PDE3 cyclic nucleotide phosphodiesterases are used clinically to overcome a reduction in receptor- mediated cAMP generation in patients with heart failure. In the short term, PDE3 inhibitors have beneficial inotropic effects, but their long-term use has been associated with an increase in mortality that may be attributable to pro-apoptotic actions. Finding a way to attain the inotropic actions of PDE3 inhibition without the adverse long-term consequences would represent a significant therapeutic advance. In cardiac myocytes, PDE3 activity is found in intracellular compartments represented in cytosolic and microsomal fractions. Inotropic actions are likely to result from the inhibition of membrane-associated PDE3 activity and the consequent increase in cAMP-stimulated Ca2+ cycling, while pro-apoptotic changes in gene transcription are more likely to result from the inhibition of cytosolic PDE3 activity. Selective inhibition of membrane-associated activity might have inotropic actions without pro-apoptotic effects, but the three isoforms of PDE3 in cardiac myocytes - PDE3A1, PDE3A2 and PDE3A3 - are identical in their sensitivity to conventional catalytic-site inhibitors. Recent experiments, however, indicate that membrane-associated PDE3 isoforms integrate into multiprotein complexes with other membrane-associated proteins, providing a mechanism through which they can selectively regulate the phosphorylation of proteins involved in cAMP- mediated signaling in a specific compartment of cardiac myocytes. Blocking the interactions of PDE3 isoforms with these binding proteins may be a novel mechanism through which membrane-associated PDE3 activity can be targeted without inhibiting cytosolic activity. The availability of an agent that can selectively block the interactions of PDE3 isoforms with their membrane-associated binding proteins would allow us to test this hypothesis. With this goal in mind, our specific aims are: 1. To identify the proteins that interact with membrane-associated PDE3 isoforms in human myocardium: PDE3A-binding proteins in purified high-density microsomes from human left ventricular myocardium will be co-immunoprecipitated with anti-PDE3 antibodies and co-affinity purified with tagged recombinant PDE3 isoforms. PDE3A-binding proteins will be identified by Western blotting and mass spectrometry. Interactions with PDE3 isoforms will be confirmed by reverse co-immunoprecipitation in preparations from native tissue and co-transfected cells and by quantitation of protein-protein interactions by ELISA and surface plasmon resonance. 2. To identify the amino-acid sequences in these PDE3 isoforms that are involved in these interactions: Arrays of overlapping 25-mer peptides generated from the complete amino-acid sequences of PDE3A isoforms will be probed with the PDE3A-binding proteins identified in Aim 1 to determine the specific amino-acid sequences in PDE3A with which these proteins interact. 3. To determine whether peptides generated from these amino-acid sequences can be used as competitive inhibitors to disrupt these interactions: Synthetic peptides incorporating the PDE3A- derived amino-acid sequences identified in Aim 2 will be tested for their ability to inhibit protein-protein interactions and to block co-immunoprecipitation or co-affinity purification of PDE3A-binding proteins from cardiac microsomes. Achieving these aims will yield new insight into the mechanisms through which PDE3 isoforms regulate cAMP-mediated signaling in intracellular compartments of cardiac myocytes, and will provide us with prototypical agents that will be used in future experiments to test the feasibility of membrane-selective PDE3 inhibition as a therapeutic strategy for heart failure.
Heart failure has been diagnosed in ~5 % of VA patients. Its annual mortality among veterans is now 12.6%, and morality and hospitalization rates are rising. In his testimony to Congress, Joel Kupersmith, Chief Research and Development Officer of the Veterans Health Administration, noted that 'heart failure is the most common diagnosis causing hospitalization of veterans, with resulting high costs and resource utilization over time'. Advanced therapeutic options such as heart transplants and left-ventricular assist devices are often unfeasible, especially in aging veterans, because of comorbidities that are contraindications to surgery. Improvements in the nonsurgical treatment of heart failure are thus especially important to VA patients. The research proposed in this application, which focuses on a novel molecular mechanism through which a new pharmacological strategy for the treatment of heart failure can be based, has the potential to lead to such improvements.