Abstract Body: Downregulation of genes involved in mitochondrial energy production is a hallmark of heart failure, which associates with inhibition of RNA polymerase II (Pol II) recruitment to the gene promoters. Sirt1 is a protein deacetylase regulating transcription of metabolic genes. However, how Sirt1 regulates metabolic genes in heart failure is controversial. Here we show that Sirt1 promotes pressure overload (PO)-induced downregulation of metabolic genes possibly through inhibition of the Pol II recruitment. Cardiac-specific Sirt1 overexpression in mice (Tg-Sirt1) promoted PO-induced cardiac dysfunction (Ejection fraction (%): wild type (WT) sham 76, WT PO 68, Tg-Sirt1 sham 76, Tg-Sirt1 PO 48*, p<0.05 vs WT PO) and downregulation of metabolic genes, including Idh3a (relative Idh3a mRNA levels; WT Sham 1.0, WT PO 0.55, Tg-Sirt1 0.98, Tg-Sirt1 PO 0.41, p<0.05 vs WT PO). In cultured cardiomyocytes, Sirt1 overexpression suppressed metabolic enzymes, reporter gene activity driven by endogenous metabolic gene promoters, and mitochondrial respiration. Chromatin immunoprecipitation and in vitro DNA binding assays, using 500bp of endogenous Idh3a promoter sequence, showed that Sirt1 inhibits the Pol II recruitment to the promoter. Unbiased screening using mass spectrometry identified Sub1 as a Sirt1 binding protein. Sub1 is an evolutionally conserved DNA binding protein that mediates the Pol II recruitment via the preinitiation complex formation. We confirmed direct binding of Sub1 to the Sirt1 catalytic domain (235-499aa) and Sub1-dependent Sirt1 recruitment to the Idh3a promoter using the in vitro reconstitution system. In vitro DNA binding assays with the Idh3a promoter sequence demonstrated that Sirt1 inhibits preinitiation complex formation, but not Sub1 recruitment. These results suggest that Sirt1 inhibits Sub1-induced preinitiation complex formation, thereby suppressing transcription of metabolic genes in the failing heart.