SIRT3 is identified as the major mitochondrial deacetylase. Two distinct isoforms of the murine SIRT3 have been identified with the short isoform having no recognizable mitochondrial localization sequence (MLS) and the long isoform having a putative MLS. A recent study questions the mitochondrial deacetylase activity of this short isoform. In contrast, the long isoform has been shown to be predominantly mitochondrial with robust deacetylase activity. Here we investigate whether the amino-terminus of the long SIRT3 isoform is a legitimate MLS and evaluate in-situ mitochondrial deacetylase activity of both isoforms. We confirm the presence of long and short isoforms in murine liver and kidney. The long isoform is generated via intra-exon splicing creating a frame-shift to expose a novel upstream translation start site. Mitochondrial localization is significantly more robust following transfection of the long compared to the short isoform. Insertion of this alternatively spliced novel 5’ sequence upstream of a GFP-reporter plasmid shows greater than 80% enrichment in mitochondria, confirming this region as a legitimate mitochondrial localization sequence. Despite lower mitochondrial expression of the short isoform, the capacity to deacetylate mitochondrial proteins and to restore mitochondrial respiration is equally robust following transient transfection of either isoform into SIRT3 knockout embryonic fibroblasts. How these alternative transcripts are regulated and whether they modulate distinct targets is unknown. Furthermore, in contrast to exclusive mitochondrial enrichment of endogenous SIRT3, overexpression of both isoforms show nuclear localization. This overexpression effect, may partially account for previously observed divergent phenotypes attributed to SIRT3.

Prolonged cardiac overexpression of the mitochondrial biogenesis regulatory transcriptional coactivator PGC-1α disrupts cardiac contractile function and its genetic ablation limits cardiac capacity to enhance work-load. In contrast, transient induction of PGC-1α alleviates neuronal cell oxidative stress and enhances skeletal myotube antioxidant defenses. We explored whether transient upregulation of PGC-1α in the heart protects against ischemia-reperfusion injury. The transient induction of PGC-1α in the cardiac-restricted inducible PGC-1α transgenic mouse, increased PGC-1α protein levels 5-fold. Following 25 minutes of ischemia and 2 hours of reperfusion on a Langendorff perfusion apparatus, contractile recovery and the rate pressure product was significantly blunted in mice overexpressing PGC-1α vs. controls. Affymetrix gene array analysis showed a 3-fold PGC-1α-mediated upregulation of adenine nucleotide translocase 1 (ANT1). As ANT1 upregulation induces cardiomyocyte cell death we investigated whether the induction of ANT1 by PGC-1α contributes to this enhanced ischemia-stress susceptibility. Infection with adenovirus harboring PGC-1α into cardiac-derived H9c2 cells significantly upregulates ANT1 without changing basal cell viability. In response to anoxia-reoxygenation injury cell death is significantly increased following PGC-1α overexpression. This detrimental effect is abolished following siRNA knockdown of ANT1. Similarly, the attenuation of ANT-1 in the presence of PGC-1α overexpression preserves the mitochondrial membrane potential in response to hydrogen-peroxide stress. Interestingly, the isolated knockdown of ANT1 also protects H9c2 cells from anoxia-reoxygenation injury. Taken together these data suggest that transient induction of PGC-1α in the murine heart decreases ischemiareperfusion contractile recovery and diminishes anoxia-reoxygenation tolerance in H9c2 cells. These adverse phenotypes appear to be mediated, in part, by PGC-1α induced upregulation of ANT1. Keywords PGC-1α; Cardiac ischemia-reperfusion; ANT1Mitochondrial homeostasis and regulatory adaptations are recognized as central to the myocardial capacity to tolerate both ischemic and oxidative stress [1,2]. In light of our increased understanding of the molecular programs governing mitochondrial homeostasis it is postulated that modulation of mitochondrial biology may be a feasible strategy to enhance tolerance to Contact Details: Michael N. Sack, Translational Medicine Branch, NHLBI, NIH, Bld 10-CRC, Room 5-3150, 10 Center Drive, Bethesda, MD, 20892-1454, USA, sackm@nhlbi.nih.gov. Disclosures: None.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content,...

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