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Sevoflurane Postconditioning Reduces Hypoxia-Reoxygenation Injury in H9C2 Embryonic Rat Cardiomyocytes and Targets the STRADA Gene by Upregulating microRNA-107.

ABSTRACT: BACKGROUND Sevoflurane as a widely used inhalational general anesthetic that also has a cardioprotective role in hypoxia-reoxygenation (H/R) injury. This study aimed to investigate the effects of microRNA-107 (miR-107) on sevoflurane postconditioning (SpostC) in H9C2 embryonic rat cardiomyocytes and to use bioinformatics analysis to identify the molecular basis of cardioprotection from sevoflurane in human cardiac tissue. MATERIAL AND METHODS The STRADA gene was identified from the Gene Expression Omnibus (GEO) database. H9C2 embryonic rat cardiomyocytes were cultured with sevoflurane. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to measure the mRNA expression and protein expression of STRADA and miR-107 in H9C2 cells. TargetScanHuman version 7.2 was used to identify the target gene of miR-107 and to predict the STRADA 3'-UTR binding site of miR-107. The dual-luciferase reporter assay measured the relative luciferase activity. The cell proliferation rate and cell apoptosis were measured using the MTT assay and flow cytometry, respectively. RESULTS H/R injury in H9C2 cells following SpostC resulted in increased expression of miR-107 and reduced expression of STRADA. Specific binding of miR-107 was identified to STRADA 3'-UTR. Upregulation of the miR-107 in SpostC H/R injured H9C2 cells promoted cell proliferation, reduced cell apoptosis, and downregulating the protein expression of caspase-3. STRADA overexpression reduced the effects of a miR-107 mimic on SpostC. CONCLUSIONS SpostC reduced H/R injury in H9C2 embryonic rat cardiomyocytes by targeting the STRADA gene and by upregulating the expression of microRNA-107.

SUBMITTER: Jiang Q

PROVIDER: S-EPMC7197225 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Sevoflurane Postconditioning Reduces Hypoxia-Reoxygenation Injury in H9C2 Embryonic Rat Cardiomyocytes and Targets the STRADA Gene by Upregulating microRNA-107.

Jiang Qun Q Gu Shan S

Medical science monitor : international medical journal of experimental and clinical research 20200425

BACKGROUND Sevoflurane as a widely used inhalational general anesthetic that also has a cardioprotective role in hypoxia-reoxygenation (H/R) injury. This study aimed to investigate the effects of microRNA-107 (miR-107) on sevoflurane postconditioning (SpostC) in H9C2 embryonic rat cardiomyocytes and to use bioinformatics analysis to identify the molecular basis of cardioprotection from sevoflurane in human cardiac tissue. MATERIAL AND METHODS The STRADA gene was identified from the Gene Expressi ...[more]

PMID: 32332694

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Project description:Subject: Cardiovascular disease, as a very common and serious coexisting disease in diabetic patients, and is one of the risk factors that seriously affect the prognosis and complications of surgical patients. Previous studies have shown that sevoflurane post-conditioning (SPostC) exerts a protective effect against myocardial ischemia/reperfusion injury by HIF-1α, but the protective effect is weakened or even disappeared under hyperglycemia. This study aims to explore whether regulating the HIF-1α/MIF/AMPK signaling pathway can restore the protective effect and reveal the mechanism of SPostC on cardiomyocyte hypoxia/reoxygenation injury under high glucose conditions. Methods: H9c2 cardiomyocytes were cultured in normal and high-concentration glucose medium to establish a hypoxia/reoxygenation (H/R) injury model of cardiomyocytes. SPostC was performed with 2.4% sevoflurane for 15 min before reoxygenation. Cell damage was determined by measuring cell viability, lactate dehydrogenase activity, and apoptosis; Testing cell energy metabolism by detecting reactive oxygen species (ROS) generation, ATP content and mitochondrial membrane potential; Analysis of the change of HIF-1α, MIF and AMPKα mRNA expression by RT-PCR. Western blotting was used to examine the expression of HIF-1α, MIF, AMPKα and p-AMPKα proteins. HIF-1α and MIF inhibitors and agonists were administered 40 min before hypoxia. Results: 1) SPostC exerts a protective effect by increasing cell viability, reducing LDH levels and cell apoptosis under low glucose (5 μM) after undergoing H/R injury; 2) High glucose concentration (35 μM) eliminated the cardioprotective effect of SPostC, which is manifested by a significantly decrease in the protein and mRNA expression level of the HIF-1α/MIF/AMPK signaling pathway, accompanied by decreased cell viability, increased LDH levels and apoptosis, increased ROS production, decreased ATP synthesis, and decreased mitochondrial membrane potential; 3. Under high glucose (35 μM), the expression levels of HIF-1α and MIF were up-regulated by using agonists, which can significantly increase the level of p-AMPKα protein, and the cardioprotective effect of SPostC was restored. Conclusion: The signal pathway of HIF-1α/MIF/AMPK of H9c2 cardiomyocytes may be the key point of SPostC against H/R injure. The cardioprotective of SPostC could be restored by upregulating the protein expression of HIF-1α and MIF under hyperglycemia.

