-1α knockdown, which were reversed by palmitic acid. The HIF-1α activator dimethyloxalylglycine reversed the inhibitory effect of Parkin knockdown on mitophagy. In MCT-induced rats, the HIF-1α antagonist 2-methoxyestradiol (2ME) reduced RVSP, RVHI, pulmonary artery remodeling, lipid accumulation, and mitophagy. Recombinant CD36 abolished the therapeutic effect of 2ME but inhibited mitophagy. Activation of Parkin/PINK1 by salidroside (Sal) promoted mitophagy to ameliorate the pathological features of PAH-like rats, and 2ME further enhanced the therapeutic outcome of Sal. PI3K p85α/HIF-1α induced CD36-mediated fatty acid uptake and Parkin/PINK1-dependent mitophagy to accelerate the progression of experimental PAH.

Enhanced IGF-IIRα Expression Exacerbates Lipopolysaccharide-Induced Cardiac Inflammation, Hypertrophy, and Apoptosis Through Calcineurin Activation.

Cardiovascular disease is one of the leading causes of death worldwide and has a high prevalence. Insulin-like growth factor-II receptor α (IGF-IIRα) acts as a stress-inducible negative regulator. This study focused on the substantial impact of heightened expression of IGF-IIRα in cardiac myoblasts and its association with the exacerbation of cardiac dysfunction. Using lipopolysaccharide (LPS)-induced H9c2 cardiac myoblasts as a model for sepsis, we aimed to elucidate the molecular interactions between IGF-IIRα and LPS in exacerbating cardiac injury. Our findings demonstrated a synergistic induction of cardiac inflammation and hypertrophy by LPS stimulation and IGF-IIRα overexpression, leading to decreased cell survival. Excessive calcineurin activity, triggered by this combined condition, was identified as a key factor exacerbating the negative effects on cell survival. Cellular changes such as cell enlargement, disrupted actin filaments, and upregulation of hypertrophy-related and inflammation-related proteins contributed to the overall hypertrophic and inflammatory responses. Overexpression of IGF-IIRα also exacerbated apoptosis induced by LPS in H9c2 cardiac myoblasts. Inhibiting calcineurin in LPS-treated H9c2 cardiac myoblasts with IGF-IIRα overexpression effectively reversed the detrimental effects, reducing cell damage and mitigating apoptosis-related cardiac mechanisms. Our study suggests that under sepsis-like conditions in the heart with IGF-IIRα overexpression, hyperactivation of calcineurin worsens cardiac damage. Suppressing IGF-IIRα and calcineurin expression could be a potential intervention to alleviate the impact of the illness and improve cardiac function.

A Combination of Cardamonin and Doxorubicin Selectively Affect Cell Viability of Melanoma Cells: An In Vitro Study.

Treatment of the most aggressive and deadliest form of skin cancer, the malignant melanoma, still has room for improvement. Its invasive nature and ability to rapidly metastasize and to develop resistance to standard treatment often result in a poor prognosis. While the highly effective standard chemotherapeutic agent doxorubicin (DOX) is widely used in a variety of cancers, systemic side effects still limit therapy. Especially, DOX-induced cardiotoxicity remains a big challenge. In contrast, the natural chalcone cardamonin (CD) has been shown to selectively kill tumor cells. Besides its anti-tumor activity, CD exhibits anti-oxidative, anti-inflammatory and anti-bacterial properties. In this study, we investigated the effect of the combinational treatment of DOX with CD on A375 melanoma cells compared to normal human dermal fibroblasts (NHDF) and rat cardiac myoblasts (H9C2 cells). DOX-induced cytotoxicity was unselective and affected all cell types, especially H9C2 cardiac myoblasts, demonstrating its cardiotoxic effect. In contrast, CD only decreased the cell viability of A375 melanoma cells, without harming normal (healthy) cells. The addition of CD selectively protected human dermal fibroblasts and rat cardiac myoblasts from DOX-induced cytotoxicity. While no apoptosis was induced by the combinational treatment in normal (healthy) cells, an apoptosis-mediated cytotoxicity was demonstrated in A375 melanoma cells. CD exhibited thiol reactivity as it was able to directly interact with N-acetylcysteine (NAC) in a cell-free assay and to induce heme oxygenase-1 (HO-1) in all cell types. And that took place in a reactive oxygen species (ROS)-independent manner. DOX decreased the mitochondrial membrane potential (Δψm) in all cell types, whereas CD selectively decreased mitochondrial respiration, affecting basal respiration, maximal respiration, spare respiratory capacity and ATP production in A375 melanoma cells, but not in healthy cardiac myoblasts. The DOX-induced cytotoxicity seen in melanoma cells was ROS-independent, whereas the cytotoxic effect of CD was associated with CD-induced ROS-formation and/or its thiol reactivity. This study highlights the beneficial properties of the addition of CD to DOX treatment, which might protect patients from DOX-induced cardiotoxicity. Future experiments with other tumor cell lines or a mouse model should substantiate this hypothesis.

