Oxidative Stress In Cardiomyocytes Research Articles

Abstract 12931: Myocardial Regeneration in End-stage Heart Failure After Left Ventricular Unloading

Introduction: Left Ventricular Assist Devices (LVAD) decrease cardiac mechanical work and metabolic demand and may decrease cardiomyocyte oxidative stress, followed by cell cycle re-entry and cell regeneration. We tested whether LVAD long-term support is associated with myocardial regeneration. Methods: Viability fluorodeoxyglucose (18FDG) - positron-emission tomography scans were performed >3 months after LVAD implantation and every 6 months thereafter. 4 patients aged 18-70 with end-stage non-ischemic (NICM, n=3) or ischemic cardiomyopathy (ICM, n=1) were included. Low metabolic activity myocardial regions of interest (ROI) at <15th percentile of relative 18FDG signal intensity were identified on the baseline scan. Global signal and co-registered low-intensity ROIs were analyzed on subsequent scans vs. baseline. Results: LV ejection fraction ranged 0-35% at baseline. Two patients (LVAD speed 2660 rotations per minute - RPM, output 3.4-4.1 and 5.6-5.9 liters per minute (LPM), respectively) underwent 2 scans before cardiac transplantation, exhibiting borderline increased global signal (1-4%) and significantly increased 18FDG signal in the baseline low-intensity ROI on the follow-up scan (67.7, 75.7%). The other 2 patients (LVAD parameters: 5600 RPM/ 4.6-5.8 LPM; 9200 RPM/ 4.3-4.8 LPM) exhibited decreased global 18FDG signal (10-16%) and increased signal in the co-registered baseline low-intensity ROIs (1.7-21.5%) (Figure). Conclusions: LVAD long-term support is associated with a global decrease in myocardial 18FDG uptake signal over time, and with increased viability signal in baseline low-intensity ROIs, suggestive of myocardial regeneration in metabolically inactive regions. Future research is required to identify patient subgroups that are most likely to exhibit high regeneration potential upon long-term LVAD support.

Melphalan induces cardiotoxicity through oxidative stress in cardiomyocytes derived from human induced pluripotent stem cells

BackgroundTreatment-induced cardiotoxicity is a leading noncancer-related cause of acute and late onset morbidity and mortality in cancer patients on antineoplastic drugs such as melphalan—increasing clinical case reports have documented that it could induce cardiotoxicity including severe arrhythmias and heart failure. As the mechanism by which melphalan impairs cardiac cells remains poorly understood, here, we aimed to use cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) to investigate the cellular and molecular mechanisms of melphalan-induced cardiotoxicity.MethodshiPSC-CMs were generated and treated with clinically relevant doses of melphalan. To characterize melphalan-induced cardiotoxicity, cell viability and apoptosis were quantified at various treatment durations. Ca2+ transient and contractility analyses were used to examine the alterations of hiPSC-CM function. Proteomic analysis, reactive oxygen species detection, and RNA-Sequencing were conducted to investigate underlying mechanisms.ResultsMelphalan treatment of hiPSC-CMs induced oxidative stress, caused Ca2+ handling defects and dysfunctional contractility, altered global transcriptomic and proteomic profiles, and resulted in apoptosis and cell death. The antioxidant N-acetyl-l-cysteine attenuated these genomic, cellular, and functional alterations. In addition, several other signaling pathways including the p53 and transforming growth factor-β signaling pathways were also implicated in melphalan-induced cardiotoxicity according to the proteomic and transcriptomic analyses.ConclusionsMelphalan induces cardiotoxicity through the oxidative stress pathway. This study provides a unique resource of the global transcriptomic and proteomic datasets for melphalan-induced cardiotoxicity and can potentially open up new clinical mechanism-based targets to prevent and treat melphalan-induced cardiotoxicity.

Downregulation of FOXO6 alleviates hypoxia-induced apoptosis and oxidative stress in cardiomyocytes by enhancing Nrf2 activation via upregulation of SIRT6.