Project description:Anesthetic postconditioning is a cellular protective approach whereby exposure to a volatile anesthetic renders a tissue more resistant to subsequent ischemic/reperfusion event. Sevoflurane postconditioning (SPostC) has been shown to exert cardioprotection against ischemia/reperfusion injury, but the underlying mechanism is unclear. We hypothesized that SPostC protects cardiomyocytes against hypoxia/reoxygenation (H/R) injury by maintaining/restoring mitochondrial morphological integrity, a critical determinant of cell fate.Primary cultures of neonatal rat cardiomyocytes (NCMs) were subjected to H/R injury (3 h of hypoxia followed by 3 h reoxygenation). Intervention with SPostC (2.4% sevoflurane) was administered for 15 min upon the onset of reoxygenation. Cell viability, Lactate dehydrogenase (LDH) level, cell death, mitochondrial morphology, mitochondrial membrane potential and mitochondrial permeability transition pore (mPTP) opening were assessed after intervention. Mitochondrial fusion and fission regulating proteins (Drp1, Fis1, Mfn1, Mfn2 and Opa1) were assessed by immunofluorescence staining and western blotting was performed to determine the level of protein expression.Cardiomyocyte H/R injury resulted in significant increases in LDH release and cell death that were concomitant with reduced cell viability and reduced mitochondrial interconnectivity (mean area/perimeter ratio) and mitochondrial elongation, and with reduced mitochondrial membrane potential and increased mPTP opening. All the above changes were significantly attenuated by SPostC. Furthermore, H/R resulted in significant reductions in mitochondrial fusion proteins Mfn1, Mfn2 and Opa1 and significant enhancement of fission proteins Drp1 and Fis1. SPostC significantly enhanced Mfn2 and Opa1 and reduced Drp1, without significant impact on Mfn1 and Fis1.Sevoflurane postconditioning attenuates cardiomyocytes hypoxia/reoxygenation injury (HRI) by restoring mitochondrial fusion/fission balance and morphology.

Project description:BackgroundMitochondrial dysfunction would ultimately lead to myocardial cell apoptosis and death during ischemia-reperfusion injuries. Autophagy could ameliorate mitochondrial dysfunction by autophagosome forming, which is a catabolic process to preserve the mitochondrial's structural and functional integrity. HO-1 induction and expression are important protective mechanisms. This study in order to investigate the role of HO-1 during mitochondrial damage and its mechanism.Methods and resultsThe H9c2 cardiomyocyte cell line were incubated by hypoxic and then reoxygenated for the indicated time (2, 6, 12, 18, and 24 h). Cell viability was tested with CCK-8 kit. The expression of endogenous HO-1(RT-PCR and Western blot) increased with the duration of reoxygenation and reached maximum levels after 2 hours of H/R; thereafter, the expression gradually decreased to a stable level. Mitochondrial dysfunction (Flow cytometry quantified the ROS generation and JC-1 staining) and autophagy (The Confocal microscopy measured the autophagy. RFP-GFP-LC3 double-labeled adenovirus was used for testing.) were induced after 6 hours of H/R. Then, genetic engineering technology was employed to construct an Lv-HO1-H9c2 cell line. When HO-1 was overexpressed, the LC3II levels were significantly increased after reoxygenation, p62 protein expression was significantly decreased, the level of autophagy was unchanged, the mitochondrial membrane potential was significantly increased, and the mitochondrial ROS level was significantly decreased. Furthermore, when the HO-1 inhibitor ZnPP was applied the level of autophagy after reoxygenation was significantly inhibited, and no significant improvement in mitochondrial dysfunction was observed.ConclusionsDuring myocardial hypoxia-reoxygenation injury, HO-1 overexpression induces autophagy to protect the stability of the mitochondrial membrane and reduce the amount of mitochondrial oxidation products, thereby exerting a protective effect.

Dataset Information

Sevoflurane Postconditioning Reduces Hypoxia-Reoxygenation Injury in H9C2 Embryonic Rat Cardiomyocytes and Targets the STRADA Gene by Upregulating microRNA-107.

Publications

Sevoflurane Postconditioning Reduces Hypoxia-Reoxygenation Injury in H9C2 Embryonic Rat Cardiomyocytes and Targets the STRADA Gene by Upregulating microRNA-107.

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