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CTRP13 Protects H9c2 Cells Against Hypoxia/Reoxygenation (H/R)-Induced Injury Via Regulating the AMPK/Nrf2/ARE Signaling Pathway.

ABSTRACT: Myocardial infarction (MI) is identified as the myocardial necrosis due to myocardial ischemia/reperfusion (I/R) injury and remains a leading cause of mortality. C1q/TNF-related protein 13 (CTRP13) is a member of CTRP family that has been found to be involved in coronary artery disease (CAD). However, the role of CTRP13 in MI remains unclear. We aimed to explore the functional role of CTRP13 in H9c2 cells exposed to hypoxia/reoxygenation (H/R). Our results demonstrated that H/R stimulation significantly decreased the expression of CTRP13 in H9c2 cells. H/R-induced an increase in ROS production and reductions in activities of SOD and CAT were prevented by CTRP13 overexpression but were aggravated by CTRP13 silencing. Moreover, CTRP13 overexpression could reverse the inductive effect of H/R on caspase-3 activity and bax expression, as well as the inhibitory effect of H/R on bcl-2 expression in H9c2 cells. However, CTRP13 silencing presented opposite effects with CTRP13 overexpression. Furthermore, CTRP13 overexpression enhanced the H/R-stimulated the expression levels of p-AMPK and nuclear Nrf2, and Nrf2 transcriptional activity. However, inhibition of AMPK reversed the CTRP13-mediated activation of Nrf2/ARE signaling and the cardiac-protective effect in H/R-exposed H9c2 cells. Additionally, silencing of Nrf2 reversed the protective effects of CTRP13 against H/R-stimulated oxidative stress and apoptosis in H9c2 cells. Finally, recombinant CTRP13 protein attenuated myocardial I/R-induced injury in rats. Taken together, these findings indicated that CTRP13 protected H9c2 cells from H/R-stimulated oxidative stress and apoptosis via regulating the AMPK/Nrf2/ARE signaling pathway. Our results provided evidence for the therapeutic potential of CTRP13 in myocardial I/R injury.

SUBMITTER: Jiang W

PROVIDER: S-EPMC8335840 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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.

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CTRP13 Protects H9c2 Cells Against Hypoxia/Reoxygenation (H/R)-Induced Injury Via Regulating the AMPK/Nrf2/ARE Signaling Pathway.

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