Cardiac Phospholipase D Signal Transduction And Functional Analysis In A Transgenic Mouse Model With Cardiac-specific Overexpression Of Human Phospholipase D₂

Phospholipase D (PLD) is distributed widely in animals, plants, fungi and bacteria. PLD is an enzyme that catalyses the hydrolysis of the terminal phosphodiester bond of phosphatidylcholine (PC), the most prevalent membrane phospholipid, into phosphatidic acid (PA) and choline. PC hydrolysis by PLD is a response to a large number of extracellular signals.PA is an important lipid second messenger that has been implicated in many cellular functions including the regulation of cell differentiation and proliferation and membrane vesicle trafficking. PA can be further metabolized to form diacylglycerol (DAG) by phosphatidic acid phosphohydrolase. DAG is the major endogenous regulator of protein kinase C (PKC) isoforms. PA also canbe derived to form lyso-phosphatidic acid (LPA) by phospholipase A2 . LPA induces a variety of biological effects such as proliferation in epidermal cells, stress fiber formation, and contraction in smooth muscles.Recently, two isoforms of PLD, PLD₁ and PLD₂, have been cloned. PLD₁ is a 124KDa protein. PLD2 is a 933-amino-acid 106KDa protein that shares 50%-55% amino acid identity with PLDi and has a different chromosomal location. Human PLD2 (hPLD2) is 90% identical at the amino acid level with mouse PLD2 (mPLDa) isoform and is more distantly related to PLDi protein.Both PLDi and PLD2 have two highly conserved HKD motifs or PLD domains that are essential for catalysis and dimerization. They also have other functional domains such as Px domain which might be implicated in protein-protein interaction and a pleckstrin homology(PH)-like domain which binds P1P2 and may also be involved hi protein-protein interaction.PLD has implications in a wide range of physiological processes and diseases including inflammation, secretion, mitogenesis, neuronal stimulation, cytoskeletal reorganization, and the respiratory burst in neutrophils. PLD also is a regulator in multiple settings hi the heart which include stimulating inositol 1,4,5-triphosphate production in cardiomyocytes, phosphorylation of cardiac protein and an increase of intracellular free Ca2+ and contractile force. Prolonged activation of cardiac PLD has also been linked to cardiac hypertrophy, potentially through the conversion of PA to DAG. Many hormones, neurotransmitters, and growth factors produce cardiac effects through PLD signal transduction, such as catecholamine, atrial natriuretic factor, bradykinin, angiotensin n, endothelin-1, platelet-derived growth factor, and adenosine.In 1991 Panagia et al. demonstrated by using isolated subcellular membranes from rat ventricular myocardium that active PLD is bound to thesarcolemmal membranes. Recently, Park et al. found PLD2 is the major myocardial PLD and it localizes primarily at sarcolemmal membranes.However, regulation of PLD in vivo and the influence of prolonged overexpression of cardiac PLD on the heart in vivo remain unknown. In this study, using transgenic mice with cardiac-specific overexpression of human PLDa we measured cardiac performance under different loading conditions, different content norepinephrine and assessed ischemia-reperfusion injury to examine the cardiac PLD singnal transduction in vivo and the influence of prolonged overexpression of cardiac PLD on heart.1. Methods1.1 AnimalsTo generate a transgenic construct expressing the polyhistidine labeled human PLD2 under the control of cardiac-specific mouse d-myosin heavy chain (a -MHC) promoter, the 3441-bp cDNA of hPLD2 cut with SacH and BsrBI, blunted, was ligated in the MCS, PvuH of pRSETC (Invitrogen) in frame. The 3881-bp cDNA of 6His- hPLD2 was cut with Ndel and BstEH , blunt-ended and ligated into Hindm restriction site of pBlue- d- MHC-SV40polyA downstream from the mouse cardiac-specific ?-MHC promoter and upstream from SV40polyA. The final construct (12.2kb) was double cut with Notl and a 9.4kb Notl DNA fragment was isolated, purified, and injected into the pronuclei of fertilized FVB mouse ova (Eurogentec, Belgium). The presence of the transgene was determi...