Forkhead box protein O6 (FOXO6) has been recently identified as a novel regulator of oxidative stress in multiple pathological processes. However, whether FOXO6 participates in the regulation of oxidative stress of myocardial infarction is unclear. The present study was performed to evaluate the potential role of FOXO6 in regulating hypoxia-induced apoptosis and oxidative stress in cardiomyocytes in vitro. Our results demonstrated that FOXO6 expression was highly elevated in cardiomyocytes exposed to hypoxia. Downregulation of FOXO6 expression by the siRNA-mediated gene knockdown in hypoxia-exposed cardiomyocytes increased cell viability, while repressing apoptosis and reactive oxygen species (ROS) production. In contrast, overexpression of FOXO6 enhanced the sensitivity of cardiomyocytes to hypoxia-induced injury. Further, in-depth research revealed that knockdown of FOXO6 promoted the expression of sirtuin6 (SIRT6) and enhanced the activation of nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant signaling. Moreover, SIRT6 inhibition markedly blocked the FOXO6 knockdown-induced promotion effect on Nrf2 activation. In addition, Nrf2 inhibition partially reversed the FOXO6 knockdown-mediated protective effect against hypoxia-induced cardiomyocyte injury. Taken together, the findings of our study demonstrate that knockdown of FOXO6 is capable of protecting cardiomyocytes from hypoxia-induced apoptosis and oxidative stress by enhancing Nrf2 activation via upregulation of SIRT6. Our study highlights a potential role of FOXO6 in myocardial infarction and suggests it as an attractive target for cardioprotection.

Stratified Treatment of Heart Failure with preserved Ejection Fraction: rationale and design of the STADIA-HFpEF trial.

AimsHigh myocardial stiffness in heart failure with preserved ejection fraction (HFpEF) is attributed to comorbidity‐induced structural and functional remodelling through inflammation and oxidative stress affecting coronary microvascular endothelial cells and cardiomyocytes, which augments interstitial fibrosis and cardiomyocyte stiffness. In murine and human HFpEF myocardium, sodium glucose co‐transporter 2 (SGLT2) inhibition ameliorates cardiac microvascular endothelial cell and cardiomyocyte oxidative stress, while enhancing myocardial protein kinase G activity and lowering titin‐based cardiomyocyte stiffness. Failure of previous HFpEF outcome trials refocuses attention to improving pathophysiological insight and trial design with better phenotyping of patients and matching of therapeutic targets to prevailing pathogenetic mechanisms. SGLT2 inhibition could represent a viable therapeutic option especially in HFpEF patients in whom high diastolic left ventricular (LV) stiffness is predominantly caused by elevated cardiomyocyte stiffness and associated endothelial dysfunction, whereas HFpEF patients with extensive myocardial fibrosis might be less responsive. This study aims to investigate a stratified treatment approach, using dapagliflozin in heart failure patients with preserved ejection fraction without evidence of significant myocardial fibrosis.Methods and resultsThe Stratified Treatment to Ameliorate DIAstolic left ventricular stiffness in early Heart Failure with preserved Ejection Fraction (STADIA‐HFpEF) is a Phase II, randomized, 2 × 2 crossover trial, evaluating the efficacy of 13 weeks of treatment with dapagliflozin 10 mg od in 26 patients with HFpEF, with normal cardiac magnetic resonance imaging‐derived extracellular volume. The co‐primary endpoint is echocardiographically derived change in E/e'/LV end‐diastolic volume index and change in mean LV e'.ConclusionsThe STADIA‐HFpEF trial will be the first study to evaluate the direct effects of dapagliflozin on amelioration of LV stiffness, using histological phenotyping to discern early HFpEF.

Parkin overexpression reduces inflammation-mediated cardiomyocyte apoptosis through activating Nrf2/ARE signaling pathway

Inflammation has been acknowledged as one of the pathological alterations in various cardiovascular disorders. Parkin has been found to be associated with mitochondrial protection. In the present study, we explored the influence of Parkin overexpression on cardiomyocyte induced by LPS-mediated inflammation response. Our results demonstrated that cardiomyocyte viability was reduced and apoptotic rate was increased upon LPS treatment, an effect that may be caused by cardiomyocyte oxidative stress. At the molecular levels, LPS treatment promoted ROS production, a result that was followed by a drop in the levels of anti-oxidants. Interestingly, Parkin overexpression significantly promoted cardiomyocyte survival and this cardioprotective was attributable to the anti-oxidative property. Parkin overexpression enhanced the expression of anti-oxidative factors such as GSH, SOD and GPX, resulting into depressed ROS production. Further, we found that Parkin modulated cellular anti-oxidative capacity through the Nrf2/ARE signaling pathway. This finding demonstrates that oxidative stress could be considered as the core of inflammation response. Further, therapeutic approaches targeting Parkin would improve cardiomyocyte anti-oxidative capacity through activating Nrf2/ARE signaling pathway.

