Reactive Oxygen Species In Cardiomyocytes Research Articles

PKM2 regulates metabolic flux and oxidative stress in the murine heart.

Cardiac metabolism ensures a continuous ATP supply, primarily using fatty acids in a healthy state and favoring glucose in pathological conditions. Pyruvate kinase muscle (PKM) controls the final step of glycolysis, with PKM1 being the main isoform in the heart. PKM2, elevated in various heart diseases, has been suggested to play a protective role in cardiac stress, but its function in basal cardiac metabolism remains unclear. We examined hearts from global PKM2 knockout (PKM2-/-) mice and found reduced intracellular glucose. Isotopic tracing of U-13C glucose revealed a shift to biosynthetic pathways in PKM2-/- cardiomyocytes. Total ATP content was two-thirds lower in PKM2-/- hearts, and functional analysis indicated reduced mitochondrial oxygen consumption. Total reactive oxygen species (ROS) and mitochondrial superoxide were also increased in PKM2-/- cardiomyocytes. Intriguingly, PKM2-/- hearts had preserved ejection fraction compared to controls. Mechanistically, increased calcium/calmodulin-dependent kinase II activity and phospholamban phosphorylation may contribute to higher sarcoendoplasmic reticulum calcium ATPase 2 pump activity in PKM2-/- hearts. Loss of PKM2 led to altered glucose metabolism, diminished mitochondrial function, and increased ROS in cardiomyocytes. These data suggest that cardiac PKM2 acts as an important rheostat to maintain ATP levels while limiting oxidative stress. Although loss of PKM2 did not impair baseline contractility, its absence may make hearts more sensitive to environmental stress or injury.

Development and Application of a Mitochondrial Genetically Encoded Voltage Indicator in Narcosis.

Mitochondrial membrane potential (MMP) plays a crucial role in the function of cells and organelles, involving various cellular physiological processes, including energy production, formation of reactive oxygen species (ROS), unfolded protein stress, and cell survival. Currently, there is a lack of genetically encoded fluorescence indicators (GEVIs) for MMP. In our screening of various GEVIs for their potential monitoring MMP, the Accelerated Sensor of Action Potentials (ASAP) demonstrated optimal performance in targeting mitochondria and sensitivity to depolarization in multiple cell types. However, mitochondrial ASAPs also displayed sensitivity to ROS in cardiomyocytes. Therefore, two ASAP mutants resistant to ROS were generated. A double mutant ASAP3-ST exhibited the highest voltage sensitivity but weaker fluorescence. Overall, four GEVIs capable of targeting mitochondria were obtained and named mitochondrial potential indicators 1-4 (MPI-1-4). In vivo, fiber photometry experiments utilizing MPI-2 revealed a mitochondrial depolarization during isoflurane-induced narcosis in the M2 cortex.

Bardoxolone methyl attenuates doxorubicin-induced cardiotoxicity by inhibiting the TXNIP-NLRP3 pathway through Nrf2 activation.

Bardoxolone methyl, which triggers nuclear factor erythroid 2-related factor (Nrf2), has therapeutic effects against myocardial infarction, heart failure, and other diseases. Nrf2 can inhibit the activation of the thioredoxin-interacting protein (TXNIP)/NLR family pyrin domain-containing protein 3 (NLRP3) pathway. Doxorubicin is an anthracycline chemotherapeutic drug associated with cardiotoxicity, limiting its clinical use. In this study, we explored the specific mechanism of the Nrf2-TXNIP-NLRP3 pathway in doxorubicin-induced cardiotoxicity using bardoxolone methyl in animal and cell models. Using in vivo and in vitro experiments, we show that doxorubicin can induce oxidative stress and pyroptosis in the heart. Western blot and co-immunoprecipitation experimental results found that doxorubicin can reduce the interaction between TXNIP and TRX, increase the interaction between TXNIP and NLRP3, and activate the pyroptosis process. Bardoxolone methyl reduces the accumulation of reactive oxygen species in cardiomyocytes through the Nrf2 pathway, inhibits the interaction between TXNIP and NLRP3, and alleviates the progression of myocardial damage and cardiac fibrosis. Bardoxolone methyl lost its therapeutic effect when the expression of Nrf2 was decreased. Additionally, repressing the expression of TXNIP can inhibit the activation of NLRP3 and alleviate myocardial damage caused by doxorubicin. Collectively, our findings confirm that bardoxolone methyl alleviates doxorubicin-induced cardiotoxicity by activating Nrf2 and inhibiting the TXNIP-NLRP3 pathway.

