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Sestrin2 maintains OXPHOS integrity to modulate cardiac substrate metabolism during ischemia and reperfusion.

ABSTRACT: Sestrin2 (Sesn2) is a stress-inducible protein that declines with aging in the heart. We reported that rescue Sesn2 levels in aged mouse hearts through gene therapy improves the resistance of aged hearts to ischemia and reperfusion (I/R) insults. We hypothesize that Sesn2 as a scaffold protein maintains mitochondrial integrity to protect heart from ischemic injury during I/R. Young C57BL/6 J (3-6 months), aged C57BL/6 J (24-26 months), and young Sesn2 KO (3-6 months, C57BL/6 J background) mice were subjected to in vivo regional ischemia and reperfusion. The left ventricle was collected for transcriptomics, proteomics and metabolomics analysis. The results demonstrated that Sesn2 deficiency leads to aging-like cardiac diastolic dysfunction and intolerance to ischemia reperfusion stress. Seahorse analysis demonstrated that Sesn2 deficiency in aged and young Sesn2 KO versus young hearts lead to impaired mitochondrial respiration rate with defects in Complex I and Complex II activity. The Sesn2 targeted proteomics analysis revealed that Sesn2 plays a critical role in maintaining mitochondrial functional integrity through modulating mitochondria biosynthesis and assembling of oxidative phosphorylation (OXPHOS) complexes. The RNA-Seq data showed that alterations in the expression of mitochondrial compositional and functional genes and substrate metabolism related genes in young Sesn2 KO and aged versus young hearts. Further immunofluorescence and immunoprecipitation analysis demonstrated that Sesn2 is translocated into mitochondria and interacts with OXPHOS components to maintain mitochondrial integrity in response to I/R stress. Biochemical analysis revealed that Sesn2 is associated with citrate cycle components to modulate pyruvate dehydrogenase and isocitrate dehydrogenase activities during I/R stress. Thus, Sesn2 serves as a scaffold protein interacting with OXPHOS components to maintain mitochondrial integrity under I/R stress. Age-related downregulation of cardiac Sesn2 fragilizes mitochondrial functional integrity in response to ischemic stress.

SUBMITTER: Ren D

PROVIDER: S-EPMC7734306 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Sestrin2 maintains OXPHOS integrity to modulate cardiac substrate metabolism during ischemia and reperfusion.

Ren Di D He Zhibin Z Fedorova Julia J Zhang Jingwen J Wood Elizabeth E Zhang Xiang X Kang David E DE Li Ji J

Redox biology 20201201

Sestrin2 (Sesn2) is a stress-inducible protein that declines with aging in the heart. We reported that rescue Sesn2 levels in aged mouse hearts through gene therapy improves the resistance of aged hearts to ischemia and reperfusion (I/R) insults. We hypothesize that Sesn2 as a scaffold protein maintains mitochondrial integrity to protect heart from ischemic injury during I/R. Young C57BL/6 J (3-6 months), aged C57BL/6 J (24-26 months), and young Sesn2 KO (3-6 months, C57BL/6 J background) mice w ...[more]

PMID: 33316744

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Project description:A novel stress-inducible protein, Sestrin2 (Sesn2), declines in the heart with aging. AMPK has emerged as a pertinent stress-activated kinase that has been shown to have cardioprotective capabilities against myocardial ischemic injury. We identified the interaction between Sesn2 and AMPK in the ischemic heart. To determine whether ischemic AMPK activation-modulated by the Sesn2-AMPK complex in the heart-is impaired in aging that sensitizes the heart to ischemic insults, young C57BL/6 mice (age 3-4 mo), middle-aged mice (age 10-12 mo), and aged mice (age 24-26 mo) were subjected to left anterior descending coronary artery occlusion for in vivo regional ischemia. The ex vivo working heart system was used for measuring substrate metabolism. The protein level of Sesn2 in hearts was gradually decreased with aging. Of interest, ischemic AMPK activation was blunted in aged hearts compared with young hearts (P < 0.05); the AMPK downstream glucose uptake and the rate of glucose oxidation were significantly impaired in aged hearts during ischemia and reperfusion (P < 0.05 vs. young hearts). Myocardial infarction size was larger in aged hearts (P < 0.05 vs. young hearts). Immunoprecipitation with Sesn2 Ab revealed that cardiac Sesn2 forms a complex with AMPK and upstream liver kinase B1 (LKB1) during ischemia. Of interest, the binding affinity between Sesn2 and AMPK upstream LKB1 is impaired in aged hearts during ischemia (P < 0.05 vs. young hearts). Furthermore, Sesn2-knockout hearts demonstrate a cardiac phenotype and response to ischemic stress that is similar to wild-type aged hearts (i.e., impaired ischemic AMPK activation and higher sensitivity to ischemia- and reperfusion- induced injury). Adeno-associated virus-Sesn2 was delivered to aged hearts via a coronary delivery approach and significantly rescued the protein level of Sesn2 and the ischemic tolerance of aged hearts; therefore, Sesn2 is a scaffold protein that mediates AMPK activation in the ischemic myocardium via an interaction with AMPK upstream LKB1. Decreased Sesn2 levels in aging lead to a blunted ischemic AMPK activation, alterations in substrate metabolism, and an increased sensitivity to ischemic insults-Quan, N., Sun, W., Wang, L., Chen, X., Bogan, J. S., Zhou, X., Cates, C., Liu, Q., Zheng, Y., Li J. Sestrin2 prevents age-related intolerance to ischemia and reperfusion injury by modulating substrate metabolism.

