Hypoxia-induced Myocardial Injury Research Articles

The cytoprotective effect of Gymnema inodorum leaf extract against hypoxia-induced cardiomyocytes injury

Ischemic heart disease stands out as a major global contributor to mortality, with the initiation of hypoxia, marked by reduced oxygen availability, disrupting the balance of reactive oxygen species (ROS), leading to cellular injury. Exploring antioxidants derived from medicinal plants is becoming more interesting as a potential alternative treatment, especially for mitigating myocardial injury. Thus, this study aimed to assess the cytoprotective efficacy of Gymnema inodorum leaf extract (GIE) in a rat cardiac myoblast, H9c2, subjected to an in vitro hypoxia. The cell viability, intracellular ROS production and the expression of inflammatory cytokines were quantified, and hypoxia-induced cell morphology changes were observed using confocal fluorescence microscopy. The results showed that GIE notably enhanced cell viability, preserving membrane integrity, when compared with the hypoxic group. Remarkably, GIE significantly reduced hypoxia-induced intracellular ROS production, attributable to its inherent antioxidant properties. Furthermore, GIE significantly reduced interleukin (IL)-1β, interleukin (IL)-6 mRNA expression level and tended to reduce tumor necrosis factor-α (TNF-α) mRNA expression. In conclusion, these findings underscore the potential of GIE in mitigating hypoxia-induced myocardial injury, highlighting its robust antioxidant and anti-inflammatory attributes.

16α-OHE1 alleviates hypoxia-induced inflammation and myocardial damage via the activation of β2-Adrenergic receptor

ObjectiveMyocardial injuries resulting from hypoxia are a significant concern, and this study aimed to explore potential protective strategies against such damage. Specifically, we sought to investigate the cardioprotective effects of 16α-hydroxyestrone (16α-OHE1). MethodsMale Sprague‒Dawley (SD) rats were subjected to hypoxic conditions simulating high-altitude exposure at 6000 m in a low-pressure chamber for 7 days. Before and during hypoxic exposure, estradiol (E2) and various doses of 16α-OHE1 were administered for 14 days. Heart weight/body weight (HW/BW), myocardial structure, Myocardial injury indicators and inflammatory infiltration in rats were measured. H9C2 cells cultured under 5% O2 conditions received E2 and varying doses of 16α-OHE1; Cell viability, apoptosis, inflammatory infiltration, and Myocardial injury indicators were determined. Expression levels of β2AR were determined in rat hearts and H9C2 cells. The β2AR inhibitor, ICI 118,551, was employed to investigate β2AR's role in 16α-OHE1's cardioprotective effects. ResultsHypoxia led to substantial myocardial damage, evident in increased heart HW, CK-MB, cTnT, ANP, BNP, structural myocardial changes, inflammatory infiltration, and apoptosis. Pre-treatment with E2 and 16α-OHE1 significantly mitigated these adverse changes. Importantly, the protective effects of E2 and 16α-OHE1 were associated with the upregulation of β2AR expression in both rat hearts and H9C2 cells. However, inhibition of β2AR by ICI 118,551 in H9C2 cells nullified the protective effect of 16α-OHE1 on myocardium. ConclusionOur findings suggest that 16α-OHE1 can effectively reduce hypoxia-induced myocardial injury in rats through β2ARs, indicating a promising avenue for cardioprotection.

Mechanistic insights into the ameliorative effects of hypoxia-induced myocardial injury by Corydalis yanhusuo total alkaloids: based on network pharmacology and experiment verification.

Introduction: Corydalis yanhusuo total alkaloids (CYTA) are the primary active ingredients in yanhusuo, known for their analgesic and cardioprotective effects. However, the mechanisms underlying the treatment of Myocardial ischemia (MI) with CYTA have not been reported. The purpose of this study was to explore the protective effect of CYTA on MI and its related mechanisms. Methods: A network pharmacology was employed to shed light on the targets and mechanisms of CYTA's action on MI. The protective effect of CYTA against hypoxia damage was evaluated in H9c2 cells. Furthermore, the effects of CYTA on L-type Ca2+ current (ICa-L), contractile force, and Ca2+ transient in cardiomyocytes isolated from rats were investigated using the patch clamp technique and IonOptix system. The network pharmacology revealed that CYTA could regulate oxidative stress, apoptosis, and calcium signaling. Cellular experiments demonstrated that CYTA decreased levels of CK, LDH, and MDA, as well as ROS production and Ca2+ concentration. Additionally, CYTA improved apoptosis and increased the activities of SOD, CAT, and GSH-Px, along with the levels of ATP and Ca2+-ATPase content and mitochondrial membrane potential. Moreover, CYTA inhibited ICa-L, cell contraction, and Ca2+ transient in cardiomyocytes. Results: These findings suggest that CYTA has a protective effect on MI by inhibiting oxidative stress, mitochondrial damage, apoptosis and Ca2+ overload. Discussion: The results prove that CYTA might be a potential natural compound in the field of MI treatment, and also provide a new scientific basis for the its utilization.