Echinacoside ameliorates doxorubicin‑induced cardiac injury by regulating GPX4 inhibition‑induced ferroptosis.

Echinacoside (ECH) is a compound derived from the natural herbs Cistanche and Echinacea, which has considerable protective effects on heart failure (HF). HF is characterized by myocardial damage and abnormal ferroptosis. Glutathione peroxidase 4 (GPX4) is an important regulator of ferroptosis, which plays a role in ferroptosis-related diseases. Despite this, the therapeutic mechanisms of ECH against HF remain unknown. Therefore, the aim of the present study was to investigate the cardioprotective effect and underlying mechanisms of ECH in the treatment of doxorubicin (DOX)-induced chronic HF (CHF). Cell proliferation was assessed using a CCK-8 assay. Furthermore, cardiac cell injury and oxidative stress were determined by measuring the lactate dehydrogenase (LDH), malondialdehyde (MDA), and glutathione (GSH) levels. The levels of Fe2+ and lipid reactive oxygen species (ROS), and expression of the biomarkers of ferroptosis, including GPX4 and prostaglandin-endoperoxide synthase 2 (PTGS2), were measured to examine cardiomyocyte ferroptosis. Additionally, RNA interference was used to silence Gpx4. In vitro and in vivo, ECH considerably reduced the MDA and LDH levels and increased the GSH level, thereby attenuating DOX-induced cardiac injury and oxidative stress. Meanwhile, ECH treatment decreased the lipid ROS levels and PTGS2 expression while increasing GPX4 expression, thereby alleviating DOX-induced cardiomyocyte ferroptosis. Moreover, knockdown of Gpx4 inhibited the protective effects of ECH on DOX-induced accumulation of lipid ROS in cardiomyocytes. These findings indicate that ECH can reduce DOX-induced cardiac injury by inhibiting ferroptosis via GPX4, highlighting its value as a potentially valuable therapeutic target in the management of CHF.

Dapagliflozin attenuates high glucose-and hypoxia/reoxygenation-induced injury via activating AMPK/mTOR-OPA1-mediated mitochondrial autophagy in H9c2 cardiomyocytes

This study investigated the protective effect of dapagliflozin on H9c2 cardiomyocyte function under high glucose and hypoxia/reoxygenation (HG-H/R) conditions and identified the underlying molecular mechanisms. Dapagliflozin reduced the level of lactate dehydrogenase and reactive oxygen species in cardiomyocytes under HG-H/R conditions and was accompanied by a decrease in caspase-3/9 activity. In addition, Dapagliflozin significantly reduced mitochondrial permeability transition pore opening and increased ATP content, accompanied by upregulation of OPA1 with autophagy-related protein molecules and activation of the AMPK/mTOR signalling pathway in HG-H/R treated cardiomyocytes. OPA1 knockdown or compound C treatment attenuated the protective effects of dapagliflozin on the cardiomyocytes under HG-H/R conditions. Downregulation of OPA1 expression increased mitochondrial intolerance in cardiomyocytes during HG-H/R injury and the AMPK-mTOR-autophagy signalling is a key mechanism for protecting mitochondrial function and reducing cardiomyocyte apoptosis. Collectively, dapagliflozin exerted protective effects on the cardiomyocytes under HG-H/R conditions. Dapagliflozin attenuated myocardial HG-H/R injury by activating AMPK/mTOR-OPA1-mediated mitochondrial autophagy.

Reduction of intracellular Mg2+ caused by reactive oxygen species in rat ventricular myocytes.

The concentration of intracellular free Mg2+ ([Mg2+]i) should be maintained strictly for the regulation of cellular functions. Since reactive oxygen species (ROS) are liable to increase in various pathological conditions and induce cellular damage, we investigated whether ROS affect intracellular Mg2+ homeostasis. We measured [Mg2+]i in ventricular myocytes from Wistar rats using the fluorescent indicator, mag-fura-2. The administration of hydrogen peroxide (H2O2) decreased [Mg2+]i in Ca2+-free Tyrode's solution. Intracellular free Mg2+ was also reduced by endogenous ROS as generated by pyocyanin, which was inhibited by pretreatment with n-acetyl cysteine (NAC). The rate of change in [Mg2+]i by 500 μM H2O2 in 5 min (on average, -0.61 μM/s) was independent of extracellular Na+, and intra- and extracellular Mg2+ concentrations. When extracellular Ca2+ was present, the rate of Mg2+ decrease was significantly reduced, on average, by ∼60%. The half-maximal effective concentration of H2O2 on the Mg2+ decrease was estimated to be between 400 and 425 μM. The Mg2+ decrease by H2O2 in the absence of Na+ was inhibited by 200 μM imipramine, a known inhibitor of Na+/Mg2+ exchange. We perfused rat hearts with the Ca2+-free Tyrode's solution containing H2O2 (500 μM, 5 min) on the Langendorff apparatus,. H2O2 stimulation increased Mg2+ concentration in the perfusate, suggesting the H2O2-induced decrease in [Mg2+]i was caused by Mg2+ extrusion. Collectively, these results suggest the existence of a Na+-independent Mg2+ efflux system activated by ROS in cardiomyocytes. The lower [Mg2+]i may in part be attributed to ROS-mediated cardiac dysfunction.

