Methods for modulating cyclic nucleotide-mediated signaling in cardiac myocytes and compositions

Abstract

The invention provides a polypeptide possessing anti-hypertrophic activity in a cardiac myocyte, wherein the polypeptide is a mutant variant derived from wild-type PDE3A1 protein and wherein the wild-type PDE3A1 protein has the amino acid sequence given in SEQ ID NO:1 at amino acid position 146 to 1141.

1 .- 55 . (canceled) 
     
     
         56 . A method for preventing, inhibiting or reversing myocardial hypertrophy comprising administering a vector having a nucleic acid molecule encoding an isolated or purified polypeptide possessing anti-hypertrophic activity in a cardiac myocyte, wherein the polypeptide is a mutant derived from wild-type PDE3A1 protein and wherein the wild-type PDE3A1 protein has the amino-acid sequence given in SEQ ID NO:1 at amino acid position 146 to 1141 in a cardiac myocyte, said vector being genetically modified by insertion of at least one therapeutic gene into said vector to produce functional molecules in a sufficient amount to prevent, inhibit or reverse myocardial hypertrophy in the cell. 
     
     
         57 .- 103 . (canceled) 
     
     
         104 . The method of  claim 56 , wherein the mutant derived from wild-type PDE3A1 protein is a catalytically compromised mutant of PDE3A1 protein. 
     
     
         105 . The method of  claim 104 , wherein the catalytically compromised mutant of PDE3A1 protein is a catalytically inactive mutant of PDE3A1 protein or a catalytically reduced mutant of PDE3A1 protein. 
     
     
         106 . The method of  claim 104  or  105 , wherein the catalytically compromised, reduced or inactive mutant of PDE3A1 protein is a result of a mutation affecting tyrosine-751 (Y751), histidine-836 (H836), histidine-840 (H840), glutamic acid-866 (E866), aspartic acid-950 (D950), or phenylalanine-1004 (F1004). 
     
     
         107 . The method of  claim 105 , wherein the catalytically inactive mutant of PDE3A1 protein has a mutation at phenylalanine-1004 (F1004). 
     
     
         108 . The method of  claim 106  or  107 , wherein the mutation is an amino acid change to an alanine. 
     
     
         109 . The method of  claim 106  or  107 , wherein the mutation is an amino acid change to any amino acid other than an alanine. 
     
     
         110 . The method of  claim 109 , wherein the amino acid change to any amino acid other than an alanine is selected from a group of amino acids consisting of glycine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, tyrosine, tryptophan, phenylalanine, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine or arginine, and wherein not selected from the group is amino acid present in the wild-type PDE3A1 at a position to be mutated. 
     
     
         111 . The method of  claim 56 , wherein the mutant derived from wild-type PDE3A1 protein is a deletion mutant of PDE3A1 protein. 
     
     
         112 . The method of  claim 111 , wherein the deletion mutant of PDE3A1 protein lacks an intact C-terminal catalytic region. 
     
     
         113 . The polypeptide of  claim 111 , wherein the deletion mutant of PDE3A1 protein comprises an amino-terminal sequence without the C-terminal catalytic region. 
     
     
         114 . The polypeptide of  claim 112  or  113 , wherein the C-terminal catalytic region is given in SEQ ID NO:1 at amino acid position 669 to 1108. 
     
     
         115 . The polypeptide of  claim 113 , wherein the amino-terminal sequence comprises ten or more amino acids having a sequence identical or homologous to a sequence at amino acid position 146 to 668 in SEQ ID NO:1. 
     
     
         116 . The polypeptide of  claim 113 , wherein the amino-terminal sequence comprises ten or more amino acids having a sequence identical or homologous to a sequence in the N-terminal amino-acid sequence present in PDE3A1 but not in PDE3A3, corresponding to amino acid position 146 to 483 in SEQ ID NO:1. 
     
     
         117 . The polypeptide of  claim 113 , wherein the amino-terminal sequence comprises ten or more amino acids having a sequence identical or homologous to a sequence in the N-terminal amino-acid sequence present in PDE3A1 but not in PDE3A2, corresponding to amino acid position 146 to 299 in SEQ ID NO:1. 
     
     
         118 . The method of  claim 111  or  112 , wherein the polypeptide comprises a sequence of amino acids present in both PDE3A1 and PDE3A2 isoforms but having a different conformation in the two isoforms. 
     
     
         119 . The method of  claim 111  or  112 , wherein the polypeptide comprises a sequence of amino acids present in both PDE3A1 and PDE3A2 isoforms but having differential accessibility for protein-protein interaction in the two isoforms. 
     
     
         120 . The polypeptide of  claim 119 , wherein the sequence of amino acids present in both PDE3A1 and PDE3A2 isoforms has greater accessibility for protein-protein interactions in the PDE3A1 isoform than in the PDE3A2 isoform. 
     
     
         121 . The method of  claim 111  or  112 , wherein the polypeptide comprises a sequence of amino acids comprising a serine amino acid that is differentially phosphorylated between PDE3A1 and PDE3A2 isoforms or a serine amino acid that is unique to PDE3A1 which maybe phosphorylated. 
     
     
         122 . The method of  claim 121 , wherein phosphorylation of the serine amino acid in PDE3A1 isoform influences protein-protein interactions.