The long-term goals of this project are to 1) understand the role of mitochondria in ischemia-reperfusion injury and cardioprotection ; 2) to understand the role of altered ion homeostasis and altered metabolism in ischemia-reperfusion and cardioprotection and 3) to understand changes in cytosolic and mitochondrial signaling involved in cardioprotection and cell death. It is proposed that ischemic preconditioning (PC) initiates signaling that converges on mitochondria and results in cardioprotection. It has been proposed that cells die following ischemia and reperfusion because of a rise in mitochondrial calcium which leads to activation of the mitochondrial permeability transition pore (MPT). The increase in mitochondrial calcium is proposed to occur via calcium uptake by the mitochondrial calcium uniporter. The composition of the MPT is unknown, but cyclophilin D has been shown to be a regulator of the MPT. We examined the role of calcium uptake by mitochondrial calcium uniporter (MCU) in regulating cell death and metabolism. In collaboration with Dr. Toren Finkel, we characterize a mouse model that lacks expression of the recently discovered mitochondrial calcium uniporter (MCU). Mitochondria derived from MCU(-/-) mice have no apparent capacity to rapidly uptake calcium. Whereas basal metabolism seems unaffected, the skeletal muscle of MCU(-/-) mice exhibited alterations in the phosphorylation and activity of pyruvate dehydrogenase. In addition, MCU(-/-) mice exhibited marked impairment in their ability to perform strenuous work. We further show that mitochondria from MCU(-/-) mice lacked evidence for calcium-induced permeability transition pore (PTP) opening. Surprisingly, the lack of PTP opening does not seem to protect MCU(-/-) cells and tissues from cell death. Following global I/R in a Langendorff perfused heart model infarct size was indistinguishable between WT hearts and hearts from MCU-/- mice. To further complicate the picture, cyclosporine A, an inhibitor of the mPTP, reduced infarct size in WT hearts, but not in MCU-/- hearts. We have been testing whether loss of MCU leads to alterations in other cell death pathways. We also examined the response of the mouse to addition of isoproterenol. We also examined the role of succinylation in regulating cardiac function and response to ischemia and reperfusion. Succinylation refers to modification of lysine residues with succinyl groups donated by succinyl-CoA. Sirtuin5 (Sirt5) is a mitochondrial NAD+-dependent deacylase that catalyzes the removal of succinyl groups from proteins. Sirt5 and protein succinylation are conserved across species, suggesting functional importance of the modification. Sirt5 loss impacts liver metabolism but the role of succinylation in the heart has not been explored. We combined affinity enrichment with proteomics and mass spectrometry to analyze total succinylated lysine content of mitochondria isolated from WT and Sirt5-/- mouse hearts. We identified 887 succinylated lysine residues in 184 proteins. 44 peptides (5 proteins) occurred uniquely in WT samples, 289 (46 proteins) in Sirt5-/- samples, and 554 (133 proteins) were common to both groups. The 46 unique proteins in Sirt5-/- heart participate in metabolic processes such as fatty acid β-oxidation (Eci2) and branched chain amino acid catabolism, and include respiratory chain proteins (Ndufa7, 12, 13, Dhsa). We performed label-free analysis of the peptides common to WT and Sirt5-/- hearts. 16 peptides from 9 proteins were significantly increased in Sirt5-/- by at least 30%. The adenine nucleotide transporter 1 showed the highest increase in succinylation in Sirt5-/- (108.4 fold). The data indicates that succinylation is widespread in the heart and enriched in metabolic pathways. We examined whether the loss of Sirt5 would impact ischemia-reperfusion (I/R) injury and we found an increase in infarct size in Sirt5-/- hearts compared to WT littermates (68.5+/-1.1% Sirt5-/- vs 39.6+/- 6.8% WT) following 20 minutes of ischemia and 90 minutes reperfusion. We further demonstrate that the degree of I/R injury in Sirt5-/- heart is restored to WT levels by pretreatment with dimethyl malonate, a competitive inhibitor of succinate dehydrogenase (SDH), implicating alteration in SDH activity as causative of the injury.

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U.S. National Heart Lung and Blood Inst
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