An injectable and conductive TEMPOL/polypyrrole integrated peptide co-assembly hydrogel promotes functional maturation of cardiomyocytes for myocardial infarction repair

A cell-implantation strategy offers a promising paradigm for cardiac repair after myocardial infarction (MI). However, the hostile reactive oxygen species (ROS) microenvironment in infarcted zone hinders the survival and maturation of implanted cells and affects the ultimate therapeutic outcome. Besides, the generation of fibrosis scarring deprives the myocardium of electrical communications and induces cardiac dysfunction, eventually resulting in heart failure. Herein, an injectable ROS-scavenging/conductive composite hydrogel (R&C-Gel) was constructed via specific binding between conductive polypyrrole (PPy) and multi-component co-assembly peptides. Integrating antioxidant TEMPOL into the peptide endows it with strong ROS-eliminating ability, and the nanoengineering of polypyrrole (PPy) by wet milling facilitates PPy uniformly binding to the target T59 peptide in nanofibers . The thixotropy of co-assembly hydrogels confirmed by rheology and the macroporous structure of the R&C-Gel surface observed by SEM together suggest that the R&C-Gel could be an ideal vehicle for cell engraftment. The antioxidant ability and electroconductivity of the R&C-Gel were verified. In vitro experiments demonstrated that the R&C-Gel could efficiently remove ROS in cardiomyocytes and reduce apoptosis under oxygen-glucose deprivation (OGD) conditions. The rhythmic intracellular Ca 2+ puffs and expression of Cx-43 protein further proved the R&C-Gel could enhance the electrical and contractile performance of cardiomyocytes. Echocardiography and histological analysis convincingly revealed that the R&C-Gel-combined cardiomyocytes significantly promoted cardiac repair and rebuilt cardiac function, such as reducing apoptosis, accelerating gap junction formation , increasing ejection fraction , and decreasing the fibrosis area. This study provides new insight into the design and construction of multifunctional conductive hydrogels for cardiac tissue engineering . • An injectable composite hydrogel (R&C-Gel) is prepared via specific binding between conductive PPy and multi-component peptides. • Integrating the TEMPOL endows the R&C-Gel with strong ROS-eliminating ability to reduce the apoptosis of CMs under hypoxia. • Nanoengineering of vapor-polymerized PPy membrane to conductive NanoClusters facilitates PPy uniformly binding to nanofibers of R&C-gel. • R&C-Gel promotes rhythmic intracellular Ca 2+ puffs and the expression of Cx-43 protein in the maturation of CMs. • Injection of CMs@R&C-Gel is capable of restoring cardiac function post-MI.

Resveratrol activates PI3K/AKT to reduce myocardial cell apoptosis and mitochondrial oxidative damage caused by myocardial ischemia/reperfusion injury

Resveratrol is a kind of iPolyphenols widely existing in herbal medicine. Here we aim to investigate whether resveratrol can reduce the degree of myocardial ischemia/reperfusion (IR) injury and inhibit the development of oxidative stress, and elucidate the molecular mechanism of resveratrol in protecting myocardial cells. The primary rat cardiomyocytes were used to establish an ischemia/reperfusion model in vitro, and a series of routine biochemical experiments were conducted to explore the antioxidant and anti-apoptotic effects of resveratrol in myocardial ischemia-reperfusion injury. Compared with that of the simulated ischemia-refusion (SIR) group, cell viability in the SIR and resveratrol co-treatment groups increased significantly (P < 0.001), the release of lactate dehydrogenase (LDH) and creatine kinase MB (CKMB) decreased, the positive rate of reactive oxygen species (ROS) in cardiomyocytes decreased, and the concentration of catalase and glutathione peroxidase increased significantly (P < 0.001). Besides, resveratrol can activate PI3K/AKT signaling pathway. PI3K siRNA can inhibit the PI3K/AKT signaling mediated by resveratrol. The addition of resveratrol can significantly increase the activity of mitochondrial superoxide dismutase (SOD) and reduce the malondialdehyde (MDA), which indicates that the oxidative damage of mitochondria induced by resveratrol was significantly weakened. The mitochondrial functional changes induced by resveratrol can be reversed by PI3K siRNA. In conclusion, our study shows that resveratrol can reduce ROS in cardiomyocytes by PI3K/AKT signaling pathway activation, and effectively inhibit the apoptosis of cardiomyocytes, thus having a direct protective effect on cardiomyocytes under SR.