Protective effects of epigallocatechin-3-gallate counteracting the chronic hypobaric hypoxia-induced myocardial injury in plain-grown rats at high altitude

Exposure to hypobaric hypoxia (HH) environment causes stress to the body, especially the oxygen-consuming organs. Chronic HH conditions have adverse effects on the myocardium. Thus, we conducted this experiment and aim to evaluate such adverse effects and explore the therapeutic role of epigallocatechin-3-gallate (EGCG) in rats’ heart under chronic HH conditions. For that purpose, we transported rats from plain to a real HH environment at high altitude for establishing the HH model. At high altitude, animals were treated with EGCG while the salidroside was used as the positive control. General physiological data were collected, and routine blood test results were analyzed. Cardiac magnetic resonance (CMR) was examined to assess the structural and functional changes of the heart. Serum levels of cardiac enzymes and pro-inflammatory cytokines were examined. Oxidative markers in the left ventricle (LV) were detected. Additionally, ultrastructural and histopathological changes and apoptosis of the LV were assessed. Furthermore, the antioxidant stress-relevant proteins nuclear factor E2-related factor 2 (Nrf2) and the heme oxygenase-1 (HO-1) were detected. The experiment revealed that EGCG treatment decreased HH-induced elevation of cardiac enzymes and relieved mitochondrial damage of the LV. Notably, EGCG treatment significantly alleviated oxidative stress in the LV and inflammatory response in the blood. Western blot confirmed that EGCG significantly upregulated Nrf2 and HO-1. Therefore, EGCG may be considered a promising natural compound for treating the HH-induced myocardial injuries.

Intermedin induces autophagy and attenuates hypoxia-induced injury in cardiomyocytes by regulation of MALAT1/ULK1

Myocardial infarction is a predominant cause of cardiovascular diseases with high incidence and death rate worldwide. Although growing evidence has suggested that IMD has significant protective influences on the cardiovascular system, the molecular regulatory mechanism of IMD in hypoxia-induced injury caused by myocardial infarction is urgent to be elucidated. In the present study, we found hypoxia led to a noteworthy enhancement in IMD expression and IMD alleviated hypoxia-induced myocardial injury of NRCMs. Furthermore, IMD was proved to inhibit hypoxia-induced injury by regulating MALAT1. Our findings suggested MALAT1 positively regulated the mRNA and protein expression level of ULK1 and hypoxia induced autophagy of NRCMs. MALAT1 stimulated autophagy to block hypoxia-induced cell injury in NRCMs via upregulation of ULK1 expression. Autophagy suppression abolished the protective capability of IMD overexpression against hypoxia-induced myocardial injury in NRCMs. In a word, our study shed light on the central mechanism of IMD in preventing hypoxia-induced injury caused by myocardial infarction. We confirmed IMD induced autophagy and attenuated hypoxia-induced injury in cardiomyocytes via MALAT1/ULK1, which may contribute to designing effective therapeutic approaches of myocardial infarction.

The role of ferroptosis and endoplasmic reticulum stress in intermittent hypoxia-induced myocardial injury.

