Abstract Body: Prostaglandin E2 (PGE2) is an autacoid that acts through G-protein coupled receptors (EP1-EP4) in various cell types within the heart, including cardiomyocytes in which EP3 and EP4 predominate. We previously reported that mice with cardiomyocyte-specific overexpression of the EP3 receptor (EP3 TG) demonstrate reduced cardiac function and diminished cardiomyocyte contractility in isolated cardiomyocytes. We therefore hypothesized that PGE2 via the EP3 receptor regulates calcium handling proteins to reduce contractility. To test our hypothesis, we performed RNA sequencing (RNA seq) on the left ventricle of fifteen-week-old male EP3 TG mice along with their WT controls. Western blots were used to confirm changes in the expression of calcium-handling proteins such as Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a), Ryanodine receptor 2 (RYR2), Phospholamban (PLB), Troponins (C, I, T) and voltage-gated calcium channel (CACNB2). For RNA seq, all data is reported as Log2Fold change, adjusted P value, and western blot data is reported as mean ± standard error (SE) of the mean. Additionally, isolated cardiomyocyte contractility was assessed using the IonOptix system, and data is reported as the mean ± SE. RNA seq data shows that SERCA2a and RYR2 expression were significantly downregulated in TG mice (SERCA2a -0.44, p=4.72 x 10^-6; RYR2: -0.86, p=4.08 x 10^-5) in addition to significant reductions in Troponin I (-0.43, p=0.038) and the Voltage-gated calcium channel (CACNB2: -0.71,p=0.0055). Western blot analyses confirmed the reductions in SERCA2a, RYR2, and CACNB2 although these changes were only significant for CACNB2 (1.0 ± 0.03 for WT vs 0.59 ± 0.04 for TG, p = 0.016). Expression of Troponins did not change in TG mice. Consistent with our previous data, IonOptix analysis shows reduced contractility of myocytes from EP3 TG mice (bl% peak h is 3.47 ± 0.20 for WT vs 1.0 ± 0.14 for TG, p<0.0001) despite a similar increase in the Ca²⁺ transients (Ca²⁺ peak h for WT= 0.86 ± 0.04 and TG=0.86 ± 0.07) suggesting potential changes in the sensitivity of the myofilament to Ca²⁺. In conclusion, the reduced contractility observed in EP3 TG mice may be due to alterations in calcium handling proteins and/or changes in calcium sensitivity at the level of the myofilament.