Dexmedetomidine attenuates H2O2-induced apoptosis of rat cardiomyocytes independently of antioxidant enzyme expression

Introduction and ObjectivesDexmedetomidine is a highly selective alpha-2 adrenoceptor agonist that has sedative and analgesic properties and myocardial protective effects. However, the mechanism underlying the protective effect of dexmedetomidine on cardiomyocytes remains unknown. This study mainly aimed to investigate the effects of dexmedetomidine on the generation of reactive oxygen species (ROS) in cardiomyocytes and whether it inhibits the apoptosis of cardiomyocytes by affecting antioxidant enzyme expression. MethodsNeonatal rat cardiomyocytes were pretreated with dexmedetomidine (100 nM) for 24 h. The cardiomyocytes were then incubated with 200 μM hydrogen peroxide solution (H2O2) for 4 h. PCR assay was used to determine the mRNA expression of antioxidant enzymes. Western blot assay was used to determine the protein expression of antioxidant enzymes. Fluorescence microscopy with the MitoSOX probe was used to detect the formation of ROS in cardiomyocytes, and fluorescence-activated cell sorting with annexin V/PI was used to determine the number of apoptotic cardiomyocytes. ResultsDexmedetomidine reduced ROS generation and antioxidant enzymes levels in cardiomyocytes before H2O2 stimulation (p<0.05). However, ROS generation and apoptosis in cardiomyocytes were significantly increased after H2O2 treatment, and dexmedetomidine pretreatment markedly inhibited the changes (p<0.05). ConclusionFor the first time, to the best of our knowledge, our study shows that dexmedetomidine has a protective effect on cardiomyocytes through inhibition of ROS-induced apoptosis, and more importantly, this effect is independent of antioxidant enzyme mRNA and protein expression.

Dapagliflozin suppresses ER stress and protects doxorubicin-induced cardiotoxicity in breast cancer patients.

Cancer patients with diabetes have an increasing risk of Dox-induced cardiotoxicity. Despite previous studies reporting benefits of dapagliflozin on the cardiovascular system, it remains unknown whether dapagliflozin has a cardioprotective effect in cancer patients with diabetes. We aimed to investigate the potential of dapagliflozin for preventing doxorubicin (Dox)-induced cardiotoxicity. Using Taiwan National Health Insurance Database, the incidence of heart failure of cancer patients with or without diabetes was investigated. Streptozotocin (STZ)-induced diabetic rats were pretreated with oral dapagliflozin for 6weeks followed by Dox for 4weeks via intraperitoneal injection. Sequential echocardiography was applied to assess cardiac function. For in vitro analysis, cardiomyocytes cultured in high glucose were treated with dapagliflozin at 10μM and subsequently exposed to Dox at 1μM. Apoptosis and endoplasmic reticulum (ER) stress-related protein expression were measured. Among the studied patients, those with diabetes had a higher risk of major adverse cardiovascular events including the development of heart failure. In diabetic rats, dapagliflozin reduced cardiac fibrosis and significantly improved cardiac function. Dapagliflozin effectively inhibited Dox-induced apoptosis and reactive oxygen species in cardiomyocytes under high glucose. Mechanistically, we showed that dapagliflozin decreased the cardiac expression of Bax and cleaved caspase 3 but increased Bcl-2. Dapagliflozin also significantly reduced ER stress-associated proteins including GRP78, PERK, eIF-2α, ATF-4, and CHOP. Our study revealed for the first time that dapagliflozin mitigated Dox-induced cardiomyocyte apoptosis in diabetes. These results indicate that dapagliflozin could be useful for preventing cardiotoxicity in diabetic cancer patients receiving Dox treatment.