Obstructive sleep apnea (OSA) is related to increased risk of cardiovascular disease. Ferroptosis is a form of programmed cell death characterized by iron overload and plays critical roles in myocardial injury. This study aimed to investigate the role of ferroptosis in intermittent hypoxia (IH)-induced myocardial injury involving endoplasmic reticulum stress (ERS). AC16 human cardiomyocytes were exposed to IH or normoxia conditions. Mice were randomly grouped as follows: normal control (NC), IH, ferrostatin-1 + IH (FIH), and N-acetylcysteine + IH (AIH). The mRNA levels of GPX4, xCT, FTH1, and FACL4 in AC16 cells were detected by qRT-PCR. The protein levels of GPX4, xCT, NOX4, ATF4, CHOP, Bcl-2, and Bax in myocardial tissue were detected by Western blot analysis. The mRNA expression levels of GPX4 and xCT in AC16 cells were significantly lower in IH group than that of NC group. In IH mice, myocardial tissues were injured accompanied by increased level of ferroptosis and ERS. Inhibition of ferroptosis and treatment of N-acetylcysteine reduced ERS and myocardial injury in mice exposed to IH. In addition, compared to ferrostatin-1, N-acetylcysteine exerted a greatereffect in relieving IH-induced myocardial damage and ERS. Ferroptosis was involved in IH-related myocardial injury accompanied by the activation of ERS. Inhibition of ferroptosis and acetylcysteine treatment alleviated IH-related myocardial injury, which may be a potential target for therapeutic approaches to OSA-induced myocardial injury.

Ginsenoside Rh2 Inhibits NLRP3 Inflammasome Activation and Improves Exosomes to Alleviate Hypoxia-Induced Myocardial Injury.

The inflammatory microenvironment after acute myocardial infarction (MI) is a key limiting factor in the clinical application of stem cell transplantation and paracrine exosome therapy. Qishen Yiqi Pills contain a saponin ingredient called Ginsenoside Rh2 (Rh2) which exhibits a certain therapeutic effect on MI. However, the mechanism by which Rh2 alleviates the inflammatory microenvironment and improves the therapeutic efficiency of exosomes remains enigmatic. Here, we found that Rh2 attenuated the adverse effect of oxygen-glucose deprivation (OGD)-induced cellular injury, an in vitro pathological model of MI. Confocal microscopy revealed that DiI-labeled BMSCs-derived exosomes exhibited an increased homing ability of cardiomyocytes, which, in turn, inhibited the nuclear translocation of NF-κB p65 and NLRP3 inflammasome activation, thereby alleviating the inflammatory microenvironment and further facilitating the homing of exosomes to cardiomyocytes by forming a feed-forward enhancement loop. Additionally, we found that Rh2 could regulate the HMGB1/NF-κB signaling pathway to improve the OGD environment of cardiomyocytes, increasing the efficiency of the feed-forward loop. In conclusion, we found that Rh2 can improve the inflammatory microenvironment by enhancing the protection of exosomes against myocardial injury, providing new insights into the indirect modification of exosomes by Rh2 in MI treatment.

CircSLC8A1 Exacerbates Hypoxia-Induced Myocardial Injury via Interacting with MiR-214-5p to Upregulate TEAD1 Expression

Circular RNAs (circRNAs) act as important regulators in myocardial infarction (MI). This study aimed to explore the regulatory mechanism of circRNA solute carrier family 8 member A1 antisense RNA 1 (circSLC8A1) in hypoxia-induced myocardial injury.Exosomes were isolated by ultracentrifugation and identified by microscopic observation or protein detection. Protein levels were examined by Western blot. CircSLC8A1, microRNA-214-5p (miR-214-5p), and TEA domain transcription factor 1 (TEAD1) levels were determined via quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability and apoptosis were analyzed by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyl tetrazolium bromide (MTT) and flow cytometry, respectively. Inflammatory cytokines were measured using enzyme-linked immunosorbent assay (ELISA). Oxidative stress was assessed by reactive oxygen species (ROS) production, malondialdehyde (MDA) level, and superoxide dismutase (SOD) activity through the corresponding detection kits. Target analysis was performed by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay, and pull-down assay.Exosomes released circSLC8A1 from hypoxic cardiomyocytes. Exosomal circSLC8A1 exacerbated hypoxia-induced repression of cell viability but promotion of cell apoptosis, inflammation, and oxidative stress. Knockdown of circSLC8A1 ameliorated hypoxia-mediated cell injury. CircSLC8A1 directly targeted miR-214-5p and miR-214-5p downregulation reverted the effects of si-circSLC8A1 on hypoxia-treated cardiomyocytes. TEAD1 was a target of miR-214-5p and circSLC8A1 upregulated TEAD1 level via targeting miR-214-5p. In addition, miR-214-5p inhibited hypoxia-caused cell injury by downregulating the expression of TEAD1.These results suggested that circSLC8A1 aggravated cell damages in hypoxia-treated cardiomyocytes by the regulation of TEAD1 via sponging miR-214-5p.