FULLEROL alleviates myocardial ischemia-reperfusion injury by reducing inflammation and oxidative stress in cardiomyocytes via activating the Nrf2/HO-1 signaling pathway.

Myocardial ischemia-reperfusion injury (MIRI) is myocardial tissue injury caused by blood supply returning to myocardial tissue after ischemia or hypoxia. The purpose of this study was to investigate the protective effect of FULLEROL on myocardial tissue in MIRI rats and its mechanism. We use rats and ligate their left anterior descending coronary artery to make a MIRI model, and we also subcutaneously injected some MIRI rats with FULLEROL daily for two weeks before modeling. We determined the effects of model building and the therapeutic effect of FULLEROL on MIRI by detecting the changes of myocardial tissue morphology, myocardial injury markers and cardiac function in rats. In addition, we detected the expression changes of inflammatory factors and antioxidative molecules in rat myocardial tissue and serum to determine the effect of FULLEROL on inflammation and oxidative stress in myocardial tissue. Finally, we detected the activity of the Nrf2/HO-1 signaling pathway in rat cardiomyocytes to determine the mechanism of action of FULLEROL. The structure of myocardial tissue in MIRI rats was remarkably damaged, and the range of myocardial infarction was increased. In addition, the concentrations of creatine kinase and lactate dehydrogenase were increased, and the heart function was reduced, while FULLEROL could reverse these conditions. In addition, FULLEROL was found to reduce the concentration of the inflammatory factors in rat myocardial tissue and serum, and to increase the expression of antioxidative molecules in myocardial tissue. The Nrf2/HO-1 signaling pathway was found to be related to MIRI and FULLEROL could increase the activity of the Nrf2/HO-1 signaling pathway in cardiomyocytes. FULLEROL can alleviate MIRI by promoting the activity of the Nrf2/HO-1 signaling pathway to reduce the expression of inflammatory factors in rats and increase the antioxidative capacity of cardiomyocytes.

AMPKα2 Overexpression Reduces Cardiomyocyte Ischemia-Reperfusion Injury Through Normalization of Mitochondrial Dynamics.

Cardiac ischemia-reperfusion (I/R) injury is associated with mitochondrial dysfunction. Recent studies have reported that mitochondrial function is determined by mitochondrial dynamics. Here, we hypothesized that AMPKα2 functions as an upstream mediator that sustains mitochondrial dynamics in cardiac I/R injury and cardiomyocyte hypoxia-reoxygenation (H/R) in vitro. To test this, we analyzed cardiomyocyte viability and survival along with mitochondrial dynamics and function using western blots, qPCR, immunofluorescence, and ELISA. Our results indicated that both AMPKα2 transcription and translation were reduced by H/R injury in cardiomyocytes. Decreased AMPKα2 levels were associated with cardiomyocyte dysfunction and apoptosis. Adenovirus-mediated AMPKα2 overexpression dramatically inhibited H/R-mediated cardiomyocyte damage, possibly by increasing mitochondrial membrane potential, inhibiting cardiomyocyte oxidative stress, attenuating intracellular calcium overload, and inhibiting mitochondrial apoptosis. At the molecular level, AMPKα2 overexpression alleviated abnormal mitochondrial division and improved mitochondrial fusion through activation of the Sirt3/PGC1α pathway. This suggests AMPKα2 contributes to maintaining normal mitochondrial dynamics. Indeed, induction of mitochondrial dynamics disorder abolished the cardioprotective effects afforded by AMPKα2 overexpression. Thus, cardiac I/R-related mitochondrial dynamics disorder can be reversed by AMPKα2 overexpression in a manner dependent on the activation of Sirt3/PGC1α signaling.

Irisin ameliorates high glucose‐induced cardiomyocytes injury via AMPK/mTOR signal pathway