Genetic enhancement of mitochondrial Ca2+ buffering capacity prevents apoptotic signaling activation in response to cytosolic Ca2+ elevation

Introduction: Over the past decade, multiple groups have used genetically engineered mitochondrial Ca2+ (mtCa2+)-sensitive biosensors to delineate the role of mtCa2+ uptake in various cell types/tissues. It has been widely shown that inhibition of mtCa2+ influx via the main mtCa2+ channel, mtCa2+ uniporter (MCU), can mitigate increases in free mitochondrial-matrix Ca2+ concentration ([Ca2+]mt) in response to cytosolic Ca2+ elevation, which prevents overproduction of reactive oxygen species, opening of mitochondrial transition pore (mPTP), and activation of apoptotic signaling. However, chronic interruption of the mtCa2+ handling system may be unfavorable for maintaining physiological cellular functions. Inhibiting mtCa2+ influx may secondarily increase cytosolic Ca2+ concentration ([Ca2+]c) in the absence of MCU activity for cytosolic Ca2+ clearance, activating signaling pathways that cause cellular damage. [Ca2+]mt is determined by the balance of mtCa2+ influx, efflux, and Ca2+ buffering capacity at the mitochondrial matrix. However, the molecular identity of mitochondrial Ca2+ buffering machinery is still unknown. Hypothesis: Enhancement of mtCa2+ buffering capacity by overexpressing mtCa2+ biosensors can prevent mtCa2+ overload by chelating excessive free Ca2+ at the mitochondrial matrix and providing anti-apoptotic effects during cytosolic Ca2+ elevation. Methods: HEK293T cells and H9c2 cardiac myoblasts transfected with mtCa2+ biosensors underwent biochemical, cell biological, and physiological assays. In this experiment, we tested Mitycam, a mitochondria-targeted inverse pericam (Kettlewell S, et al, 2009), and its mutant Mitycam-E31Q, a lower Ca2+-binding affnity version of the original Mitycam (Haviland S, et al, 2014). Results: First, we used Western blotting with an anti-GFP antibody to confirm that the original and mutant Mitycams were expressed at a similar protein size in HEK293T cells and H9c2 cells. The expression level of Mitycam-E31Q was significantly higher than the original Mitycam (55.4±12.9% increase, n=3, p<0.05) incresedespite the equal amount of plasmids transfected. Next, we observed that both Mitycams were exclusively localized in mitochondria by confocal microscopy after co-transfection with mitochondrial matrix-targeted DsRed (mt-RFP) in HEK293T cells and H9c2 cells. A decrease in Mitycam fluorescence in response to transient [Ca2+]c elevation was detected in H9c2 cells following treatment with thapsigargin, an inhibitor of sarco/endoplasmic Ca2+-ATPase (SERCA). These changes were abolished when cells were co-transfected with a dominant-negative pore-forming subunit of MCU, MCUb, confirming that Mitycam and Mitycam-E31Q are capable of sensing [Ca2+]mt elevation by mtCa2+ uptake via MCU. Lastly, H9c2 cells were transfected with Mitycams or mitochondrial matrix-targeted GFP (mt-GFP) as a control, and exposed to prolonged [Ca2+]c elevation by ionomycin, a membrane-permeable Ca2+ ionophore. Cells expressing mt-GFP showed apoptotic signaling activation after ionomycin treatment as assessed by the increased the amount of cleaved caspase-3 by Western blotting (40.1 ±9.4% increase after treatment compared to before treatment, n=4, p<0.05). Importantly, both Mitycams significantly inhibited the increase in cleaved caspase-3 after ionomycin stimulation, with the largest inhibition from Mitycam-E31Q (25.0±9.8% and 47.1±10.1% inhibition in Mitycam and Mitycam-E31Q, respectively. n=4). Conclusion: Overexpressing mitochondrial Ca2+ biosensors may serve as additional artificial buffering machinery in the mitochondrial matrix, which can reduce mtCa2+ overload and prevent apoptotic signaling activation in response to [Ca2+]c elevation. NIH R01HL136757 (to J.O.-U.), R01HL160699 (to B.S.J.), P30GM1114750 (Targeted project to J.O.-U.), P30GM110759 (Targeted project to J.O.-U.), U54GM115677 (Targeted project to B.S.J.), and American Heart Association (AHA) 18CDA34110091 (to B.S.J.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Activation of BKα channel provented uremic cardiomyopathy by attenuating oxidative stress