Lotusine, an alkaloid from Nelumbo nucifera (Gaertn.), attenuates doxorubicin-induced toxicity in embryonically derived H9c2 cells.

Cardiotoxicity is the major challenge in chemotherapy with doxorubicin (DOX) or adriamycin. Doxorubicin manifests oxidative stress via an uncontrolled progression of reactive oxygen species in cardiomyocytes; thereby, dysregulation and dysfunction of myocardium thus lead to apoptosis. Several attempts have been made to overcome this side effect in patients with antioxidant-rich supplements to control the free radicals. Plant-based or plant-derived compounds pay more attention to cure such complications in patients for supporting the treatment, revitalizing or regulating the normal metabolism. Hence, our study focused on pretreatment of embryonically derived rat cardiomyocytes (H9c2) with phytocompound lotusine to prevent DOX-mediated oxidative stress. From the experiment, the DOX-exposed cells have shown morphological abnormalities such as reduced cell size, shrinkage, blebbing, and chromatin condensation, whereas no such deformities were observed in lotusine-pretreated cells even after the exposure to DOX. Increased endogenous antioxidants with reduced lipid peroxidation were observed in lotusine-pretreated cells, whereas the antioxidants were reduced along with increased lipid peroxidation in doxorubicin-exposed cells. A decreased reactive oxygen species generation was evidenced with the 2',7'-dichlorofluorescein diacetate (DCF-DA) staining method. In qPCR analysis, the lotusine-pretreated cells have mitigated doxorubicin-mediated apoptosis by downregulating the pro-apoptotic gene Bax and apoptotic executor caspase-3. It was further confirmed with the luminometric assay, which resulted in lesser luminescence in lotusine-pretreated cells, whereas higher luminescence was recorded in doxorubicin-alone-treated cells. In conclusion, the present study revealed that the lotusine pretreatment has exhibited potential cardioprotective activity against DOX-induced oxidative stress by increasing the intracellular antioxidant defense.

Melatonin attenuates doxorubicin-induced cardiotoxicity through preservation of YAP expression.

There are increasing concerns related to the cardiotoxicity of doxorubicin in the clinical setting. Recently, melatonin has been shown to exert a cardioprotective effect in various cardiovascular diseases, including cardiotoxic conditions. In this study, we examined the possible protective effects of melatonin on doxorubicin‐induced cardiotoxicity and explored the underlying mechanisms related to this process. We found that in vitro doxorubicin treatment significantly decreased H9c2 cell viability and induced apoptosis as manifested by increased TUNEL‐positive cells, down‐regulation of anti‐apoptotic protein Bcl‐2, as well as up‐regulation of pro‐apoptotic protein Bax. This was associated with increased reactive oxygen species (ROS) levels and decreased mitochondrial membrane potentials (MMP). In vivo, five weeks of doxorubicin treatment significantly decreased cardiac function, as evaluated by echocardiography. TUNEL staining results confirmed the increased apoptosis caused by doxorubicin. On the other hand, combinational treatment of doxorubicin with melatonin decreased cardiomyocyte ROS and apoptosis levels, along with increasing MMP. Such doxorubicin‐melatonin co‐treatment alleviated in vivo doxorubicin‐induced cardiac injury. Western Blots, along with in vitro immunofluorescence and in vivo immunohistochemical staining confirmed that doxorubicin treatment significantly down‐regulated Yes‐associated protein (YAP) expression, while YAP levels were maintained under co‐treatment of doxorubicin and melatonin. YAP inhibition by siRNA abolished the protective effects of melatonin on doxorubicin‐treated cardiomyocytes, with reversed ROS level and apoptosis. Our findings suggested that melatonin treatment attenuated doxorubicin‐induced cardiotoxicity through preserving YAP levels, which in turn decreases oxidative stress and apoptosis.

Papel del estrés oxidativo y nitrosativo en la hipertrofia cardiaca y remodelación ventricular