High glucose (HG)-induced cardiomyocytes (CMs) injury is a leading cause of diabetic cardiomyopathy with little treatment options. Irisin, a new myokine, which is cleaved from its precursor fibronectin type III domain-containing protein 5 (FNDC5), has aroused great attention as an essential cardioprotective factor and glucose metabolism regulator but little was known on diabetic cardiomyopathy yet. Here, we aim to clarify the role of irisin in the HG-induced CMs injury. Neonatal Sprague-Dawley rat CMs were cultured in a normal or HG medium for 12, 24, and 48 hr, respectively before exposing to irisin. The apoptosis level was determined by terminal-deoxynucleotidyl transferase-mediated-dUTP nick end-labeling assay. Cell viability was measured with the conventional methyl thiazolyl tetrazolium assay. Moreover, reactive oxygen species production was evaluated by dihydroethidium staining. Inflammatory factors, namely tumor necrosis factor-α, interleukin-6, interleukin-1β were determined by enzyme-linked immunosorbent assay kits. Furthermore, protein and messenger RNA (mRNA) expressions were measured by western blot and quantitative real-time polymerase chain reaction, respectively. HG increases the apoptosis of CMs and activated the inflammatory responses and oxidative stress in CMs. Meanwhile, the mRNA and protein expressions of FNDC5 are decreased after HG exposure. Nevertheless, the increased apoptosis is alleviated by irisin treatment. Notably, irisin suppresses the inflammatory responses and oxidative stress in injured CMs. Mechanically, after the administration of Compound C, AMP-activated protein kinase (AMPK) inhibitor, these cardioprotective effects resulting from irisin are reversed. Irisin plays a significant role in antiapoptosis, anti-inflammation, antioxidative stress in HG-induced CMs via AMPK/mammalian target of the rapamycin signaling pathway.

GSKIP protects cardiomyocytes from hypoxia/reoxygenation-induced injury by enhancing Nrf2 activation via GSK-3β inhibition

Glycogen synthase kinase (GSK)-3β interaction protein (GSKIP), a key regulator of signaling transduction, is implicated in multiple pathological processes. However, whether GSKIP is involved in myocardial infarction is unknown. The present study was designed to determine the potential involvement of GSKIP in myocardial hypoxia/reoxygenation (H/R) injury, as an in vitro model for the study of myocardial infarction. Our data showed that H/R treatment triggered a marked decrease in GSKIP expression in cardiomyocytes. The upregulation of GSKIP significantly rescued the decreased viability of H/R-exposed cardiomyocytes and attenuated H/R-induced apoptosis and reactive oxygen species (ROS) generation. On the contrary, the depletion of GSKIP enhanced the sensitivity of cardiomyocytes to H/R-induced injury. Further data exhibited that GSKIP overexpression upregulated the nuclear expression of nuclear factor-erythroid-derived 2-related factor 2 (Nrf2) and increased Nrf2/antioxidant response element (ARE)-mediated transcription activity associated with upregulation of GSK-3β phosphorylation. Interestingly, inhibition of GSK-3β by a chemical inhibitor markedly enhanced Nrf2/ARE activation and abrogated GSKIP depletion-exacerbated sensitivity to H/R-induced injury. In addition, Nrf2 inhibition markedly reversed GSKIP overexpression-induced cardioprotective effect against H/R injury. Overall, these results demonstrate that overexpression of GSKIP alleviates H/R-induced apoptosis and oxidative stress in cardiomyocytes by enhancing Nrf2/ARE antioxidant signaling via GSK-3β inhibition. Our study indicates a potential role of GSKIP in myocardial infarction and GSKIP may serve as a promising molecular target for cardioprotection.

Resolvin E1 protects against doxorubicin-induced cardiotoxicity by inhibiting oxidative stress, autophagy and apoptosis by targeting AKT/mTOR signaling

Doxorubicin (DOX)-induced cardiotoxicity impairs the quality of life of cancer patients during or after DOX treatment, and it is imperative to explore a novel strategy to address this problem. Resolvin E1 (RvE1) is derived from eicosapentaenoic acid (EPA), which has been reported to exert beneficial effects on DOX-induced oxidative stress in cardiomyocytes. This study was designed to investigate whether RvE1 protects against DOX-induced cardiotoxicity, and the underlying mechanism was explored. DOX (20 mg/kg, one injection, i.p.) was used to induce DOX-induced cardiotoxicity in C57BL/6 mice. At 5 days after DOX administration, the effect of RvE1 was assessed by measuring cardiac function, oxidative stress, autophagy and apoptosis in cardiac tissue. We used an AKT inhibitor and rapamycin to investigate the underlying mechanisms. Our results showed that RvE1 inhibited the DOX-induced decrease in body weight and heart weight, the reduction in left ventricular ejection fraction and fractional shortening, and the increase in lactate dehydrogenase, creatine kinase myocardial bound and cardiomyocyte vacuolization. Compared to the control group, the DOX group exhibited increased oxidative stress, autophagy and apoptosis in cardiac tissue, which were alleviated by treatment with RvE1. The AKT/mTOR signaling pathways were responsible for RvE1-mediated regulation of DOX-induced oxidative stress, autophagy and myocardial apoptosis. In conclusion, RvE1 protected against DOX-induced cardiotoxicity via the regulation of AKT/mTOR signaling.