Inhibition of GARS1-DT Protects Against Hypoxic Injury in H9C2 Cardiomyocytes via Sponging miR-212-5p.

The present study aimed to elucidate the function of long noncoding RNA GARS1-DT in hypoxia-induced injury in ex-vivo cardiomyocytes and explore its underlying mechanism. Hypoxic injury was confirmed in H9C2 cells by the determination of cell viability, migration, invasion, and apoptosis. GARS1-DT expression was estimated in H9C2 cells after hypoxia. We then measured the effects of GARS1-DT knockdown on hypoxia-induced H9C2 cells. The interaction between GARS1-DT and miR-212-5p was also investigated. Hypoxia treatment led to cell damage in H9C2 cardiomyocytes, accompanied with the upregulation of GARS1-DT expression. Transfection of GARS1-DT small interfering RNA remarkably attenuated hypoxia-induced injury by enhancing cell viability, migration, and invasion, and reducing apoptosis. Furthermore, GARS1-DT served as an endogenous sponge for miR-212-5p, and its expression was negatively regulated by GARS1-DT. The effects of GARS1-DT knockdown on hypoxia-induced injury were significantly abrogated by miR-212-5p silence. Besides, suppression of GARS1-DT activated PI3K/AKT pathway in hypoxia-treated H9C2 cells, which were reversed by inhibition of miR-212-5p. Our findings demonstrated the novel molecular mechanism of GARS1-DT/miR-212-5p/PI3K/AKT axis on the regulation of hypoxia-induced myocardial injury in H9C2 cells, which may provide potential therapeutic targets for acute myocardial infarction treatment.

The microRNA-210/Casp8ap2 Axis Alleviates Hypoxia-Induced Myocardial Injury by Regulating Apoptosis and Autophagy

Coronary heart disease (CHD) is a serious condition comprising atherosclerosis-mediated ischaemic and hypoxic myocardial injury. This study aimed to investigate the mechanism of the miR-210/Casp8ap2 signalling pathway in hypoxic myocardial cells. mRNA and protein expression levels were determined by quantitative real-time PCR and western blotting, respectively. MTT was used to evaluate cell survival, and flow cytometry was used to assess apoptosis and the cell cycle distribution. The interaction between miR-210 and ­Casp8ap2 was detected by dual-luciferase reporter assay. As a result, overexpression of miR-210 significantly inhibited apoptosis and reduced the proportion of cells in G1 phase. Moreover, miR-210 suppressed autophagy by upregulating p62 levels and reducing the LC3-II/I ratio in hypoxic cardiomyocytes. miR-210 regulated apoptosis and autophagy by directly targeting Casp8ap2. Furthermore, the expression levels of Casp8ap2, Cleaved caspase 8, Cleaved caspase 3and Beclin-1 were all decreased in response to miR-210. In short, our results suggest that miR-210 exerts anti-apoptotic and anti-autophagic effects in hypoxic cardiomyocytes, which alleviates myocardial injury in response to hypoxia.

Mechanism of salidroside relieving the acute hypoxia-induced myocardial injury through the PI3K/Akt pathway.