Background: Uremic cardiomyopathy and renal fibrosis contribute to chronic kidney disease (CKD)-induced morbidity and mortality. Our previous study found that activation of the large conductance calcium-activated potassium channels (BK channels) attenuated renal fibrosis in mice (Wang et al, Kid Int, 2021). We hypothesized that upregulation of BK channels would suppress cardiac fibrosis in CKD. Methods: CKD mice were induced by 5/6 nephrectomy. To upregulation of BKa channels, BMS-191011 (10 mg/kg BW) was given by IP injection every day for 6-8 weeks. Single channel recordings were used to analyze the activation of BKa channel. Blood pressure was measured by non-invasive photoplethysmography using the tail-cuff method. Echocardiogram was used to assess cardiac function. Cultured H9C2 cardiac myoblasts were used to detect the impact of BK opener (NS1916 20mM) on fibrosis markers and oxidative stress. The mRNA expression of BKa in heart was assayed by qPCR. The Hydrogen peroxide (H2O2) was tested by Amplex Red Hydrogen Peroxide Kit. DHE (Dihydroethidium) was assayed to detect superoxide. Superoxide Dismutase (SOD), a defense of oxidative enzyme, was measured using colorimetric activity kit. Results: The expression of BKa mRNA and protein were decreased and cardiac fibrosis was increased in the heart of CKD mice. Single channel recordings showed that human uremic serum (User) attenuated the activation of BKa channel. Upregulation of BKa channel by BSM-191011 decreased the CKD-induced increase in systolic and diastolic blood pressure. Echocardiogram showed that LV end-diastolic dimension increased in 5/6Nx mice, but back to normal after BK opener treatment. Ejection fraction also improved by this treatment. Fibronectin and vimentin were also significantly increased in CKD heart, whereas BMS treatment attenuated the amount of both proteins. In cultured cardiac myoblasts, User and TGFβ increased fibronectin and collagen I, which were in a dose-dependent manner. BKa openers (NS1916) limited the User and TGF-induced upregulation of both proteins in H9C2 cells. User also significantly increased H2O2 and superoxide production in cultured H9C2 cells. Activation of BKa significantly abolished this upregulation. In addition, User and TGFβ decrease SOD production, NS1916 blocked this decline in a dose dependent manner. Conclusions: Activation of BK channel in CKD animals attenuates cardiac fibrosis likely through reduction of uremia-induced oxidative stress. This study could provide new approaches for developing therapeutic strategies for treating uremic cardiomyopathy in chronic kidney diseases. the Department of Veteran Affairs MERIT Award 5I01BX000994 to HC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Serelaxin Protects H9c2 Cardiac Myoblasts against Hypoxia and Reoxygenation-Induced Damage through Activation of AMP Kinase/Sirtuin1: Further Insight into the Molecular Mechanisms of the Cardioprotection of This Hormone.

Serelaxin (RLX), namely the human recombinant Relaxin-2 hormone, protects the heart from ischemia/reperfusion (I/R)-induced damage due to its anti-inflammatory, anti-apoptotic and antioxidant properties. RLX acts by binding to its specific RXFP1 receptor whereby it regulates multiple transduction pathways. In this in vitro study, we offer the first evidence for the involvement of the AMP kinase/Sirtuin1 (AMPK/SIRT1) pathway in the protection by RLX against hypoxia/reoxygenation (H/R)-induced damage in H9c2 cells. The treatment of the H/R-exposed cells with RLX (17 nmol L-1) enhanced SIRT1 expression and activity. The inhibition of SIRT1 signaling with EX527 (10 µmol L-1) reduced the beneficial effect of the hormone on mitochondrial efficiency and cell apoptosis. Moreover, RLX upregulated the AMPK pathway, as shown by the increase in the expression of phospho-AMPK-activated protein. Finally, AMPK pathway inhibition by Compound C (10 and 20 μmol L-1) abrogated the increase in SIRT1 expression induced by RLX, thus suggesting the involvement of the AMPK pathway in this effect of RLX. These results strengthen the concept that RLX exerts its cardioprotective effects against H/R-induced injury through multiple pathways which also include AMPK/SIRT1. These new findings support the use of RLX or RLX-derived molecules as a promising therapeutic for those diseases in which I/R and oxidative stress play a pathogenic role.

Kynurenic acid, a key L-tryptophan-derived metabolite, protects the heart from an ischemic damage.