Modulation of the acute defence reaction by eplerenone prevents cardiac disease progression in viral myocarditis.

AimsLeft ventricular (LV) dysfunction in viral myocarditis is attributed to myocardial inflammation and fibrosis, inducing acute and long‐time cardiac damage. Interventions are not established. On the basis of the link between inflammation, fibrosis, aldosterone, and extracellular matrix regulation, we aimed to investigate the effect of an early intervention with the mineralocorticoid receptor antagonist (MRA) eplerenone on cardiac remodelling in a murine model of persistent coxsackievirus B3 (CVB3)‐induced myocarditis.Methods and resultsSWR/J mice were infected with 5 × 104 plaque‐forming units of CVB3 (Nancy strain) and daily treated either with eplerenone (200 mg/kg body weight) or with placebo starting from Day 1. At Day 8 or 28 post infection, mice were haemodynamically characterized and subsequently sacrificed for immunohistological and molecular biology analyses. Eplerenone did not influence CVB3 load. Already at Day 8, 1.8‐fold (P < 0.05), 1.4‐fold (P < 0.05), 3.2‐fold (P < 0.01), and 2.1‐fold (P < 0.001) reduction in LV intercellular adhesion molecule 1 expression, presence of monocytes/macrophages, oxidative stress, and apoptosis, respectively, was observed in eplerenone‐treated vs. untreated CVB3‐infected mice. In vitro, eplerenone led to 1.4‐fold (P < 0.01) and 1.2‐fold (P < 0.01) less CVB3‐induced cardiomyocyte oxidative stress and apoptosis. Furthermore, collagen production was 1.1‐fold (P < 0.05) decreased in cardiac fibroblasts cultured with medium of eplerenone‐treated vs. untreated CVB3‐infected HL‐1 cardiomyocytes. These ameliorations were in vivo translated into prevention of cardiac fibrosis, as shown by 1.4‐fold (P < 0.01) and 2.1‐fold (P < 0.001) lower collagen content in the LV of eplerenone‐treated vs. untreated CVB3‐infected mice at Days 8 and 28, respectively. This resulted in an early and long‐lasting improvement of LV dimension and function, as indicated by reduced LV end‐systolic volume and end‐diastolic volume, and an increase in LV contractility (dP/dtmax) and LV relaxation (dP/dtmin), respectively (P < 0.05).ConclusionsEarly intervention with the MRA eplerenone modulates the acute host and defence reaction and prevents cardiac disease progression in experimental CVB3‐induced myocarditis without aggravation of viral load. The findings advocate for an initiation of therapy of viral myocarditis as early as possible, even before the onset of inflammation‐induced myocardial dysfunction. This may also have implications for coronavirus disease‐19 therapy.

Sinapine, but not sinapic acid, counteracts mitochondrial oxidative stress in cardiomyocytes

IntroductionWhen confronted to stress or pathological conditions, the mitochondria overproduce reactive species that participate in the cellular dysfunction. These organelles are however difficult to target with antioxidants. A feature of mitochondria that can be used for this is the negatively charged compartments they form. Most of mitochondrion-targeting antioxidants are therefore cationic synthetic molecules. Our hypothesis is that such mitochondriotropic traits might also exists in natural molecules. AimWe tested here whether sinapine, a natural phenolic antioxidant-bearing a permanent positive charge, can target mitochondria to modulate mitochondrial oxidative stress. MethodsExperiments were performed in-vitro, in-cellulo, ex-vivo, and in-vivo, using cardiac tissue. The sinapic acid -lacking the positively-charged-choline-moiety present in sinapine-was used as a control. Sinapine entry into mitochondria was investigated in-vivo and in cardiomyocytes. We used fluorescent probes to detect cytosolic (H2DCFDA) and mitochondrial (DHR123) oxidative stress on cardiomyocytes induced with either hydrogen peroxide (H2O2) or antimycin A, respectively. Finally, ROS production was measured with DHE 10 min after ischemia-reperfusion (IR) on isolated heart, treated or not with sinapine, sinapic acid or with a known synthetic mitochondrion-targeted antioxidant (mitoTempo). ResultsWe detected the presence of sinapine within mitochondria in-vitro, after incubation of isolated cardiomyocytes, and in-vivo, after oral treatment. The presence of sinapic acid was not detected in the mitochondria. Both the sinapine and the sinapic acid limited cytosolic oxidative stress in response to H2O2. Only sinapine was able to blunt oxidative stress resulting from antimycin A-induced mtROS. Both mitoTempo and sinapine improved cardiac functional recovery following IR. This was associated with lower ROS production within the cardiac tissue. ConclusionSinapine, a natural cationic hydrophilic phenol, commonly and substantially found in rapeseed species, effectively (i) enters within the mitochondria, (ii) selectively decreases the level of mitochondrial oxidative stress and, (iii) efficiently limits ROS production during cardiac ischemia-reperfusion.