ObjectiveThe objective was to investigate the anti-inflammatory effects of salidroside through the PI3K/Akt signaling pathway and its protective effects on acute hypoxia-induced myocardial injury in rats.MethodsA total of 24 healthy Sprague-Dawley male rats were selected as the experimental subjects. All rats were divided into 4 groups by using the random number table method, with 6 rats in each group. The groups included the normal control group, the salidroside group, the hypobaric hypoxia group, and the hypobaric hypoxia + salidroside group. Rats in the salidroside group were fed in the original animal laboratory and were intragastrically administered with salidroside every morning at a dosage of 35 mg/kg. Rats in the normal control group were intragastrically administered with an equal dosage of saline. Rats in the hypobaric hypoxia + salidroside group were intragastrically administered with salidroside every morning at a dosage of 35 mg/kg, who were fed in the hypoxic experiment module for animals. The altitude was increased to 4000 m, and the rats were kept in the module for 24 h. Rats in the hypobaric hypoxia group were intragastrically administered with an equal dosage of saline in the same environment, and the altitude was increased to 4000 m after administration. Parameters of blood gas analysis, histopathological changes in cardiac tissues, cardiac indexes, and inflammatory factors IL-6 and TNF-α levels of rats in groups were compared.Results1. The cardiac indexes of rats in groups were compared. The differences between the hypobaric hypoxia group and the hypobaric hypoxia + salidroside group were statistically significant (P < 0.05). 2. The results of blood gas analysis of rats in groups were compared. The differences between the hypobaric hypoxia group and the hypobaric hypoxia + salidroside group were significantly different (P < 0.05). 3. In the hypobaric hypoxia group, the myocardial cells of rats were arranged disorderly and shaped differently, with cases such as edema, degeneration, necrosis, nucleus pyknosis, and massive infiltration of inflammatory cells. In the hypobaric hypoxia + salidroside group, the above-mentioned pathological changes in myocardial cells were relieved. 4. Compared with the hypobaric hypoxia group, in the hypobaric hypoxia + salidroside group, the concentrations of IL-6 and TNF-α in rats decreased apparently, and the differences were statistically significant (P < 0.05).ConclusionSalidroside had the repairing and protective effects on the hypobaric hypoxia-induced myocardial injuries in rats. The application of salidroside could reduce the inflammatory responses of rats with hypobaric hypoxia-induced myocardial injuries through PI3K/Akt signaling pathway, thereby protecting the myocardial cells.

Astragaloside IV attenuates chronic intermittent hypoxia-induced myocardial injury by modulating Ca2+ homeostasis.

Obstructive sleep apnea syndrome (OSAS) is an important consequence of chronic intermittent hypoxia (CIH). Astragaloside IV (AS-IV) exerts multiple protective effects in diverse diseases. However, whether AS-IV can attenuate CIH-induced myocardial injury is unclear. In this study, rats exposed to CIH were established and treated with AS-IV for 4 weeks. In vitro, H9C2 cardiomyocytes subjected to CIH exposure were treated with AS-IV for 48 hours. Then the cardiac function, morphology, fibrosis, apoptosis and Ca2+ homeostasis were determined to assess cardiac damage. Results showed that AS-IV attenuated cardiac dysfunction and histological lesions in CIH rats. The increased TUNEL-positive cells and activated apoptotic proteins in CIH rats were reduced by AS-IV. We also noticed that AS-IV reversed the accumulation of Ca2+ and altered expressions of Ca2+ handling proteins (decreases of SERCA2a and RYR2, and increases of p-CaMKII and NCX1) under CIH exposure. Furthermore, CIH-induced reduction of SERCA2a activity was increased by AS-IV in rats. Similar results were also observed in H9C2 cells. Altogether, these findings indicate that AS-IV modulates Ca2+ homeostasis to inhibit apoptosis, protecting against CIH-induced myocardial injury eventually, suggesting it may be a potential agent for cardiac damage of OSAS patients. SIGNIFICANCE OF THE STUDY: Chronic intermittent hypoxia (CIH) is a great contributor of OSAS, which is closely associated with cardiovascular diseases. It is necessary for developing a promising drug to attenuate CIH-induced myocardial injury. This work suggests that AS-IV can attenuate myocardial apoptosis and calcium disruption, thus protecting against CIH-induced myocardial injury. It may represent a novel therapeutic for cardiac damage of OSAS.

Apigenin protects against ischemia-/hypoxia-induced myocardial injury by mediating pyroptosis and apoptosis.

Apigenin is a traditional Chinese medicine found in many plants that plays critical roles in several diseases, including cardiovascular diseases. We investigated the protective effect of apigenin against ischemia/hypoxia (I/H)-induced myocardium injury in vitro and explored the potential molecular mechanisms. Together with the flow cytometry results, our results indicated that apigenin attenuated the pyroptosis and apoptosis of myoblastic H9c2 cells that were induced by I/H injury. Furthermore, the pro-inflammatory cytokines (interleukin 18, IL-18, and IL-1β) were investigated for the roles of apigenin in I/H-induced myocardium injury. It was observed that increases in IL-1β and IL-18 levels were significantly reduced by apigenin treatment of I/H-induced H9C2 cells. The results demonstrated that apigenin protected H9c2 cells against I/H-induced myocardium injury. The protective effects were most likely related to the reduction of pyroptosis, apoptosis, and pro-inflammatory cytokines.