Renal injury induces major changes in plasma and cardiac metabolites. Using a small- animal in vivo model, we sought to identify a key metabolite whose levels are significantly modified following an acute kidney injury (AKI) and to analyze whether this agent could offer cardiac protection once an ischemic event has occurred. Metabolomics profiling of cardiac lysates and plasma samples derived from rats that underwent AKI 1 or 7 days earlier by 5/6 nephrectomy versus sham-operated controls was performed. We detected 26 differential metabolites in both heart and plasma samples at the two selected time points, relative to sham. Out of which, kynurenic acid (kynurenate, KYNA) seemed most relevant. Interestingly, KYNA given at 10 mM concentration significantly rescued the viability of H9C2 cardiac myoblast cells grown under anoxic conditions and largely increased their mitochondrial content and activity as determined by flow cytometry and cell staining with MitoTracker dyes. Moreover, KYNA diluted in the drinking water of animals induced with an acute myocardial infarction, highly enhanced their cardiac recovery according to echocardiography and histopathology. KYNA may represent a key metabolite absorbed by the heart following AKI as part of a compensatory mechanism aiming at preserving the cardiac function. KYNA preserves the in vitro myocyte viability following exposure to anoxia in a mechanism that is mediated, at least in part, by protection of the cardiac mitochondria. A short-term administration of KYNA may be highly beneficial in the treatment of the acute phase of kidney disease in order to attenuate progression to reno-cardiac syndrom and to reduce the ischemic myocardial damage following an ischemic event.

Pimobendan prevents cardiac dysfunction, mitigates cardiac mitochondrial dysfunction, and preserves myocyte ultrastructure in a rat model of mitral regurgitation

BackgroundPimobendan has been proven to delay the onset of congestive heart failure (CHF) in dogs with mitral regurgitation (MR); however, molecular underlying mechanisms have not been fully elucidated. This study aimed to investigate (1) the effects of pimobendan on cardiac function, cardiac mitochondrial quality and morphology, and cardiac ultrastructure in a rat model of chronic MR and (2) the direct effect of pimobendan on intracellular reactive oxygen species (ROS) production in cardiac cells. MR was surgically induced in 20 Sprague-Dawley rats, and sham procedures were performed on 10 rats. Eight weeks post-surgery, the MR rats were randomly divided into two groups: the MR group and the MR + pimobendan group. Pimobendan (0.15 mg/kg) was administered twice a day via oral gavage for 4 weeks, whereas the sham and MR groups received equivalent volumes of drinking water. Echocardiography was performed at baseline (8 weeks post-surgery) and at the end of the study (4 weeks after treatment). At the end of the study protocol, all rats were euthanized, and their hearts were immediately collected, weighed, and used for transmission electron microscopy and mitochondrial quality assessments. To evaluate the role of pimobendan on intracellular ROS production, preventive or scavenging properties were tested with H2O2-induced ROS generation in rat cardiac myoblasts (H9c2).ResultsPimobendan preserved cardiac functions and structure in MR rats. In addition, pimobendan significantly improved mitochondrial quality by attenuating ROS production and depolarization (P < 0.05). The cardiac ultrastructure and mitochondrial morphology were significantly preserved in the MR + pimobendan group. In addition, pimobendan appeared to play as a ROS scavenger, but not as a ROS preventer, in H2O2-induced ROS production in H9c2 cells.ConclusionsPimobendan demonstrated cardioprotective effects on cardiac function and ultrastructure by preserving mitochondrial quality and acted as an ROS scavenger in a rat model of MR.

Abstract P1155: P62 Overexpression Rescues Defective Autophagy Flux In Cardiomyocytes Deficient In P21 Activated Kinase 1

P21-Activated Kinase 1 (PAK1) is a serine-threonine kinase that plays a critical role in cardiomyocyte survival under numerous stressful conditions. However, the underlying mechanisms for PAK1’s cardioprotective effects remain unclear. Autophagy is a lysosome-dependent degradation pathway for eliminating and recycling long-lived proteins and unwanted cellular components, which is essential for cellular homeostasis. In this study, we investigated whether and how PAK1 regulates autophagy in cardiomyocytes. PAK1 was either silenced by siRNA knockdown or overexpressed by adenovirus-mediated gene transfer in H9c2 cardiac myoblast cells. Autophagy flux was determined by measuring LC3-II protein levels or by a fluorescent autophagy reporter with and without the lysosomal protease inhibitors pepstatin A (pepA) and Aloxistatin (E64d). PAK1 downregulation reduced LC3-II levels in the total cell lysates, which were not affected by pepA and E64d, suggesting that knockdown of PAK1 inhibited autophagy flux. Inversely, PAK1 overexpression increased LC3-II levels, which were further elevated by pepA and E64d, suggesting that PAK1 was sufficient to accelerate autophagy flux. Interestingly, the expression of p62, a ubiquitin-binding autophagic adaptor, was concurrently upregulated by PAK1 overexpression or downregulated by PAK1 knockdown, suggesting that p62 may mediates PAK1-dependent autophagy. Indeed, overexpression of p62 restored autophagy flux in the absence of PAK1 (Figure 1). Together, these results suggest that PAK1 is both sufficient and necessary for maintaining normal autophagy function in cardiomyocytes, which is likely mediated by p62.

Indoxyl sulfate induces left ventricular hypertrophy via the AhR-FGF23-FGFR4 signaling pathway.

