Abstract
Introduction: SIRT3 deficiency leads to increased mitochondrial acetylation, and enhances sensitivity of the heart to stress. Recently we demonstrated that a worse outcome of ischemia-reperfusion (IR) injury in aged hearts was associated with increased protein acetylation. The aim of this study was to investigate the mechanism of acetylation and the identity of acetylated targets in aged hearts in the context of IR injury. Methods: Mitochondria were isolated from WT adult (7 mo.), SIRT3 +/- adult (7 mo.) and WT aged (18 mo.) hearts. SIRT3 protein expression and identification of acetylated proteins from SIRT3 +/- adult and WT aged samples were investigated using proteomics (2D gel, western blot, peptide mass fingerprinting). Results: Mitochondrial acetylome analysis revealed that many hyperacetylated patterns were identical between adult SIRT3 +/- and aged WT hearts, suggesting that increased protein acetylation in the aged WT heart might be due to SIRT3 inhibition. Several targets were indentified, including complex I NADH dehydrogenase flavoprotein 1, which is known to interact with SIRT3. In this regard, we demonstrated that respiratory complex I (CxI) activity was significantly inhibited in both SIRT3 +/- adult and WT aged hearts. Analysis of SIRT3 protein revealed that negatively charged species of SIRT3 (relative to the bulk SIRT3 population), were lost in aged hearts. These alterations suggest post-translational modification of SIRT3 which may lead to loss of its deacetylase activity. Alternatively, we found that upregulation of the mitochondrial acetyltransferase GCN5L1 in aged hearts (examined by western blot) may also contribute to enhanced protein acetylation. Conclusions: These data support a connection between SIRT3 downregulation, mitochondrial protein acetylation, and exacerbation of IR injury in aging hearts. Furthermore, it is known that functional CxI is required for several modes of cardioprotection suggesting that downregulation of CxI in SIRT3 +/- or WT aged hearts may underlie their poor recovery from IR injury.
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