Objetivo: hacer una revisión de los mecanismos moleculares del estrés oxidativo y nitrosativo en la fisiopatología de la falla cardiaca.&#x0D; Metodología: se hizo una búsqueda en Medline (Pubmed) con las palabras clave: oxidative stress, ventricular remodeling, heart failure y nitrosative stress. Se consultó además bibliografía citada por autores de reconocida trayectoria en investigación en este tema.&#x0D; Resultados: se seleccionaron los 112 artículos más relevantes en el tema de estrés oxidativo/ nitrosativo que se relacionarán con falla cardiaca.&#x0D; Conclusiones: las respuestas de estrés de los cardiomiocitos y del tejido miocárdico es muy probable que constituyan un aspecto significativo del desarrollo de patologías cardiacas que desencadenan como evento final su progresión hacia falla cardiaca. El estrés oxidativo es un tema común en la fisiopatología de la cardiomiopatía isquémica y no isquémica. Patologías cardiacas como la falla cardiaca son usualmente precedidas de hipertrofia cardiaca secundaria, al menos en parte, a la generación de especies reactivas del oxígeno en los cardiomiocitos. Varios son los mecanismos implicados en la remodelación ventricular y progresión de la falla cardiaca que dependen de alteraciones homeostáticas en los sistemas que generan estrés oxidativo y/o nitrosativo. La discusión se centra en la reciente evidencia derivada de investigaciones llevadas a cabo tanto in vitro en cardiomiocitos cultivados como in vivo en modelos experimentales de patologías cardiacas. Las implicaciones clínicas de los recientes descubrimientos aunque son muy prometedoras para la terapéutica de la falla cardiaca, aun no han logrado trasladarse con total éxito a la práctica clínica en los ensayos clínicos realizados hasta el momento

Reduction of miR-29a-3p induced cardiac ischemia reperfusion injury in mice via targeting Bax.

The current study mainly aimed to evaluate the expression and the potential mechanism of miR-29a-3p in the hearts of mice after cardiac ischemia reperfusion (CIR) injury. Quantitative PCR was carried out to assess the relative levels of miR-29a-3p in the hearts of a CIR injury mouse model. To the best of our knowledge, the current study is the first to show that the level of miR-29a-3p was significantly decreased in the hearts of CIR injury mouse models compared with that of sham controls. Moreover, the authors found that decreased miR-29a-3p levels enhanced the production of reactive oxygen species in cardiomyocytes. Meanwhile, the inhibition of miR-29a-3p induced substantial cardiomyocyte apoptosis. Further study showed that the inhibition of miR-29a-3p decreased the activation of Akt and p38, suggesting a stress-induced self-regulatory mechanism after CIR injury in primary cardiomyocytes. A dual luciferase assay and western blot analysis showed that Bax was a target gene of miR-29a-3p. The authors also measured the level of miR-29a-3p in the plasma of 100 acute myocardial infarction (AMI) patients and found that circulating miR-29a-3p was significantly decreased in AMI patients. Receiver operating characteristic curve analysis showed that miR-29a-3p could be used to screen AMI patients from healthy controls. Hence, the authors of the current study propose that reduced miR-29a-3p levels in primary cardiomyocytes contribute to CIR injury-related apoptosis mainly by targeting Bax.

The role of endogenous reactive oxygen species in cardiac myocyte autophagy.

Autophagy is implicated in the maintenance of cardiac homeostasis. Autophagy is activated in heart failure, in which reactive oxygen species (ROS) are increased. Exogenous ROS have been shown to induce cardiomyocyte autophagy alterations. However, little is known about the influences of physiological levels of endogenous ROS on cardiomyocyte autophagy. In the present study, we tested the hypothesis that endogenous ROS in cardiomyocytes play an important role in inducing autophagy. Cultured H9C2 cardiomyocytes or Sprague-Dawley rats were treated with the antioxidant N-acetyl-cysteine (NAC) or the superoxide dismutase mimic tempol under the basal or nutrient deprivation conditions. The autophagic flux was assessed by the lysosomal inhibitor chloroquine. In H9C2 cardiomyocytes, under a basal condition, NAC or tempol increased the ratio of LC3 II/I proteins and reduced LC3 II autophagic flux. Under nutrient deprivation, NAC increased the LC3 II/I ratio and reduced LC3 II autophagic flux. In vivo studies in rats, NAC treatment increased the LC3 II/I ratio and p-Akt protein expression in myocardium. We concluded that the antioxidants reduced autophagic flux in cardiomyocytes under the basal or nutrient deprivation conditions, suggesting that endogenous ROS promote autophagy flux under physiological conditions, and this effect is mediated, at least in part, through Akt inhibition.