Patients with chronic kidney disease (CKD) have a high risk of left ventricular hypertrophy (LVH). Fibroblast growth factor 23 (FGF23) and indoxyl sulfate (IS) are associated with LVH in patients with CKD, but the interactions between these molecules remain unknown. We investigated whether IS contributes to LVH associated with FGF23 in cultured cardiomyocytes and CKD mice. In cultured rat cardiac myoblast H9c2 cells incubated with IS, mRNA levels of the LVH markers atrial natriuretic factor, brain natriuretic peptide, and β-myosin heavy chain were significantly upregulated. Levels of mRNA of the polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3), which regulates FGF23 O-glycosylation, and FGF23 were also upregulated in H9c2 cells. Intact FGF23 protein expression and fibroblast growth factor receptor 4 (FGFR4) phosphorylation were increased in cell lysates by IS administration. In C57BL/6J mice with heminephrectomy, IS promoted LVH, whereas the inhibition of FGFR4 significantly reduced heart weight and left ventricular wall thickness in IS-treated groups. While there was no significant difference in serum FGF23 concentrations, cardiac FGF23 protein expression was markedly increased in IS-injected mice. GALNT3, hypoxia-inducible factor 1 alpha, and FGF23 protein expression was induced in H9c2 cells by IS treatment and suppressed by the inhibition of Aryl hydrocarbon receptor which is the receptor for IS. This study suggests that IS increases FGF23 protein expression via an increase in GALNT3 and hypoxia-inducible factor 1 alpha expression, and activates FGF23-FGFR4 signaling in cardiomyocytes, leading to LVH.

Metformin Inhibits Autophagy, Mitophagy and Antagonizes Doxorubicin-Induced Cardiomyocyte Death.

The antidiabetic drug metformin has been shown to reduce cardiac injury under various pathological conditions, including anticancer drug doxorubicin (DOX)-induced cardiotoxicity, which makes metformin a prime candidate for repurposing. However, the mechanisms that mediate the cardioprotective effects of metformin remain highly controversial. In this study, we tested a prevailing hypothesis that metformin activates autophagy/mitophagy to reduce DOX cardiotoxicity. FVB/N mice and H9C2 cardiac myoblasts were treated with metformin, respectively. Autophagy/mitophagy was determined by Western blot analysis of microtubule-associated protein light chain 3, form-II (LC3-II), a well-established marker of autophagic vesicles. Although metformin had minimal effects on basal LC3-II levels, it significantly inhibited the accumulation of LC3-II levels by the lysosomal protease inhibitors pepstatin A and E64d in both total cell lysates and mitochondrial fractions. Also, dual fluorescent autophagy/mitophagy reporters demonstrated that metformin slowed the degradation rate of autophagic cargos or mitochondrial fragments in the lysosomes. These surprising results suggest that metformin inhibits rather than stimulates autophagy/mitophagy, sharply contrasting the popular belief. In addition, metformin diminished DOX-induced autophagy/mitophagy as well as cardiomyocyte death. Together, these results suggest that the cardioprotective effects of metformin against DOX cardiotoxicity may be mediated by its ability to inhibit autophagy and mitophagy, although the underlying molecular mechanisms remain to be determined.

Regulation of mineralocorticoid receptor activation by circadian protein TIMELESS.

The mineralocorticoid receptor (MR) is a ligand-activated transcription factor that regulates cardiorenal physiology and disease. Ligand-dependent MR transactivation involves a conformational change in the MR and recruitment of coregulatory proteins to form a unique DNA-binding complex at the hormone response element in target gene promoters. Differences in the recruitment of coregulatory proteins can promote tissue-, ligand- or gene-specific transcriptional outputs. The goal of this study was to evaluate the circadian protein TIMELESS as a selective regulator of MR transactivation. TIMELESS has an established role in cell cycle regulation and DNA repair. TIMELESS may not be central to mammalian clock function and does not bind DNA; however, RNA and protein levels oscillate over 24 h. Co-expression of TIMELESS down-regulated MR transactivation of an MR-responsive reporter in HEK293 cells, yet enhanced transactivation mediated by other steroid receptors. TIMELESS markedly inhibited MR transactivation of synthetic and native gene promoters and expression of MR target genes in H9c2 cardiac myoblasts. Immunofluorescence showed aldosterone induces colocalisation of TIMELESS and MR, although a direct interaction was not confirmed by coimmunoprecipitation. Potential regulation of circadian clock targets cryptochrome 1 and 2 by TIMELESS was not detected. However, our data suggest that these effects may involve TIMELESS coactivation of oestrogen receptor alpha (ERα). Taken together, these data suggest that TIMELESS may contribute to MR transcriptional outputs via enhancing ERα inhibitory actions on MR transactivation. Given the variable expression of TIMELESS in different cell types, these data offer new opportunities for the development of MR modulators with selective actions.

Ginsenoside Rb1 from Panax notoginseng Suppressed TNF-α-Induced Matrix Metalloproteinase-9 via the Suppression of Double-Strand RNA-Dependent Protein Kinase (PKR)/NF-κB Pathway.

