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Cardiomyocyte specific deletion of p53 decreases cell injury during ischemia-reperfusion: Role of Mitochondria.

ABSTRACT: p53 is a tumor suppressor protein with a very low content in the basal condition, but the content rapidly rises during stress conditions including ischemia-reperfusion. An increase in p53 content increases cardiac injury during ischemia-reperfusion. Since mitochondrial damage plays a key role in cardiac injury during ischemia-reperfusion, we asked if genetic ablation of p53 decreases cardiac injury by protecting mitochondria. Isolated, perfused hearts from cardiac specific p53 deletion or wild type underwent 25 min global ischemia at 37 °C and 60 min reperfusion. At the end of reperfusion, hearts were harvested for infarct size measurement. In separate groups, cardiac mitochondria were isolated at 30 min reperfusion. Time control hearts were buffer-perfused without ischemia. Compared to wild type, deletion of p53 improved cardiac functional recovery and decreased infarct size following ischemia-reperfusion. Oxidative phosphorylation was improved in p53 deletion mitochondria following ischemia-reperfusion compared to wild type. The net release of ROS generation from wild type but not in p53 deletion mitochondria was increased following ischemia-reperfusion. Peroxiredoxin 3 (PRDX 3) content was higher in p53 deletion than that in wild type, indicating that p53 deletion increases a key antioxidant. Ischemia-reperfusion led to increased spectrin cleavage (a marker of cytosolic calpain1 activation) in wild type but not in p53 deletion mice. Ischemia-reperfusion increased the truncation of mature AIF (apoptosis inducing factor, an indicator of mitochondrial calpain1 activation) in wild type but not in p53 deletion mice. The loss of cytochrome c from mitochondria was also decreased in p53 deletion following ischemia-reperfusion. Bcl-2 content was decreased in wild type but not in p53 deletion following reperfusion, suggesting that depletion of bcl-2 contributes to permeabilization of the mitochondrial outer membrane. Thus, deletion of p53 decreases cardiac injury by protecting mitochondria through attenuation of oxidative stress and calpain activation during ischemia-reperfusion.

SUBMITTER: Chen Q

PROVIDER: S-EPMC7484321 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Aims:A longevity gene, Sirtuin 1 (SIRT1) and energy sensor AMP-activated protein kinase (AMPK) have common activators such as caloric restriction, oxidative stress, and exercise. The objective of this study is to characterize the role of cardiomyocyte SIRT1 in age-related impaired ischemic AMPK activation and increased susceptibility to ischemic insults. Methods and results:Mice were subjected to ligation of left anterior descending coronary artery for in vivo ischemic models. The glucose and fatty acid oxidation were measured in a working heart perfusion system. The cardiac functions by echocardiography show no difference in young wild-type C57BL/6?J (WT, 4-6?months), aged WT C57BL/6?J (24-26?months), and young inducible cardiomyocyte-specific SIRT1 knockout (icSIRT1 KO) (4-6?months) mice under physiological conditions. However, after 45 mins ischaemia and 24-h reperfusion, the ejection fraction of aged WT and icSIRT1 KO mice was impaired. The aged WT and icSIRT1 KO hearts vs. young WT hearts also show an impaired post-ischemic contractile function in a Langendorff perfusion system. The infarct size of aged WT and icSIRT1 KO hearts was larger than that of young WT hearts. The immunoblotting data demonstrated that aged WT and icSIRT1 KO hearts vs. young WT hearts had impaired phosphorylation of AMPK and downstream acetyl-CoA carboxylase during ischaemia. Intriguingly, AMPK upstream LKB1 is hyper-acetylated in both aged WT and icSIRT1 KO hearts; this could blunt activation of LKB1, leading to an impaired AMPK activation. The working heart perfusion results demonstrated that SIRT1 deficiency significantly impaired substrate metabolism in the hearts; fatty acid oxidation is augmented and glucose oxidation is blunted during ischaemia and reperfusion. Adeno-associated virus (AAV9)-Sirt1 was delivered into the aged hearts via a coronary delivery approach, which significantly rescued the protein level of SIRT1 and the ischemic tolerance of aged hearts. Furthermore, AMPK agonist can rescue the tolerance of aged heart and icSIRT1 KO heart to ischemic insults. Conclusions:Cardiac SIRT1 mediates AMPK activation via LKB1 deacetylation, and AMPK modulates SIRT1 activity via regulation of NAD+ level during ischaemia. SIRT1 and AMPK agonists have therapeutic potential for treatment of aging-related ischemic heart disease.

Project description:BackgroundIn myocardial ischemia-reperfusion injury, myocardial damage is aggravated when blood perfusion is restored in myocardial infarction. Ubiquitin-specific protease 11 (USP11), a deubiquitinating enzyme, could remove the ubiquitination of substrate proteins and regulate protein stability, thereby affecting multiple pathological processes.AimsTo investigate the potential function of USP11 in myocardial ischemia-reperfusion injury and its underlying mechanisms.Study designIn vivo and in vitro experimental study.MethodsThe ischemia-reperfusion rat model in vivo was evolved, wherein the left anterior descending coronary artery was ligated for 30 min, followed by ligature release for 120 min. Meanwhile, H9C2 cells were brought to hypoxia for 6 h and then reoxygenated for 18 h to establish a cell hypoxia-reoxygenation (H/R) injury in vitro. Then, the loss-of-function experiments of USP11 were performed. Triphenyltetrazolium chloride and hematoxylin and eosin staining were performed to observe myocardial injury. The MTT assay was utilized to detect H9C2 cell viability. Pyroptosis was analyzed by TUNEL staining and flow cytometry. Pyroptosis-related protein expression and TRAF3 were analyzed via Western blot. The content of inflammatory factors was examined by enzyme-linked immunoassay. Co-immunoprecipitation and ubiquitination assays were performed to analyze for USP11 interacting with TRAF3.ResultsUSP11 was upregulated in the ischemic heart tissue. Ischemia-reperfusion and H/R injuries increased USP11 expression. USP11 loss-of-function assays showed that USP11 knockdown alleviated ischemia-reperfusion- and H/R-induced myocardial cell damage, pyroptosis, pro-inflammatory factor secretion, and IKKβ/NF-κB pathway activation. In H9C2 cells, USP11 stabilized TRAF3 by deubiquitination. Furthermore, rescue experiments revealed that TRAF3 overexpression reversed the protection of silencing USP11 on H/R-induced H9C2 cell injury.ConclusionThis study confirmed that USP11 knockdown ameliorated myocardial ischemia-reperfusion injury by downregulating TRAF3, suggesting that USP11 silencing can be a novel target of myocardial infarction.

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Cardiomyocyte specific deletion of p53 decreases cell injury during ischemia-reperfusion: Role of Mitochondria.

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