Transient upregulation of PGC-1alpha diminishes cardiac ischemia tolerance via upregulation of ANT1.

Prolonged cardiac overexpression of the mitochondrial biogenesis regulatory transcriptional coactivator PGC-1alpha disrupts cardiac contractile function and its genetic ablation limits cardiac capacity to enhance workload. In contrast, transient induction of PGC-1alpha alleviates neuronal cell oxidative stress and enhances skeletal myotube anti-oxidant defenses. We explored whether transient upregulation of PGC-1alpha in the heart protects against ischemia-reperfusion injury. The transient induction of PGC-1alpha in the cardiac-restricted inducible PGC-1alpha transgenic mouse, increased PGC-1alpha protein levels 5-fold. Following 25 min of ischemia and 2h of reperfusion on a Langendorff perfusion apparatus, contractile recovery and the rate pressure product was significantly blunted in mice overexpressing PGC-1alpha vs. controls. Affymetrix gene array analysis showed a 3-fold PGC-1alpha-mediated upregulation of adenine nucleotide translocase 1 (ANT1). As ANT1 upregulation induces cardiomyocyte cell death we investigated whether the induction of ANT1 by PGC-1alpha contributes to this enhanced ischemia-stress susceptibility. Infection with adenovirus harboring PGC-1alpha 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-1alpha overexpression. This detrimental effect is abolished following siRNA knockdown of ANT1. Similarly, the attenuation of ANT-1 in the presence of PGC-1alpha 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-1alpha in the murine heart decreases ischemia-reperfusion contractile recovery and diminishes anoxia-reoxygenation tolerance in H9c2 cells. These adverse phenotypes appear to be mediated, in part, by PGC-1alpha induced upregulation of ANT1.

Characterization of the murine SIRT3 mitochondrial localization sequence and comparison of mitochondrial enrichment and deacetylase activity of long and short SIRT3 isoforms.

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. In this study, 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 with 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 shows nuclear localization. This overexpression effect, may partially account for previously observed divergent phenotypes attributed to SIRT3.