Chronic inflammation is commonly accompanied by the stimulation of matrix metalloproteinases (MMPs) production and the degradation of the extracellular matrix. The overexpression of MMP-9 (Gelatinase B) highly participates in the progression of pathetic cardiac remodeling and liver cancer metastasis. Panax notoginseng (Burkill) F. H. Chen (Sanqi), a widely used traditional Chinese medicinal herb, shows myocardial protective and anti-tumor effects. In this study, we examined the inhibitory effect of different PNG extracts on tumor necrosis factor (TNF)-α-induced MMP-9 expression in cardiac myoblast H9c2 cells. Using a bioassay-guided fractionation scheme, the most active extract was fractionated by silica gel column chromatography and high-performance liquid chromatography until an active compound was obtained. The compound was identified as Ginsenoside Rb1 by nuclear magnetic resonance. Ginsenoside Rb1 inhibited TNF-α-induced MMP-9 production in both H9c2 and liver carcinoma HepG-2 cells. Interestingly, it did not affect the MMP-2 (Gelatinase A) level and the cell proliferation of the two cell lines. The inhibitory effects of Ginsenoside Rb1 may be due to its modulation of double-strand RNA-dependent protein kinase and nuclear factor kappa B signaling pathways. The results reveal the potential use of Ginsenoside Rb1 for the treatment of inflammatory and MMP-9-related cardiac remodeling and metastasis of hepatocellular carcinomas.

Disruption of mitochondrial bioenergetics, calcium retention capacity and cell viability caused by D-2-hydroxyglutaric acid in the heart

Accumulation of D-2-hydroxyglutaric acid (D-2-HG) is the biochemical hallmark of D-2-hydroxyglutaric aciduria type I and, particularly, of D-2-hydroxyglutaric aciduria type II (D2HGA2). D2HGA2 is a metabolic inherited disease caused by gain-of-function mutations in the gene isocitrate dehydrogenase 2. It is clinically characterized by neurological abnormalities and a severe cardiomyopathy whose pathogenesis is still poorly established. The present work investigated the potential cardiotoxicity D-2-HG, by studying its in vitro effects on a large spectrum of bioenergetics parameters in heart of young rats and in cultivated H9c2 cardiac myoblasts. D-2-HG impaired cellular respiration in purified mitochondrial preparations and crude homogenates from heart of young rats, as well as in digitonin-permeabilized H9c2 cells. ATP production and the activities of cytochrome c oxidase (complex IV), alpha-ketoglutarate dehydrogenase, citrate synthase and creatine kinase were also inhibited by D-2-HG, whereas the activities of complexes I, II and II-III of the respiratory chain, glutamate, succinate and malate dehydrogenases were not altered. We also found that this organic acid compromised mitochondrial Ca2+ retention capacity in heart mitochondrial preparations and H9c2 myoblasts. Finally, D-2-HG reduced the viability of H9c2 cardiac myoblasts, as determined by the MTT test and by propidium iodide incorporation. Noteworthy, L-2-hydroxyglutaric acid did not change some of these measurements (complex IV and creatine kinase activities) in heart preparations, indicating a selective inhibitory effect of the enantiomer D. In conclusion, it is presumed that D-2-HG-disrupts mitochondrial bioenergetics and Ca2+ retention capacity, which may be involved in the cardiomyopathy commonly observed in D2HGA2.

OR07-4 Regulation of Mineralocorticoid Receptor Transactivation By Circadian Protein Timeless

Abstract The mineralocorticoid receptor (MR) is a ligand activated transcription factor that plays an important role in cardiovascular physiology and disease. MR transactivation is regulated by ligand-dependent conformational change in the MR and recruitment of coregulator binding partners to form a unique DNA-binding complex at the hormone response element in target gene promoters. Differences in the recruitment of coregulatory proteins can promote tissue-, ligand- or gene-specific transcriptional outputs. The goal of this study was to evaluate a candidate MR coregulator, the circadian gene timeless for ligand and cell-selective regulation of MR transactivation. While timeless is not essential for mammalian clock function and does not bind DNA, timeless RNA and protein levels oscillate over 24hr. Co-expression of timeless dose-dependently down-regulated MR transactivation of an MR-responsive reporter in HEK293 cells, yet enhanced transactivation mediated by other steroid receptors. Timeless also inhibited MR transactivation of synthetic and native gene promoters, and expression of endogenous MR target genes, in H9c2 cardiac myoblasts. Immunofluorescence staining of timeless, and MR showed colocalization to the nucleus in the presence of aldosterone and co-immunoprecipitation which suggested an interaction, although a direct interaction was not confirmed. Taken together, these data suggest timeless is a novel MR coregulatory protein that may contribute to time-of-day-dependent MR transcriptional outputs and suggest new opportunities for the development of MR antagonists with selective actions. Presentation: Saturday, June 11, 2022 12:15 p.m. - 12:30 p.m.