RETRACTED ARTICLE: Downregulation of miR-483-5p decreases hypoxia-induced injury in human cardiomyocytes by targeting MAPK3

BackgroundMiR-483-5p was recently identified as a risk factor in the early stages of acute myocardial infarction (AMI) patients. Here, we further investigated how miR-483-5p affects cardiomyocyte apoptosis and oxidative stress under hypoxic conditions.MethodsPlasma samples were collected from AMI patients and healthy volunteers. The expression of miR-483-5p was determined using quantitative real-time PCR. An in vitro hypoxic model was constructed to mimic AMI in AC16 cells. Cell viability, apoptosis and oxidative stress biomarker levels (MDA, SOD and CAT) were respectively determined using CCK-8, flow cytometry and commercial assay kits.ResultsThe expression levels of miR-483-5p were significantly higher in AMI patients than in control subjects. Circulating levels of miR-483-5p positively correlated with creatine kinase MB isoform (CK-MB) and cardiac troponin I (cTnI) levels. The in vitro experiments showed that the expression levels of miR-483-5p were also upregulated in hypoxia-induced AC16 cell injury. MiR-483-5p overexpression significantly increased hypoxia-induced cardiomyocyte apoptosis and oxidative stress, while knockdown attenuated these effects. Mechanistically, miR-483-5p directly targets MAPK3 in AC16 cells. Furthermore, the protective effects of miR-483-5p knockdown against hypoxia-induced cardiomyocyte injury are partially dependent on MAPK3.ConclusionsMiR-483-5p, which targets MAPK3, might be a potential therapeutic target for the diagnosis and prevention of hypoxia-induced myocardial injury.

Curcumin prevents chronic intermittent hypoxia-induced myocardial injury.

Background:Chronic intermittent hypoxia (IH), the hallmark feature of obstructive sleep apnoea syndrome, contributes to infarct size enhancement after myocardial ischemia–reperfusion (I/R). Curcumin (Curc), the natural pigment of Curcuma longa, has been demonstrated to be beneficial in the context of myocardial injury. In this study, we assessed the effects of Curc on the maladaptive cardiac response to IH, and particularly on IH-induced hypoxia inducible factor-1 (HIF-1) expression, oxidative stress, inflammation, endoplasmic reticulum (ER) stress and apoptosis.Methods:Swiss/SV129 mice were exposed to normoxia or IH (21–5% FiO2, 60 s cycles, 8 h per day, for 21 days) and treated orally with Curc (100 mg kg−1 day−1, oral gavage) or its vehicle. Mice were then either euthanised for heart sampling in order to perform biochemical and histological analysis, or subjected to an in vivo ischemia-reperfusion protocol in order to measure infarct size.Results:IH increased nuclear HIF-1α expression and superoxide anion (O2.–) production as well as nuclear factor kappa B (NF-kB) p65, glucose-regulated protein (Grp78) and C/EBP homologous protein (CHOP) expression. IH also induced apoptosis and increased infarct size after I/R . The IH-induced HIF-1 activation, oxidative stress, inflammation, ER stress and apoptosis were abolished by chronic Curc treatment. Curc also significantly decreased infarct size only in mice exposed to IH.Conclusion:Curc prevents IH-induced myocardial cell death signalling. Curc might be used as a combined therapy with continuous positive airway pressure in sleep apnoea patients with high cardiovascular risk.

LncRNA RMRP accelerates hypoxia-induced injury by targeting miR-214-5p in H9c2 cells

ObjectiveTo elucidate the function of lncRNA RMRP in hypoxia-induced acute myocardial infarction (AMI) in vitro and explore its underlying mechanism. MethodsHypoxic injury was confirmed by measurement of cell viability, LDH release, migration, invasion, and apoptosis in H9c2 cells. The interactions between RMRP and miR-214-5p as well as miR-214-5p and p53 were also investigated. ResultsHypoxia treatment significantly induced cell damage in H9c2 cells, accompanied with the up-regulation of RMRP expressions. Transfection of RMRP siRNA remarkably attenuated hypoxia-induced injury by enhancing cell viability, migration and invasion, and reducing cell apoptosis and LDH release; whereas, enforced expression of RMRP aggravated hypoxia-induced injury. Furthermore, RMRP served as an endogenous sponge for miR-214-5p, and its expression was negatively regulated by RMRP. The effects of RMRP knockdown on hypoxia-induced injury were further enhanced with miR-214-5p overexpression, but significantly abrogated with miR-214-5p silence. Moreover, p53 was verified as a direct target of miR-214-5p, and functional investigation revealed that RMRP regulated hypoxia-induced injury via modulating p53 signaling pathway, which was partially mediated by miR-214-5p. ConclusionOur findings demonstrated the novel molecular mechanism of RMRP/miR-214-5p/p53 axis on the regulation of hypoxia-induced myocardial injury in H9c2 cells, which might provide potential therapeutic targets for AMI treatment.

