Abstract Body: Prostaglandin E₂ (PGE2) is an autacoid that acts via 4 receptors (EP1-EP4), with EP3 and EP4 being the most prominent in the heart. We previously reported that cardiomyocyte specific knockout of EP4 (EP4 KO) results in dilated cardiomyopathy. Additionally, transgenic mice that overexpress EP3 in the cardiomyocyte (EP3 TG) develop heart failure. Recently published data from our laboratory suggests EP4 KO results in mitochondrial dysfunction, likely via EP3 receptor signaling. We therefore hypothesized that PGE2 alters energy metabolism in the heart via EP3. To test our hypothesis, we performed gene array analysis on left ventricles from adult EP4 KO and wild type (WT) mice. Significant reductions (2.1-fold) in carnitine palmitoyltransferase 2 (Cpt2) were observed in EP4 KO vs. EP4 WT. CPT2 is a key enzyme in fatty acid oxidation and deficiencies in humans have been shown to cause cardiomyopathy. Substantial reductions in CPT2 were also observed in C57 mice after 2 weeks of myocardial infarction (0.22 ± 0.04 a.u. in sham controls vs. 0.039 ± 0.06 a.u. after MI. p<0.01) by western blot analysis. Moreover, treatment with the EP3 receptor agonist, sulprostone, reduced CPT2 protein expression significantly in cardiomyocytes isolated from C57 mice (1.0 ± 0.0 a.u. in vehicle control vs. 0.67 ± 0.07 a.u. in sulprostone treated. p<0.005). These data suggest that PGE2 via its EP3 receptor reduces CPT2 expression. In fact, RNAseq analysis on left ventricles from EP3 TG and EP3 WT mice showed significant reductions in Cpt2 expression as early as 5 wks of age (log₂FC = -1.17. p<0.01). RNAseq also showed reductions in other key fatty acid oxidation/transport genes in EP3 TG mice (Acsl1; log₂FC= -0.73, Acads, log₂FC= -0.59, Cd36, log₂FC= -0.64. p<0.005). Lastly, to further investigate the role of the EP receptors on energy metabolism in the heart, we isolated cardiomyocytes from EP4 KO and EP4 WT mice and performed metabolomics. As anticipated, we observed significant reductions in L-carnitine (7.6 ± 1.13 fmol/cell in EP4 WT vs. 2.1 ± 0.32 fmol/cell in EP4 KO. p<0.005) and several acyl carnitine products. Altogether our data supports our hypothesis that PGE2 signaling via its EP3 receptor impairs mitochondrial energy metabolism in the heart by reducing fatty acid oxidation.