Hypoxia Susceptibility Related to the Expression of Long Noncoding RNA as Regulators of Abraxas2 and Camk2d in the Heart Tissue of a Hypoxic Mouse Model

Long noncoding RNAs (lncRNAs) play a regulatory role in hypoxic disease states, and identifying regulators that control gene expression in a hypoxic mouse model will introduce therapeutic targets and biomarkers of susceptibility to hypoxia. Previous studies show that CD‐1 and C57BL/6J mice are sensitive and resistant to hypoxic stress, respectively.CD‐1 and C57BL/6J mice were subjected to an intraperitoneal injection of SE‐175 (0.7 mg/kg) to induce a hypoxic response or 1x phosphate‐buffered saline (PBS) and then sacrificed after five hours in normoxic condition. RNA was extracted from heart tissue and analyzed in a microarray. Genomic BLAST was performed between the human genome and mice lncRNAs with ±2 fold change in expression (p &lt; 0.05). The expression of several genes coding sequences and their associated lncRNAs were analyzed by qPCR.We identified two lncRNA 1500002F19Rik and LOC108168899 that were found in close proximity to the genes Abraxas2 and Camk2d, respectively. Microarray results suggested that 1500002F19Rik and LOC108168899 may play a role in the expression of Abraxas2 and Camk2d, respectively. The expression levels of Abraxas2, Camk2d, and their corresponding lncRNA were analyzed using qPCR. In mice treated with SE‐175, Abraxas2 and 1500002F19Rik were both upregulated in C57BL/6J mice, but neither displayed a significant change in CD‐1 mice. Camk2d and LOC108168899 were both upregulated in C57BL/6J mice, while Camk2d was upregulated in CD‐1 mice, but LOC108168899 showed no significant change.Because of the cytoprotective effects of Abraxas2 against oxidative stress, its upregulation and that of 1500002F19Rik in C57BL/6J mice provides a possible mechanism for their resistance to hypoxic stress. Camk2d is implicated in oxidative stress in cardiomyocytes and was upregulated to a higher magnitude in CD‐1 mice than in C57BL/6J mice, while LOC108168899 only showed increased expression in C57BL/6J mice. This suggests that LOC108168899 can be involved in downregulation of Camk2d that prevents further oxidative stress in C57BL/6J mice, so its lower expression in CD‐1 mice can lead to higher expression of Camk2d which may contribute to susceptibility to hypoxia.Support or Funding InformationThis project was supported by Touro University Nevada research grant.

Suppressor of ras val-2 promotes inflammation-mediated oxidative stress and cell apoptosis in cardiomyocytes through activating Mst1-mROS signaling pathway

Context: Reperfusion injury after myocardial infarction is associated with inflammation response and oxidative stress.Aim: The aim of our study is to explore the influence of suppressor of ras val-2 (SRV2) on cardiomyocyte oxidative stress and inflammation response under hypoxia-reoxygenation (HR) injury.Methods: Cell viability was determined via MTT assay. qPCR and western blots were used to analyze the alterations of SRV2 and mammalian STE20-like protein kinases 1 (Mst1). ELISA and qPCR were used to verify the alterations of antioxidants and pro-inflammatory factors.Results: SRV2 was upregulated by HR injury in cardiomyocyte. Interestingly, loss of SRV2 attenuated HR injury-mediated cardiomyocyte damage through inhibiting cardiomyocyte apoptosis. At the molecular levels, SRV2 deletion reduced inflammation and oxidative stress induced by HR injury and thus promoted cardiomyocyte survival. Besides, SRV2 deletion inactivated the Mst1-mROS signaling pathway in cardiomyocyte and thus regulated the inflammation and oxidative stress. Interestingly, overexpression of Mst1 abolished the beneficial effects exerted by SRV2 deletion on HR-mediated cardiomyocyte death.Conclusions: SRV2 upregulation, induced by reperfusion injury, contributes to cardiomyocyte death through the Mst1-mROS signaling pathway.

