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Mitochondrial PKM2 deacetylation by procyanidin B2-induced SIRT3 upregulation alleviates lung ischemia/reperfusion injury.

ABSTRACT: Apoptosis is a critical event in the pathogenesis of lung ischemia/reperfusion (I/R) injury. Sirtuin 3 (SIRT3), an important deacetylase predominantly localized in mitochondria, regulates diverse physiological processes, including apoptosis. However, the detailed mechanisms by which SIRT3 regulates lung I/R injury remain unclear. Many polyphenols strongly regulate the sirtuin family. In this study, we found that a polyphenol compound, procyanidin B2 (PCB2), activated SIRT3 in mouse lungs. Due to this effect, PCB2 administration attenuated histological lesions, relieved pulmonary dysfunction, and improved the survival rate of the murine model of lung I/R injury. Additionally, this treatment inhibited hypoxia/reoxygenation (H/R)-induced A549 cell apoptosis and rescued Bcl-2 expression. Using Sirt3-knockout mice and specific SIRT3 knockdown in vitro, we further found that SIRT3 strongly protects against lung I/R injury. Sirt3 deficiency or enzymatic inactivation substantially aggravated lung I/R-induced pulmonary lesions, promoted apoptosis, and abolished PCB2-mediated protection. Mitochondrial pyruvate kinase M2 (PKM2) inhibits apoptosis by stabilizing Bcl-2. Here, we found that PKM2 accumulates and is hyperacetylated in mitochondria upon lung I/R injury. By screening the potential sites of PKM2 acetylation, we found that SIRT3 deacetylates the K433 residue of PKM2 in A549 cells. Transfection with a deacetylated mimic plasmid of PKM2 noticeably reduced apoptosis, while acetylated mimic transfection abolished the protective effect of PKM2. Furthermore, PKM2 knockdown or inhibition in vivo significantly abrogated the antiapoptotic effects of SIRT3 upregulation. Collectively, this study provides the first evidence that the SIRT3/PKM2 pathway is a protective target for the suppression of apoptosis in lung I/R injury. Moreover, this study identifies K433 deacetylation of PKM2 as a novel modification that regulates its anti-apoptotic activity. In addition, PCB2-mediated modulation of the SIRT3/PKM2 pathway may significantly protect against lung I/R injury, suggesting a novel prophylactic strategy for lung I/R injury.

SUBMITTER: Zhao J

PROVIDER: S-EPMC9276754 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundHydrogen peroxide (H2O2)-induced mitochondrial oxidative damage is critical to intestinal ischemia/reperfusion (I/R) injury, and PRDX3 is an efficient H2O2 scavenger that protects cells from mitochondrial oxidative damage and apoptosis. However, the function of PRDX3 in intestinal I/R injury is unclear. The aim of this study was to investigate the precise mechanism underlying the involvement of PRDX3 in intestinal I/R injury.MethodsAn intestinal I/R model was established in mice with superior mesenteric artery occlusion, and Caco-2?cells were subjected to hypoxia/reoxygenation (H/R) for the in vivo simulation of I/R.ResultsPRDX3 expression was decreased during intestinal I/R injury, and PRDX3 overexpression significantly attenuated H/R-induced mitochondrial oxidative damage and apoptosis in Caco-2?cells. The level of acetylated PRDX3 was clearly increased both in vivo and in vitro. The inhibition of SIRTs by nicotinamide (NAM) increased the level of acetylated PRDX3 and impaired the antioxidative activity of PRDX3. Furthermore, NAM did not increase the acetylation of PRDX3 in sirtuin-3 (SIRT3)-knockdown Caco-2?cells. Importantly, PRDX3 acetylation was increased in mice lacking SIRT3, and this effect was accompanied by serious mitochondrial oxidative damage, apoptosis and remote organ damage after intestinal I/R injury. We screened potential sites of PRDX3 acetylation in the previously reported acetylproteome through immunoprecipitation (IP) experiments and found that SIRT3 deacetylates K253 on PRDX3 in Caco-2?cells. Furthermore, PRDX3 with the lysine residue K253 mutated to arginine (K253R) increased its dimerization in Caco-2?cells after subjected to 12?h hypoxia and followed 4?h reoxygenation. Caco-2?cells transfected with the K253R plasmid exhibited notably less mitochondrial damage and apoptosis, and transfection of the K253Q plasmid abolished the protective effect of PRDX3 overexpression. Analysis of ischemic intestines from clinical patients further verified the correlation between SIRT3 and PRDX3.ConclusionsPRDX3 is a key protective factor for intestinal I/R injury, and SIRT3-mediated PRDX3 deacetylation can alleviate intestinal I/R-induced mitochondrial oxidative damage and apoptosis.

Project description:Mitochondrial dysfunction is a salient feature of myocardial ischemia/reperfusion injury (MIRI), while the potential mechanism of mitochondrial dynamics disorder remains unclear. This study sought to explore whether activation of Adenosine monophosphate-activated protein kinase (AMPK) could alleviate MIRI by regulating GTPase dynamin-related protein 1 (Drp1)-mediated mitochondrial dynamics. Isolated mouse hearts in a Langendorff perfusion system were subjected to ischemia/reperfusion (I/R) treatment, and H9C2 cells were subjected to hypoxia /reoxygenation (H/R) treatment in vitro. The results showed that AICAR, the AMPK activator, could significantly improve the function of left ventricular, decrease arrhythmia incidence and myocardial infarction area of isolated hearts. Meanwhile, AICAR increased superoxide dismutase (SOD) activity and decreased malondialdehyde (MDA) content in myocardial homogenate. Mechanistically, AICAR inhibited the phosphorylation of Drp1 at Ser 616 while enhanced phosphorylation of Drp1 at Ser 637. In addition, AICAR reduced the expression of inflammatory cytokines including TNF-ɑ, IL-6, and IL-1β, as well as mitochondrial fission genes Mff and Fis1, while improved the expression of mitochondrial fusion genes Mfn1 and Mfn2. Similar results were also observed in H9C2 cells. AICAR improved mitochondrial membrane potential (MMP), reduced reactive oxygen species (ROS) production, and inhibited mitochondrial damage. To further prove if Drp1 regulated mitochondrial dynamics mediated AMPK protection effect, the mitochondrial fission inhibitor Mdivi-1 was utilized. We found that Mdivi-1 significantly improved MMP, inhibited ROS production, reduced the expression of TNF-a, IL-6, IL-1β, Fis1, and Mff, and improved the expression of Mfn1 and Mfn2. However, the protection effect of Mdivi-1 was not reversed by AMPK inhibitor Compound C. In conclusion, this study confirmed that activation of AMPK exerted the protective effects on MIRI, which were largely dependent on the inhibition of Drp1-mediated mitochondrial fission.

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Mitochondrial PKM2 deacetylation by procyanidin B2-induced SIRT3 upregulation alleviates lung ischemia/reperfusion injury.

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