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.

Sestrin2（Sesn2）是一种压力诱导型蛋白质，随着心脏衰老而下降。我们报告说，通过基因疗法挽救衰老小鼠心脏中的Sesn2水平，可以提高衰老心脏对缺血和再灌注（I / R）损伤的抵抗力。我们假设Sesn2作为支架蛋白可以维持线粒体完整性，从而在I / R期间保护心脏免受缺血性损伤。在体内对C57BL / 6 J（3-6个月），C57BL / 6 J（24-26个月），Sesn2 KO（3-6个月，C57BL / 6 J）的年轻小鼠进行了体内实验局部缺血和再灌注。收集左心室用于转录组学，蛋白质组学和代谢组学分析。结果表明，Sesn2缺乏症会导致衰老样心脏舒张功能障碍和对缺血再灌注应激的耐受性。海马分析表明，老年和年轻的Sesn2 KO与年轻的心脏相比，Sesn2缺乏会导致线粒体呼吸速率受损，并伴有复合物I和复合物II活性的缺陷。Sesn2靶向蛋白质组学分析表明，Sesn2在通过调节线粒体生物合成和氧化磷酸化（OXPHOS）复合物的组装来维持线粒体功能完整性方面起着至关重要的作用。RNA-Seq数据显示，年轻的Sesn2 KO和老年与年轻的心脏中线粒体组成和功能基因以及与底物代谢相关的基因的表达发生了变化。进一步的免疫荧光和免疫沉淀分析表明，Sesn2易位到线粒体中并与OXPHOS组分相互作用，以响应I / R应激而维持线粒体完整性。生化分析表明，Sesn2与柠檬酸循环成分有关，从而在I / R应激期间调节丙酮酸脱氢酶和异柠檬酸脱氢酶的活性。因此，Sesn2充当与OXPHOS组件相互作用的支架蛋白，以在I / R应力下维持线粒体的完整性。年龄相关的心脏Sesn2下调可减轻线粒体功能完整性，以应对缺血性应激。