Melatonin receptor protects cardiomyocyte against oxidative stress-induced apoptosis through the MAPK-ERK signaling pathway

Context: The role of melatonin receptor in cardiomyocyte oxidative stress has not been investigated.Aim: The aim of our study is to verify the beneficial effects of melatonin receptors on cardiomyocyte viability under oxidative stress.Methods: Cardiomyocytes were treated with hydrogen peroxide. Cell viability was measured via MTT assay and TUNEL staining. Then, anti-oxidative factors measurement and signaling pathway analysis were performed via qPCR and ELISA.Results: Melatonin receptor activation could attenuate hydrogen peroxide-mediated cardiomyocyte death via reducing apoptosis. At the molecular levels, melatonin receptor activation reduced inflammation response and maintained mitochondrial membrane potential. In addition, melatonin receptor activation is associated with decreased oxidative stress and increased anti-oxidative factors. Finally, we found that melatonin receptor activation triggered an elevation in the activity and expression of ERK pathway and blockade of ERK pathway would abolish the beneficial effects exerted by melatonin receptors activation on cardiomyocyte survival under oxidative stress.Conclusions: Our data suggest that melatonin receptor could attenuate oxidative stress injury in cardiomyocyte through regulation of the ERK signaling pathway.

Baicalein inhibits mitochondrial apoptosis induced by oxidative stress in cardiomyocytes by stabilizing MARCH5 expression.

Abnormal mitochondrial fission and mitophagy participate in the pathogenesis of many cardiovascular diseases. Baicalein is a key active component in the roots of traditional Chinese medicinal herb Scutellaria baicalensis Georgi. It has been reported that baicalein can resist cardiotoxicity induced by several stress, but the mechanisms of baicalein operate in the protection of cardiomyocytes need to be researched further. Here we report that baicalein can promote cell survival under oxidative stress by up‐regulating the expression level of MARCH5 in cardiomyocytes. Pre‐treatment cells or mice with baicalein can stabilize the expression of MARCH5, which plays a crucial role in the regulation of mitochondrial network and mitophagy. Overexpressed MARCH5 is able to against H2O2 and ischaemia/reperfusion (I/R) stress by suppressing mitochondrial fission and enhancing mitophagy, and then attenuate cells apoptosis. Altogether, our present study investigated that baicalein exerts a protective effect through regulating KLF4‐MARCH5‐Drp1 pathway, our research also provided a novel theoretical basis for the clinical application of baicalein.

Pterostilbene Attenuates Fructose-Induced Myocardial Fibrosis by Inhibiting ROS-Driven Pitx2c/miR-15b Pathway.

Excessive fructose consumption induces oxidative stress and myocardial fibrosis. Antioxidant compound pterostilbene has cardioprotective effect in experimental animals. This study is aimed at investigating how fructose drove fibrotic responses via oxidative stress in cardiomyocytes and explored the attenuation mechanisms of pterostilbene. We observed fructose-induced myocardial hypertrophy and fibrosis with ROS overproduction in rats. Paired-like homeodomain 2 (Pitx2c) increase, microRNA-15b (miR-15b) low expression, and p53 phosphorylation (p-p53) upregulation, as well as activation of transforming growth factor-β1 (TGF-β1)/drosophila mothers against DPP homolog (Smads) signaling and connective tissue growth factor (CTGF) induction, were also detected in fructose-fed rat hearts and fructose-exposed rat myocardial cell line H9c2 cells. The results from p53 siRNA or TGF-β1 siRNA transfection showed that TGF-β1-induced upregulation of CTGF expression and p-p53 activated TGF-β1/Smads signaling in fructose-exposed H9c2 cells. Of note, Pitx2c negatively modulated miR-15b expression via binding to the upstream of the miR-15b genetic loci by chromatin immunoprecipitation and transfection analysis with pEX1-Pitx2c plasmid and Pitx2c siRNA, respectively. In H9c2 cells pretreated with ROS scavenger N-acetylcysteine, or transfected with miR-15b mimic and inhibitor, fructose-induced cardiac ROS overload could drive Pitx2c-mediated miR-15b low expression, then cause p-p53-activated TGF-β1/Smads signaling and CTGF induction in myocardial fibrosis. We also found that pterostilbene significantly improved myocardial hypertrophy and fibrosis in fructose-fed rats and fructose-exposed H9c2 cells. Pterostilbene reduced cardiac ROS to block Pitx2c-mediated miR-15b low expression and p-p53-dependent TGF-β1/Smads signaling activation and CTGF induction in high fructose-induced myocardial fibrosis. These results firstly demonstrated that the ROS-driven Pitx2c/miR-15b pathway was required for p-p53-dependent TGF-β1/Smads signaling activation in fructose-induced myocardial fibrosis. Pterostilbene protected against high fructose-induced myocardial fibrosis through the inhibition of Pitx2c/miR-15b pathway to suppress p-p53-activated TGF-β1/Smads signaling, warranting the consideration of Pitx2c/miR-15b pathway as a therapeutic target in myocardial fibrosis.