SR Ca2+-leak and disordered excitation-contraction coupling as the basis for arrhythmogenic and negative inotropic effects of acute ethanol exposure

AimsEthanol has acute negative inotropic and arrhythmogenic effects. The underlying mechanisms, however, are largely unknown. Sarcoplasmic reticulum Ca2+-leak is an important mechanism for reduced contractility and arrhythmias. Ca2+-leak can be induced by oxidative stress and Ca2+/Calmodulin-dependent protein kinase II (CaMKII). Therefore, we investigated the influence of acute ethanol exposure on excitation-contraction coupling in atrial and ventricular cardiomyocytes. Methods and resultsIsolated human atrial and murine atrial or ventricular cardiomyocytes were preincubated for 30 min and then superfused with control solution or solution containing ethanol. Ethanol had acute negative inotropic and positive lusitropic effects in human atrial muscle strips and murine ventricular cardiomyocytes. Accordingly, Ca2+-imaging indicated lower Ca2+-transient amplitudes and increased SERCA2a activity, while myofilament Ca2+-sensitivity was reduced. SR Ca2+-leak was assessed by measuring Ca2+-sparks. Ethanol induced severe SR Ca2+-leak in human atrial cardiomyocytes (calculated leak: 4.60 ± 0.45 mF/F0 vs 1.86 ± 0.26 in control, n ≥ 80). This effect was dose-dependent, while spontaneous arrhythmogenic Ca2+-waves increased ~5-fold, as investigated in murine cardiomyocytes. Delayed afterdepolarizations, which can result from increased SR Ca2+-leak, were significantly increased by ethanol. Measurements using the reactive oxygen species (ROS) sensor CM-H2DCFDA showed increased ROS-stress in ethanol treated cells. ROS-scavenging with N-acetylcysteine prevented negative inotropic and positive lusitropic effects in human muscle strips. Ethanol-induced Ca2+-leak was abolished in mice with knockout of NOX2 (the main source for ROS in cardiomyocytes). Importantly, mice with oxidation-resistant CaMKII (Met281/282Val mutation) were protected from ethanol-induced Ca2+-leak. ConclusionWe show for the first time that ethanol acutely induces strong SR Ca2+-leak, also altering excitation-contraction coupling. Acute negative inotropic effects of ethanol can be explained by reduced systolic Ca2+-release. Mechanistically, ROS-production via NOX2 and oxidative activation of CaMKII appear to play central roles. This provides a mechanism for the arrhythmogenic and negative inotropic effects of ethanol and suggests a druggable target (CaMKII).

ROS and redox signaling in myocardial ischemia-reperfusion injury and cardioprotection.

Ischemia-reperfusion (IR) injury is central to the pathology of major cardiovascular diseases, such as stroke and myocardial infarction. IR injury is mediated by several factors including the elevated production of reactive oxygen species (ROS), which occurs particularly at reperfusion. The mitochondrial respiratory chain and NADPH oxidases of the NOX family are major sources of ROS in cardiomyocytes. The first part of this review discusses recent findings and controversies on the mechanisms of superoxide production by the mitochondrial electron transport chain during IR injury, as well as the contribution of the NOX isoforms expressed in cardiomyocytes, NOX1, NOX2 and NOX4, to this damage. It then focuses on the effects of ROS on the opening of the mitochondrial permeability transition pore (mPTP), an inner membrane non-selective pore that causes irreversible damage to the heart. The second part analyzes the redox mechanisms of cardiomyocyte mitochondrial protection; specifically, the activation of the hypoxia-inducible factor (HIF) pathway and the antioxidant transcription factor Nrf2, which are both regulated by the cellular redox state. Redox mechanisms involved in ischemic preconditioning, one of the most effective ways of protecting the heart against IR injury, are also reviewed. Interestingly, several of these protective pathways converge on the inhibition of mPTP opening during reperfusion. Finally, the clinical and translational implications of these cardioprotective mechanisms are discussed.

Interleukin-33 attenuates doxorubicin-induced cardiomyocyte apoptosis through suppression of ASK1/JNK signaling pathway