Dapagliflozin Mitigates Doxorubicin-Caused Myocardium Damage by Regulating AKT-Mediated Oxidative Stress, Cardiac Remodeling, and Inflammation.

Doxorubicin (Dox) is a commonly used anthracycline chemotherapy with a side effect of cardiotoxicity, which may increase the risk of heart failure for cancer patients. Although various studies have demonstrated the cardioprotective property of dapagliflozin (DAPA), a sodium-glucose cotransporter 2 inhibitor, the detailed mechanism underlying its effect on Dox-induced cardiomyopathy is still limited. In this study, we showed that DAPA induced the activation of AKT/PI3K signaling in cardiac myoblast H9c2 cells following Dox treatment, leading to the upregulation of antioxidant HO-1, NQO1, and SOD, as well as an improved mitochondrial dysfunction via Nrf2. In addition, the reduced oxidative stress resulted in the downregulation of hypertrophy (ANP and BNP) and fibrosis (phospho-Smad3, collagen I, fibronectin, and α-SMA) markers. Furthermore, the inflammatory IL-8 concentration was inhibited after DAPA, possibly through PI3K/AKT/Nrf2/p38/NF-κB signaling. Moreover, our results were validated in vivo, and echocardiography results suggested an improved cardiac function in DAPA-receiving rats. In summary, we demonstrated that the administration of DAPA could mitigate the Dox-elicited cardiotoxicity by reducing oxidative stress, mitochondrial dysfunction, fibrosis, hypertrophy, and inflammation via PI3K/AKT/Nrf2 signaling.

An in‐Depth Analysis of the PAK1 Autophagy Signaling Pathway in H9C2 Cardiomyoblasts

P21‐activated serine/threonine Kinase 1 (PAK1) plays a critical role in cardiomyocyte survival under numerous stressful conditions. Autophagy is a cellular process that promotes homeostasis by removing and replacing antiquated or damaged cellular components. Mitophagy is a form of selective autophagy that eliminates damaged mitochondria to maintain a pool of healthy mitochondria. We have previously demonstrated that PAK1 is essential for maintaining autophagy and mitophagy activities. However, the downstream mediators have not yet been discovered. In this study, we explored the mechanisms of PAK1‐dependent autophagy and mitophagy by determining the protein expression levels of the major regulators of autophagy and mitophagy. H9c2 cardiac myoblasts were treated with siRNA to knockdown the expression of PAK1. Western blot analysis showed that PAK1 knockdown substantially reduced the protein expression levels of ATG5‐12 complex, an essential promotor of autophagosome formation, TFEB, a master regulator of lysosome biogenesis and autophagy, and p62, an autophagy receptor for ubiquitinated cargos. All these changes are expected to reduce autophagy activity. In addition, PAK1 knockdown also reduced the expression of mitophagy receptor FUNDC1 and diminished the association of p62 with mitochondria, which coincided with reduced mitophagy activity. Collectively, these results suggest that the ability of PAK1 knockdown to inhibit autophagy and mitophagy is mediated by reduced expression levels of several important regulators of autophagy and/or mitophagy pathways. Future research is warranted to determine whether restoring the expression levels of these target genes can overcome the inhibition of autophagy and mitophagy by PAK1 deficiency.

Variation in ORF3a Protein of SARS‐CoV‐2 Decreases The Severity of Host Cell Damage

