title: Homocysteine induces phosphatidylserine exposure in cardiomyocytes through inhibition of Rho kinase and flippase activity.
abstract: OBJECTIVE
  Increased levels of homocysteine (Hcy) form an independent risk factor for cardiovascular disease. In a previous study we have shown that Hcy induced phosphatidylserine (PS) exposure to the outer leaflet of the plasma membrane in cardiomyocytes, inducing a pro-inflammatory phenotype. In the present study the mechanism(s) involved in Hcy-induced PS exposure were analyzed.
  
  
  METHODS
  H9c2 rat cardiomyoblasts were subjected to 2.5 mM D,L-Hcy and analyzed for RhoA translocation and activity, Rho Kinase (ROCK) activity and expression and flippase activity. In addition, the effect of ROCK inhibition with Y27632 on Hcy-induced PS exposure and flippase activity was analyzed. Furthermore, GTP and ATP levels were determined.
  
  
  RESULTS
  Incubation of H9c2 cells with 2.5 mM D,L-Hcy did not inhibit RhoA translocation to the plasma membrane. Neither did it inhibit activation of RhoA, even though GTP levels were significantly decreased. Hcy did significantly inhibit ROCK activation, but not its expression, and did inhibit flippase activity, in advance of a significant decrease in ATP levels. ROCK inhibition via Y27632 did not have significant added effects on this.
  
  
  CONCLUSIONS
  Hcy induced PS exposure in the outer leaflet of the plasma membrane in cardiomyocytes via inhibition of ROCK and flippase activity. As such Hcy may induce cardiomyocytes vulnerable to inflammation in vivo in hyperhomocysteinaemia patients.
authors.affiliation: Department of Pathology, VU University Medical Centre, Amsterdam, The Netherlands.
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Sipkens
authors.forename: Nynke E
authors.initials: NE
authors.lastname: Hahn
authors.forename: Geerten P
authors.initials: GP
authors.lastname: van Nieuw-Amerongen
authors.forename: Coen D A
authors.initials: CD
authors.lastname: Stehouwer
authors.forename: Jan A
authors.initials: JA
authors.lastname: Rauwerda
authors.forename: Victor W M
authors.initials: VW
authors.lastname: van Hinsbergh
authors.forename: Hans W M
authors.initials: HW
authors.lastname: Niessen
authors.forename: Paul A J
authors.initials: PA
authors.lastname: Krijnen
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D006160:Guanosine Triphosphate / Q000378:metabolism; D006710:Homocysteine / Q000494:pharmacology*; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000201:enzymology*; D010718:Phosphatidylserines / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D000577:Amides; D010718:Phosphatidylserines; D049294:Phospholipid Transfer Proteins; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D006710:Homocysteine; C108830:Y 27632; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein