TY - JOUR
T1 - Inhibition of Rho - ROCK signaling induces apoptotic and non-apoptotic PS exposure in cardiomyocytes via inhibition of flippase
AU - Krijnen, Paul A. J.
AU - Sipkens, Jessica A.
AU - Molling, Johan W.
AU - Rauwerda, Jan A.
AU - Stehouwer, Coen D. A.
AU - Muller, Alice
AU - Paulus, Walter J.
AU - van Nieuw Amerongen, Geerten P.
AU - Hack, C. Erik
AU - Verhoeven, Arthur J.
AU - van Hinsbergh, Victor W. M.
AU - Niessen, Hans W. M.
PY - 2010
Y1 - 2010
N2 - 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
AB - 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
U2 - https://doi.org/10.1016/j.yjmcc.2010.07.017
DO - https://doi.org/10.1016/j.yjmcc.2010.07.017
M3 - Article
C2 - 20691698
SN - 0022-2828
VL - 49
SP - 781
EP - 790
JO - Journal of molecular and cellular cardiology
JF - Journal of molecular and cellular cardiology
IS - 5
ER -