Rationale: SERCA2 controls cardiac contractility, and its activity is negatively regulated by the cAMP phosphodiesterase PDE3A through an unknown mechanism. Recent clinical trials have shown upregulation of SERCA2 gene therapy as beneficial in heart failure.Objective: To examine whether PDE3A is physically associated with SERCA, and to further evaluate whether this protein interaction represent a novel drug target to increase SERCA2 activity.Methods and results: PDE3 inhibition increased Ca2+ transients, SR Ca2+ load and SERCA2 activity without altering global cytosolic cAMP levels in field stimulated cardiomyocytes. SERCA2 activity was increased by PDE3 inhibition in cardiomyocytes dialyzed with 5 µmol/l cAMP by patch pipettes. Active PDE3A co-purified and precipitated with SERCA2 from left ventricular myocardium, and proximity ligation assay demonstrated co-localization of PDE3A and SERCA2 in intact cardiomyocytes. A combination of immunoprecipitation and peptide interaction experiments revealed interaction between specific cytosolic regions between amino acids 277 and 493 in PDE3A and amino acid 169 to 216 in SERCA2. By whole cell voltage clamp of intact cardiomyocytes, increased SERCA2 activity was induced by dialysis with disruptor peptides of the SERCA2-PDE3A interaction. TAT-labeled PDE3A-SERCA2 disruptor peptide fragments were further able to increase SERCA2 activity in field stimulated cardiomyocytes. PDE3A-SERCA2 disruptor peptides were able to increase SERCA2 activity in cardiomyocytes in presence of either PKI or Rp-cAMP and without concomitant phospholamban phosphorylation.Conclusion: PDE3A is physically associated to SERCA2, and this direct interaction regulates SERCA2 activity in cardiomyocytes possibly by direct regulation of SERCA2. Cell permeable disruptor peptides of the PDE3A-SERCA2 protein-protein interaction is able to increase SERCA2 activity and may potentially offer a new therapeutic approach in chronic heart disease.