Interleukin-33 (IL-33), a new member of the IL-1 cytokine family, has cardiac protective effect in many circumstances. The aims of present study are to assess whether IL-33 can protect cardiomyocytes from doxorubicin (DOX)-induced apoptosis and the mechanism involved in the protection. Cardiomyocytes derived from either wild-type or c-Jun N-terminal kinase deficient (JNK−/-) mice were challenged with DOX (1 μM) with or without IL-33 (10 ng/ml). Myocyte apoptosis was assessed by measuring Caspase 3 activity, fragmented DNA and the TUNEL staining. In addition, cardiomyocyte reactive oxygen species (ROS) was assessed by measuring 2′,7′-dichlorofluorescin diacetate (DCFDA); apoptosis signal-regulating kinase 1(ASK1) and JNK phosphorylation were assessed with western blot analysis. Treatment of cardiomyocyes with DOX resulted in ROS generation, ASK1 and JNK phosphorylation and myocyte apoptosis. IL-33 inhibited the DOX-induced ROS, prevented ASK1 and JNK phosphorylation and attenuated the DOX-induced myocyte apoptosis. Genetic inhibition of ASK1 (ASK1 siRNA transfection) and JNK (JNK−/-) ameliorated the cardiac-protective effect of IL-33. Moreover, inhibition of ASK1 prevented the DOX-induced phosphorylation of JNK, while inhibition of JNK showed no effect on DOX-induced ASK1 phosphorylation. Our study indicates that: 1) ASK1/JNK signaling pathway is involved in DOX-induced cardiomyocyte apoptosis; 2) IL-33 protects cardiomyocytes from DOX-induced myocyte apoptosis through inhibition of the ASK1/JNK signaling pathway. IL-33 may have therapeutic potential for DOX-induced cardiac injury.

Molecular and Cellular Mechanisms of Cardiovascular Disorders in Diabetes.

The clinical correlations linking diabetes mellitus with accelerated atherosclerosis, cardiomyopathy, and increased post-myocardial infarction fatality rates are increasingly understood in mechanistic terms. The multiple mechanisms discussed in this review seem to share a common element: prolonged increases in reactive oxygen species (ROS) production in diabetic cardiovascular cells. Intracellular hyperglycemia causes excessive ROS production. This activates nuclear poly(ADP-ribose) polymerase, which inhibits GAPDH, shunting early glycolytic intermediates into pathogenic signaling pathways. ROS and poly(ADP-ribose) polymerase also reduce sirtuin, PGC-1α, and AMP-activated protein kinase activity. These changes cause decreased mitochondrial biogenesis, increased ROS production, and disturbed circadian clock synchronization of glucose and lipid metabolism. Excessive ROS production also facilitates nuclear transport of proatherogenic transcription factors, increases transcription of the neutrophil enzyme initiating NETosis, peptidylarginine deiminase 4, and activates the NOD-like receptor family, pyrin domain-containing 3 inflammasome. Insulin resistance causes excessive cardiomyocyte ROS production by increasing fatty acid flux and oxidation. This stimulates overexpression of the nuclear receptor PPARα and nuclear translocation of forkhead box O 1, which cause cardiomyopathy. ROS also shift the balance between mitochondrial fusion and fission in favor of increased fission, reducing the metabolic capacity and efficiency of the mitochondrial electron transport chain and ATP synthesis. Mitochondrial oxidative stress also plays a central role in angiotensin II-induced gap junction remodeling and arrhythmogenesis. ROS contribute to sudden death in diabetics after myocardial infarction by increasing post-translational protein modifications, which cause increased ryanodine receptor phosphorylation and downregulation of sarco-endoplasmic reticulum Ca(++)-ATPase transcription. Increased ROS also depress autonomic ganglion synaptic transmission by oxidizing the nAch receptor α3 subunit, potentially contributing to the increased risk of fatal cardiac arrhythmias associated with diabetic cardiac autonomic neuropathy.

Nitric Oxide Protects L-Type Calcium Channel of Cardiomyocyte during Long-Term Isoproterenol Stimulation in Tail-Suspended Rats.

The aim of this study was to investigate the effects of nitric oxide (NO) and reactive oxygen species (ROS) on L-type calcium channel (LTCC) gating properties of cardiomyocytes during long-term isoproterenol (ISO) stimulation. Expression and activity of nNOS as well as S-nitrosylation of LTCC α1C subunit significantly decreased in the myocardium of SUS rats. Long-term ISO stimulation increased ROS in cardiomyocytes of SUS rats. ISO-enhanced calcium current (I Ca,L) in the SUS group was less than that in the CON group. The maximal I Ca,L decreased to about 80% or 60% of initial value at the 50th minute of ISO treatment in CON or SUS group, respectively. Specific inhibitor NAAN of nNOS reduced maximal I Ca,L to 50% of initial value in the CON group; in contrast, NO donor SNAP maintained maximal I Ca,L in SUS group to similar extent of CON group after 50 min of ISO treatment. Long-term ISO stimulation also changed steady-state activation (P < 0.01), inactivation (P < 0.01), and recovery (P < 0.05) characteristics of LTCC in SUS group. In conclusion, NO-induced S-nitrosylation of LTCC α1C subunit may competitively prevent oxidation from ROS at the same sites. Furthermore, LTCC can be protected by NO during long-term ISO stimulation.