Abstract 164: Hypoxia-Induced Cardiomyocyte Mitophagy and Mitochondrial Permeability Transition are Inhibited by Bnip3 Phosphorylation

Bnip3 is a hypoxia-inducible initiator of cardiomyocyte cell death and has also been implicated as a mitochondrial receptor for the cellular mitophagy machinery. Previous work from our group demonstrates that the prostaglandin E1 analogue, misoprostol, prevents hypoxia-induced mitochondrial dysfunction and is a potent activator of PKA. We hypothesize that misoprostol alters the phosphorylation status of Bnip3, inhibiting its ability to induce cardiomyocyte mitophagy, mitochondrial dysfunction and cell death. Using a rodent model of neonatal hypoxia, in combination with rat primary ventricular neonatal cardiomyocytes (PVNC’s) and H9c2 cells, we assessed the effect of hypoxia and misoprostol drug treatments on mitochondrial function, mitophagy, and cell viability. In postnatal day 5 rats, hypoxia caused a 30% increase in the concentration of serum cardiac troponin-I, a clinically relevant marker for cardiomyocyte cell death, which was absent with the addition of misoprostol drug treatments (n=3). Using PVNC’s we further demonstrated that hypoxia reduced measures of mitochondrial function including membrane potential (47%) and maximal respiration (46%), which were restored back to control levels with the addition of misoprostol (p&lt;0.01). Furthermore, hypoxia induced a 36% increase in mitophagy, concurrent with a 210% increase in mitochondrial permeability transition, both of which were reversed with misoprostol drug treatment (p&lt;0.01). Using a combination of mass spectrometry and mutagenesis, we also show that PKA directly phosphorylates the transmembrane domain of Bnip3 to inhibit its function. Mechanistically, when the PKA phosphorylation site on Bnip3 was neutralized, the protective effect of misoprostol on mitochondrial membrane potential, mitophagy and permeability transition was lost. Taken together, these results demonstrate a foundational role for Bnip3 phosphorylation in the molecular regulation of cardiomyocyte mitochondrial dysfunction. These findings further identify a pharmacological mechanism, through PKA, which may ultimately be able to prevent hypoxia-induced myocardial injury.

CCAAT/enhancer binding protein homologous protein knockdown alleviates hypoxia-induced myocardial injury in rat cardiomyocytes exposed to high glucose.

Diabetic patients are more sensitive to ischemic injury than non-diabetics. Endoplasmic reticulum (ER) stress has been reported to be closely associated with the pathophysiology of ischemic injury in diabetes. The aim of the present study was to investigate the mechanisms involved in the progression of diabetes complicated by myocardial infarction (MI) and further verify the role of CCAAT/enhancer binding protein (C/EBP)-homologous protein (CHOP) using an in vitro model of diabetes/MI. The rats were exposed to 65 mg/kg streptozotocin (STZ) and left anterior descending (LAD) coronary artery ligation. ST-segment elevation, heart rate, left ventricular systolic pressure (LVSP) and LV end-diastolic pressure (LVEDP) were measured. Serum creatinine kinase-MB (CK-MB) and cardiac troponin T (cTnT) levels were examined by ELISA. Infarct size and apoptosis were measured by triphenyltetrazolium chloride staining and terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay. Pathological changes were evaluated by hematoxylin and eosin staining. H9c2 cells were used to establish an in vitro model of diabetes complicated by MI. Following CHOP knockdown, cell viability, cell cycle distribution and apoptosis were examined by Cell Counting Kit-8 assay, flow cytometry and Hoechst staining. Glucose-regulated protein 78 (GRP78), CHOP, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), endoplasmic reticulum oxidoreductase 1 (Ero1)-α, Ero1β and protein disulfide isomerase (PDI) levels in both myocardial tissues and H9c2 cells were determined by western blotting. In the present study, diabetes complicated by MI promoted ST-segment elevation and myocardial apoptosis, increased infarct size, induced pathological changes and elevated LVEDP, CK-MB, cTnT, GRP78, CHOP, Bax, Ero1α, Ero1β and PDI; however, it decreased heart rate, LVSP and Bcl-2. Additionally, high glucose combined with hypoxic treatment reduced cell viability, induced cell cycle arrest at G1 phase, promoted cell apoptosis, and activated the GRP78/CHOP and Ero1/PDI signaling pathways, which were reversed by CHOP knockdown. Thus, CHOP may be an effective therapeutic target for the treatment of diabetes complicated by MI.