Hydrogen Sulfide Attenuates High Glucose-induced Myocardial Injury in Rat Cardiomyocytes by Suppressing Wnt/beta-catenin Pathway.

Diabetic cardiomyopathy (DCM) is one of the major heart complications of diabetic patients. Hydrogen sulfide (H2S) is now recognized as an important signaling molecule and has been shown to attenuate the development of diabetic cardiomyopathy. However, the underlying mechanisms linking H2S and the development of DCM have not been fully elucidated. In the present study, we therefore sought to explore the role and mechanism of H2S in the pathogenesis of DCM by establishing high glucose-induced injury model in neonatal rat cardiomyocytes (NRCMs) and H9c2 cells. Using cystathionine gamma-lyase (CSE) overexpression and CSE interference vectors transfection, the cell viability, cell apoptosis. and oxidative stress were determined and compared between the treatment of high glucose induction and exgenous NaHS administration. Meanwhile, the relationship between the CSE/H2S system and Wnt/beta-catenin pathway was analyzed and discussed in the high glucose-induced cardiomyocytes. Our results indicated that H2S played an important protective role in high glucose-induced apoptosis and oxidative stress in cardiomyocytes, as shown by the decreased reactive oxygen species and malondialdehyde levels, and the increased activities of superoxide dismutase, catalase and glutathione peroxidase. Moreover, H2S could attenuate the Wnt/β-catenin signalling pathway and up-regulate the expression of haem oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) in the diabetic myocardium cells. Together, these results demonstrated that H2S could attenuate high glucose-induced myocardial injury in rat cardiomyocytes by suppressing Wnt/β-catenin pathway.

Diastolic dysfunction is initiated by cardiomyocyte impairment ahead of endothelial dysfunction due to increased oxidative stress and inflammation in an experimental prediabetes model

Coronary microvessel endothelial dysfunction and nitric oxide (NO) depletion contribute to elevated passive tension of cardiomyocytes, diastolic dysfunction and predispose the heart to heart failure with preserved ejection fraction. We examined if diastolic dysfunction at the level of the cardiomyocytes precedes coronary endothelial dysfunction in prediabetes. Further, we determined if myofilaments other than titin contribute to impairment. Utilizing synchrotron microangiography we found young prediabetic male rats showed preserved dilator responses to acetylcholine in microvessels. Utilizing synchrotron X-ray diffraction we show that cardiac relaxation and cross-bridge dynamics are impaired by myosin head displacement from actin filaments particularly in the inner myocardium. We reveal that increased PKC activity and mitochondrial oxidative stress in cardiomyocytes contributes to rho-kinase mediated impairment of myosin head extension to actin filaments, depression of soluble guanylyl cyclase/PKG activity and consequently stiffening of titin in prediabetes ahead of coronary endothelial dysfunction.

Aldose reductase inhibitor Epalrestat alleviates high glucose-induced cardiomyocyte apoptosis via ROS.

To clarify the role of aldose reductase inhibitor (ARI) in the high glucose-induced cardiomyocyte apoptosis and its mechanism. In this study, H9c2 cardiomyocytes were employed as objects, high-glucose medium as stimulus, and ARI Epalrestat as a therapeutic drug. The cell apoptosis and activity changes of nitric oxide synthase (NOS), NO, and reactive oxygen species (ROS) were evaluated via Hoechst staining, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and Western blotting. In addition, the mitochondrial membrane potential was measured via fluorescence counting. Epalrestat inhibited the activity of AR to improve high glucose-induced oxidative stress in cardiomyocytes, weaken ROS activity, relieve the inhibition on NO activity, alleviate mitochondrial membrane potential damage, reduce the level of high glucose-induced cardiomyocyte apoptosis, and suppress the expression and activity of Caspase-3, thereby preventing high glucose-induced cardiomyocyte apoptosis. ARI protects against high glucose-induced cardiomyocyte apoptosis.