Abstract Body: Background: Nearly half of adults have hypertension, a major risk factor for cardiovascular disease. Mitochondrial hyperacetylation is linked to hypertension, but the role of acetylation of specific proteins is not clear. We hypothesized that acetylation of mitochondrial Cyclophilin D (CypD) at K166 contributes to endothelial dysfunction and hypertension. METHODS: To test this hypothesis, we studied CypD acetylation in patients with essential hypertension, defined a pathogenic role of CypD acetylation in deacetylation mimetic CypD-K166R mutant mice and endothelial specific general control of amino acid synthesis 5 like 1 (GCN5L1) deficient mice using an angiotensin II model of hypertension. RESULTS: Arterioles from hypertensive patients had 280% higher CypD acetylation coupled with reduced Sirt3 and increased GCN5L1 levels. GCN5L1 regulates mitochondrial protein acetylation and promotes CypD acetylation which is counteracted by mitochondrial deacetylase Sirt3. In human aortic endothelial cells GCN5L1 depletion prevents superoxide overproduction. Deacetylation mimetic CypD-K166R mice were protected from vascular oxidative stress, endothelial dysfunction and angiotensin II-induced hypertension. Angiotensin II-induced hypertension increased mitochondrial GCN5L1 and reduced Sirt3 levels resulting in 250% increase in GCN5L1/Sirt3 ratio leading to imbalance between GCN5L1-mediated acetylation and Sirt3-mediated deacetylation and promoting CypD acetylation. Treatment with mitochondria-targeted scavenger of cytotoxic isolevuglandins, mito2HOBA, normalized GCN5L1/Sirt3 ratio, reduced CypD acetylation, and attenuated hypertension. The role of mitochondrial acetyltransferase GCN5L1 in endothelial function was tested in endothelial specific GCN5L1 knockout mice. Depletion of endothelial GCN5L1 prevented angiotensin II-induced mitochondrial oxidative stress, reduced maladaptive switch of vascular metabolism to glycolysis, prevented inactivation of endothelial nitric oxide, preserved endothelial-dependent relaxation, and attenuated hypertension. CONCLUSIONS: These data support the pathogenic role of CypD acetylation in endothelial dysfunction and hypertension. We suggest that targeting cytotoxic mitochondrial isolevuglandins and GCN5L1 reduces CypD acetylation which may be beneficial in cardiovascular disease.