Investigation of markers for myocardial remodeling during experimental hypobaric hypoxia in the correction with cytoflavin

To reveal the peculiarities of changes in the markers of myocardial remodeling in experimental hypobaric hypoxia. The investigation was conducted on 26 mature male Wistar rats weighing 220-310 g, which were divided into 3 groups: 1) 6 intact animals; 2) 10 rats that were exposed to 30-day simulated hypobaric hypoxia without correction; 3) 10 rats that were given once-daily intraperitoneal injections of cytoflavin solution at a rate of 0.5 ml/100 g of body weight for 30 days before immersion in a hypobaric chamber. The early stages of myocardial remodeling in hypobaric hypoxia were found to be characterized by structural re-systematization of the components of the normal ventricular wall, which was manifested by cardiomyocyte hypertrophy and ischemia; circulatory disorders; fibroblast proliferation that dramatically enhances the extracellular expression of protein matrix, which can lead to the formation of fibrous tissue and irreversible myocardial dysfunction. Elevated matrix metalloproteinase levels are evidence for collagen degradation and cardiac remodeling. In the authors' opinion, the cyto- and angioprotective effects of cytoflavin in rats treated with the latter used in combination therapy are associated with the composition of the drug, the active components of which have mutually potentiating effects, are inducers of the major metabolic pathways in the cells and are also activators of key energy-generating processes. Identification of the molecular mechanisms of myocardial remodeling is of great practical importance. This enables remodeling markers to be considered as a potential therapeutic target in the treatment of hypoxia-induced myocardial injury.

LncRNA TUG1 serves an important role in hypoxia-induced myocardial cell injury by regulating the miR‑145‑5p‑Binp3 axis.

The aim of the present study was to investigate the function of long non-coding RNA TUG1 in hypoxia-induced myocardial cell injury and to explore the potential molecular mechanisms. The cardiomyocyte cell line H9c2 was cultured under hypoxic and normoxic conditions. TUG1 expression under hypoxic conditions was then detected. The effects of TUG1 overexpression on viability, apoptosis, migration and invasion were assayed. In addition, the microRNA (miR)-145-5p expression was detected. Following H9c2 cell transfection with miR-145-5p mimics, the H9c2 cell viability, apoptosis, migration and invasion were also detected. Additionally, the target gene of miR-145-5p was assayed by Luciferase reporter assay. The protein expressions of Wnt-3a, Wnt5a, and β-catenin in H9c2 cells under hypoxic conditions were also determined. The results revealed that hypoxia induced injury in H9c2 cells, including inhibiting cell viability, migration and invasion, and promoting cell apoptosis. Overexpression of TUG1 aggravated hypoxia-induced injury in H9c2 cells. In addition, miR-145-5p was negatively regulated by TUG1, and TUG1 overexpression aggravated hypoxia-induced injury via the downregulation of miR-145-5p. Furthermore, B-cell lymphoma 2 interacting protein 3 (Bnip3) was a target of miR-145-5p, and overexpression of Bnip3 aggravated hypoxia-induced cell injury by activating Wnt/β-catenin signaling pathways in H9c2 cells. In conclusion, overexpression of TUG1 aggravated hypoxia-induced injury in cardiomyocytes by regulating the miR-145-5p-Binp3 axis. Activation of the Wnt/β-catenin signaling pathway may be a key mechanism to mediate the role of TUG1 in regulating hypoxia-induced myocardial injury. TUG1 may be an effective diagnostic marker and therapeutic target for myocardial ischemia.