Abstract Body: Prostaglandin E2 (PGE2) signals through four G protein coupled receptors (EP1-EP4), eliciting a variety of physiologic or pathologic effects. EP3 and EP4 are the most abundant in the mouse heart and we have shown that EP3 directly reduces cardiac contractility while EP4 acts to improve it. Moreover, the expression of EP3 increases in heart disease while EP4 does not. We created a cardiomyocyte (CM) specific EP4KO mouse that develops dilated cardiomyopathy with age. Similarly, the CM-specific EP3 transgenic (TG) mouse also develops heart failure (HF). Thus, cardiac EP3 signaling is deleterious, while EP4 is protective. Gene array analysis from left ventricles (LV) of EP4KO mice showed significant alterations in genes enriched for mitochondrial and myocardial metabolism pathways compared to WT littermates. We thus hypothesized that PGE2 via its EP3 receptor affects the TCA cycle, resulting in reduced cardiac function and energy output. To test our hypothesis, we employed a multi-omics strategy to examine if the absence of EP4 and/or activation of EP3 results in mitochondrial metabolic defects that contribute to heart failure. Gene array and RNAseq analysis shows genes critical for pyruvate shuttling, like Ldh and Pdh are significantly reduced in both EP4KO and EP3TG mice. Moreover, lipidomic analyses of CMs show higher lactic acid in EP4KO mice compared to WT. The phospho creatine (PCr)/ATP ratio is an important prognostic indicator in HF. Our metabolomic data suggests that PCr/ATP is lower in CMs from EP4KO mice (0.28 ± 0.07 a.u. in EP4WT vs 0.10 ± 0.04 a.u. in EP4KO, p=0.07). Furthermore, western blot shows reductions in the fatty acid translocase protein CD36 in EP3 TG LV (1.00 ± 0.16 in EP3 WT vs 0.56 ± 0.09 in EP3 TG, p≤0.05), suggesting reduced import of fatty acids. Also, EP3TG mice had reductions in Pyruvate Dehydrogenase α1 (1.00 ± 0.1 in EP3 WT vs 0.35 ± 0.06 in EP3 TG, p<0.01), Succinyl CoA Synthetase (1.00 ± 0.1 in EP3 WT vs 0.58 ± 0.08 in EP3 TG, p<0.05), and a non-significant trend towards a reduction in Isocitrate Dehydrogenase (1.00 ± 0.2 in EP3 WT vs 0.75 ± 0.04 in EP3 TG, p=0.18). Our data suggests that activation of the PGE2 EP3 receptor reduces key metabolic genes, contributing to the development of HF. This work could pave the way for future drugs targeting EP receptors.