IntroductionSevere acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) primarily targets the respiratory system. However, direct SARS‐CoV‐2 infection and viral protein expression have also been reported in other organs, which potentially contributes to multi‐organ dysfunction and increased mortality in COVID‐19. Since the original Wuhan‐type genome was sequenced, several genetic variants of SARS‐CoV‐2 have emerged with differing pathophysiological properties such as the levels of transmissibility, disease severity, and mortality. We previously reported that the protein encoded by open reading frame 3a (ORF3a), a critical protein for SARS‐CoV‐2 replication and release, is found in the mitochondria of host cells and could increase oxidative stress and apoptotic signaling. ORF3a‐Q57H is a highly recurrent variation and the most commonly found variant of ORF3a. Interestingly, the Q57H variant is associated with increased transmissibility, but lower mortality.AimTo investigate the impact of the ORF3a‐Q57H variant on host cell damage.MethodsPlasmids carrying ORF3a‐Q57H were generated by PCR‐based site mutagenesis using Wuhan‐type ORF3a (ORF3a‐WT) as a template. Whole cell lysates were prepared from HEK293T cells and H9c2 cardiac myoblasts expressing ORF3a‐WT or the mutant ORF3a‐Q57H and used for biochemical assays. Live cell imaging for assessing subcellular localization of ORF3a/ORF3a‐Q57H, mitochondrial reactive oxygen species (mROS), and caspase 3 activity in H9c2 cells was performed by confocal microscopy.ResultsIn whole cell lysates, we found that ORF3a‐Q57H exhibits significantly higher protein expression compared to ORF3a‐WT. However, there is no significant difference in the ability of mitochondrial trafficking between ORF3a‐WT and ORF3a‐Q57H assessed by live cell imaging using GFP‐tagged ORF3a/ORF3a‐Q57H with mitochondria‐targeted DsRed. Next, we investigated the effects of ORF3a expression on apoptotic and mitophagy signaling by quantifying the caspase 3 activity and LC3A/B ratio. We found that ORF3a‐Q57H has significantly lower apoptosis and mitophagy signaling compared to WT despite its higher protein expression levels. ER‐stress signaling was not activated in either ORF3a‐WT or ORF3a‐Q57H, as assessed by markers including Glucose‐regulated protein 78/94 and C/EBP‐homologous protein. Lastly, live cell imaging using the mitochondrial superoxide‐sensitive dye Mitosox Red revealed that ORF3a‐WT significantly increases mROS levels, but ORF3a‐Q57H expression does not.ConclusionSARS‐CoV‐2‐ORF3a‐Q57H causes lower oxidative stress and cell damage compared to the Wuhan‐type variant, which could help explain the decreased mortality associated with the ORF3a‐Q57H variant. These results provide novel insights on how genetic variations of SARS‐CoV‐2 influence the pathophysiology and clinical severity of COVID‐19.

Mitochondrial Toxicity Associated with Imatinib and Sorafenib in Isolated Rat Heart Fibers and the Cardiomyoblast H9c2 Cell Line.

Tyrosine kinase inhibitors (TKIs) are associated with cardiac toxicity, which may be caused by mitochondrial toxicity. The underlying mechanisms are currently unclear and require further investigation. In the present study, we aimed to investigate in more detail the role of the enzyme complexes of the electron transfer system (ETS), mitochondrial oxidative stress, and mechanisms of cell death in cardiac toxicity associated with imatinib and sorafenib. Cardiac myoblast H9c2 cells were exposed to imatinib and sorafenib (1 to 100 µM) for 24 h. Permeabilized rat cardiac fibers were treated with both drugs for 15 min. H9c2 cells exposed to sorafenib for 24 h showed a higher membrane toxicity and ATP depletion in the presence of galactose (favoring mitochondrial metabolism) compared to glucose (favoring glycolysis) but not when exposed to imatinib. Both TKIs resulted in a higher dissipation of the mitochondrial membrane potential in galactose compared to glucose media. Imatinib inhibited Complex I (CI)- and CIII- linked respiration under both conditions. Sorafenib impaired CI-, CII-, and CIII-linked respiration in H9c2 cells cultured with glucose, whereas it inhibited all ETS complexes with galactose. In permeabilized rat cardiac myofibers, acute exposure to imatinib and sorafenib decreased CI- and CIV-linked respiration in the presence of the drugs. Electron microscopy showed enlarged mitochondria with disorganized cristae. In addition, both TKIs caused mitochondrial superoxide accumulation and decreased the cellular GSH pool. Both TKIs induced caspase 3/7 activation, suggesting apoptosis as a mechanism of cell death. Imatinib and sorafenib impaired the function of cardiac mitochondria in isolated rat cardiac fibers and in H9c2 cells at plasma concentrations reached in humans. Both imatinib and sorafenib impaired the function of enzyme complexes of the ETS, which was associated with mitochondrial ROS accumulation and cell death by apoptosis.

Parkinson's disease protein 7 protected against oxidative stress of myocardial infarction direct through p47phox and nicotinamide adenine dinucleotide phosphate oxidase 4.

In the present study, we aimed to investigate the role and mechanism of Parkinson's disease protein 7 (Park7) in myocardial infarction (MI). The Park7 expression in the serum and tissues was down-regulated in mice with MI. Recombinant Park7 protein protected against MI-induced injury and reduced oxidative stress in mice model. Conversely, knockout Park7 increased injury of MI and promoted oxidative stress in MI mice model. In embryonic rat cardiac myoblasts H9c2 cells, over-expression of Park7 reduced reactive oxygen species (ROS)-induced oxidative stress, while down-regulation of Park7 increased ROS-induced oxidative stress. Park7 combined nicotinamide adenine dinucleotide phosphate (NADPH) oxidase cytoplasmic subunit p47phox protein had direct effect on inducing NADPH activator. The inhibition of p47phox reduced the effects of Park7 in ROS production of H2O2-treated H9c2 cells. The regulation of NADPH participated in the effects of Park7 on ROS production of in both MI mice model and H2O2-treated H9c2 cells. Our data demonstrated that Park7 protects against oxidative stress in MI model direct through p47phox and NADPH oxidase 4.