title: Inhibition of Rho-ROCK signaling induces apoptotic and non-apoptotic PS exposure in cardiomyocytes via inhibition of flippase.
abstract: Subsequent to myocardial infarction, cardiomyocytes within the infarcted areas and border zones expose phosphatidylserine (PS) in the outer plasma membrane leaflet (flip-flop). We showed earlier that in addition to apoptosis, this flip-flop can be reversible in cardiomyocytes. We now investigated a possible role for Rho and downstream effector Rho-associated kinase (ROCK) in the process of (reversible) PS exposure and apoptosis in cardiomyocytes. In rat cardiomyoblasts (H9c2 cells) and isolated adult ventricular rat cardiomyocytes Clostridium difficile Toxin B (TcdB), a Rho GTPase family inhibitor, C3 transferase (C3), a Rho(A,B,C) inhibitor and the ROCK inhibitors Y27632 and H1152 were used to inhibit Rho-ROCK signaling. PS exposure was assessed via flow cytometry and fluorescent digital imaging microscopy using annexin V. Akt expression and phosphorylation were analyzed via Western blot, and Akt activity was inhibited by wortmannin. The cellular concentration activated caspase 3 was determined as a measure of apoptosis, and flippase activity was assessed via flow cytometry using NBD-labeled PS. TcdB, C3, Y27632 and H1152 all significantly increased PS exposure. TcdB, Y27632 and H1152 all significantly inhibited phosphorylation of the anti-apoptotic protein Akt and Akt inhibition by wortmannin lead to increased PS exposure. However, only TcdB and C3, but not ROCK- or Akt inhibition led to caspase 3 activation and thus apoptosis. Notably, pancaspase inhibitor zVAD only partially inhibited TcdB-induced PS exposure indicating the existence of apoptotic and non-apoptotic PS exposure. The induced PS exposure coincided with decreased flippase activity as measured with NBD-labeled PS flip-flop. In this study, we show a regulatory role for a novel signaling route, Rho-ROCK-flippase signaling, in maintaining asymmetrical membrane phospholipid distribution in cardiomyocytes.
authors.affiliation: Department of Pathology, VU Medical Center, Amsterdam, The Netherlands. paj.krijnen@vumc.nl
authors.forename: Paul A J
authors.initials: PA
authors.lastname: Krijnen
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Sipkens
authors.forename: Johan W
authors.initials: JW
authors.lastname: Molling
authors.forename: Jan A
authors.initials: JA
authors.lastname: Rauwerda
authors.forename: Coen D A
authors.initials: CD
authors.lastname: Stehouwer
authors.forename: Alice
authors.initials: A
authors.lastname: Muller
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Paulus
authors.forename: Geerten P
authors.initials: GP
authors.lastname: van Nieuw Amerongen
authors.forename: C Erik
authors.initials: CE
authors.lastname: Hack
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Verhoeven
authors.forename: Victor W M
authors.initials: VW
authors.lastname: van Hinsbergh
authors.forename: Hans W M
authors.initials: HW
authors.lastname: Niessen
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D036002:ADP Ribose Transferases / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001426:Bacterial Proteins / Q000494:pharmacology; D001427:Bacterial Toxins / Q000494:pharmacology; D001905:Botulinum Toxins / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D061945:Caspase Inhibitors; D002460:Cell Line; D002469:Cell Separation; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology*; D010718:Phosphatidylserines / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: C459092:2-methyl-1-((4-methyl-5-isoquinolinyl)sulfonyl)homopiperazine; D000577:Amides; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D061945:Caspase Inhibitors; D010718:Phosphatidylserines; D049294:Phospholipid Transfer Proteins; D011725:Pyridines; C031815:toxB protein, Clostridium difficile; C108830:Y 27632; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000255:Adenosine Triphosphate; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D051057:Proto-Oncogene Proteins c-akt; D054460:rho-Associated Kinases; D053148:Caspase 3; D001905:Botulinum Toxins; D020741:rho GTP-Binding Proteins