PP2A-PR72 is a cytosolic, Ca2+-binding protein, mainly expressed in myocardium. As a regulatory subunit of protein phosphatase 2A (PP2A), PR72 forms a heterotrimeric complex together with a catalytic C and structural A subunit. It has been hypothesized that PR72 regulates activity and targeting of PP2A. But cardiac function of PP2A-PR72 remains to be elucidated. Therefore, we overexpressed PP2A-PR72 in transgenic mouse hearts under control of the αMHC promoter (TG). We measured contractile function and biochemical parameters in isolated, Langendorff-perfused (LD) hearts under basal conditions and after β-adrenergic stimulation with 1 μM isoprenaline (ISO). Under basal conditions, TG vs. wild-type (WT) spontaneously beating LD hearts showed an unchanged contractile function. Under ISO stimulation, contractile response increased in both groups. However, TG vs. WT hearts developed higher left ventricular pressure (205.9±11.8 vs. 170.3±26.3 mmHg, respectively, n=9-14, P<0.05) and higher contraction velocity (15725±1693 vs. 11590±2126 mmHg/s, respectively, n=9-14, P<0.05) at comparable beating rates. Biochemical evaluation of LD heart homogenates revealed higher total phosphatase and PP2A activity in ISO treated vs. unstimulated LD hearts without differences between genotypes. Protein kinase A (PKA) activity was diminished in TG vs. WT LD hearts under basal conditions (10.9±4.2 vs. 34.9±8.6 pmol/min/mg, respectively, n=8-9, P<0.05), while basal cAMP levels remained unchanged. ISO treated vs. unstimulated LD hearts showed a reduced PKA activity in WT, but preserved PKA activity in TG. In LD heart homogenates of both genotypes, PKA activity was stimulable by cAMP. Ca2+-Calmodulin-dependent protein kinase II (CaMKII) activity was basally reduced by 55% in TG vs. WT LD hearts as well (n=10, P<0.0.5). Consistent with basally decreased kinase activities, phosphorylation levels of myosin light chain 2 (MLC2) at Ser-15 and Ser-18 were reduced in TG vs. WT LD hearts (e.g. by 38% at Ser-18, n=10, P<0.05). ISO treatment led to reduced MLC2 phosphorylation levels in WT, but not in TG LD hearts. Moreover, ISO treatment led to a 5-fold increased troponin I phosphorylation at Ser-23/24 in both genotypes and to about 77% higher (n=7-8, P<0.05) phospholamban (PLN) phosphorylation levels at Thr-17 in TG vs. WT LD hearts. In summary, PP2A-PR72 overexpression did neither affect basal contractile function of LD hearts nor PP2A activity in LD heart homogenates. However, basal activities of PKA and CaMKII as well as basal phosphorylation levels of MLC2 were decreased. Under β-adrenergic stimulation, higher phosphorylation levels of the Ca2+-regulating protein PLN were detected in TG LD hearts, possibly contributing to the enhanced contractile function of TG LD hearts, observed under ISO stimulation. So, our study reveals for the first time the potential role of PP2A-PR72 for the contractile function of the myocardium. Public funding This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.