Project description:Reperfusion injury after extended ischemia accounts for approximately 50% of myocardial infarct size, and there is no standard therapy. HDAC inhibition reduces infarct size and enhances cardiomyocyte autophagy and PGC1α-mediated mitochondrial biogenesis when administered at the time of reperfusion. Furthermore, a specific autophagy-inducing peptide, Tat-Beclin 1 (TB), reduces infarct size when administered at the time of reperfusion. However, since SAHA affects multiple pathways in addition to inducing autophagy, whether autophagic flux induced by TB maintains mitochondrial homeostasis during ischemia/reperfusion (I/R) injury is unknown. We tested whether the augmentation of autophagic flux by TB has cardioprotection by preserving mitochondrial homeostasis both in vitro and in vivo. Wild-type mice were randomized into two groups: Tat-Scrambled (TS) peptide as the control and TB as the experimental group. Mice were subjected to I/R surgery (45 min coronary ligation, 24 h reperfusion). Autophagic flux, mitochondrial DNA (mtDNA), mitochondrial morphology, and mitochondrial dynamic genes were assayed. Cultured neonatal rat ventricular myocytes (NRVMs) were treated with a simulated I/R injury to verify cardiomyocyte specificity. The essential autophagy gene, ATG7, conditional cardiomyocyte-specific knockout (ATG7 cKO) mice, and isolated adult mouse ventricular myocytes (AMVMs) were used to evaluate the dependency of autophagy in adult cardiomyocytes. In NRVMs subjected to I/R, TB increased autophagic flux, mtDNA content, mitochondrial function, reduced reactive oxygen species (ROS), and mtDNA damage. Similarly, in the infarct border zone of the mouse heart, TB induced autophagy, increased mitochondrial size and mtDNA content, and promoted the expression of PGC1α and mitochondrial dynamic genes. Conversely, loss of ATG7 in AMVMs and in the myocardium of ATG7 cKO mice abolished the beneficial effects of TB on mitochondrial homeostasis. Thus, autophagic flux is a sufficient and essential process to mitigate myocardial reperfusion injury by maintaining mitochondrial homeostasis and partly by inducing PGC1α-mediated mitochondrial biogenesis.

Project description:Search for new cardioprotective therapies is of great importance since no cardioprotective drugs are available on the market. In line with this need, several natural biomolecules have been extensively tested for their potential cardioprotective effects. Previously, we have shown that biglycan, a member of a diverse group of small leucine-rich proteoglycans, enhanced the expression of cardioprotective genes and decreased ischemia/reperfusion-induced cardiomyocyte death via a TLR-4 dependent mechanism. Therefore, in the present study we aimed to test whether decorin, a small leucine-rich proteoglycan closely related to biglycan, could exert cardiocytoprotection and to reveal possible downstream signaling pathways. Methods: Primary cardiomyocytes isolated from neonatal and adult rat hearts were treated with 0 (Vehicle), 1, 3, 10, 30 and 100 nM decorin as 20 h pretreatment and maintained throughout simulated ischemia and reperfusion (SI/R). In separate experiments, to test the mechanism of decorin-induced cardio protection, 3 nM decorin was applied in combination with inhibitors of known survival pathways, that is, the NOS inhibitor L-NAME, the PKG inhibitor KT-5823 and the TLR-4 inhibitor TAK-242, respectively. mRNA expression changes were measured after SI/R injury. Results: Cell viability of both neonatal and adult cardiomyocytes was significantly decreased due to SI/R injury. Decorin at 1, 3 and 10 nM concentrations significantly increased the survival of both neonatal and adult myocytes after SI/R. At 3nM (the most pronounced protective concentration), it had no effect on apoptotic rate of neonatal cardiac myocytes. No one of the inhibitors of survival pathways (L-NAME, KT-5823, TAK-242) influenced the cardiocytoprotective effect of decorin. MYND-type containing 19 (Zmynd19) and eukaryotic translation initiation factor 4E nuclear import factor 1 (Eif4enif1) were significantly upregulated due to the decorin treatment. In conclusion, this is the first demonstration that decorin exerts a direct cardiocytoprotective effect possibly independent of NO-cGMP-PKG and TLR-4 dependent survival signaling.

Dataset Information

Sestrin2 maintains OXPHOS integrity to modulate cardiac substrate metabolism during ischemia and reperfusion.

Publications

Sestrin2 maintains OXPHOS integrity to modulate cardiac substrate metabolism during ischemia and reperfusion.

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