Homocysteine induces phosphatidylserine exposure in cardiomyocytes through inhibition of Rho kinase and flippase activity

J.A. Sipkens, N.E. Hahn, G.P. van Nieuw Amerongen, C.D.A. Stehouwer, J.A. Rauwerda, V.W.M. van Hinsbergh, H.W.M. Niessen, P.A.J. Krijnen

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Abstract

AIMS: 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.

CONCLUSION: 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.

Original languageEnglish
Pages (from-to)53-62
Number of pages10
JournalCellular Physiology and Biochemistry
Volume28
Issue number1
DOIs
Publication statusPublished - 2011

Cite this

Sipkens, J.A. ; Hahn, N.E. ; van Nieuw Amerongen, G.P. ; Stehouwer, C.D.A. ; Rauwerda, J.A. ; van Hinsbergh, V.W.M. ; Niessen, H.W.M. ; Krijnen, P.A.J. / Homocysteine induces phosphatidylserine exposure in cardiomyocytes through inhibition of Rho kinase and flippase activity. In: Cellular Physiology and Biochemistry. 2011 ; Vol. 28, No. 1. pp. 53-62.
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title = "Homocysteine induces phosphatidylserine exposure in cardiomyocytes through inhibition of Rho kinase and flippase activity",
abstract = "AIMS: 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.CONCLUSION: 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.",
keywords = "Adenosine Triphosphate, Amides, Animals, Cells, Cultured, Guanosine Triphosphate, Homocysteine, Myocytes, Cardiac, Phosphatidylserines, Phospholipid Transfer Proteins, Protein Kinase Inhibitors, Pyridines, Rats, rho-Associated Kinases, rhoA GTP-Binding Protein, Journal Article",
author = "J.A. Sipkens and N.E. Hahn and {van Nieuw Amerongen}, G.P. and C.D.A. Stehouwer and J.A. Rauwerda and {van Hinsbergh}, V.W.M. and H.W.M. Niessen and P.A.J. Krijnen",
note = "Copyright {\circledC} 2011 S. Karger AG, Basel.",
year = "2011",
doi = "10.1159/000331713",
language = "English",
volume = "28",
pages = "53--62",
journal = "Cellular Physiology and Biochemistry",
issn = "1015-8987",
publisher = "S. Karger AG",
number = "1",

}

Homocysteine induces phosphatidylserine exposure in cardiomyocytes through inhibition of Rho kinase and flippase activity. / Sipkens, J.A.; Hahn, N.E.; van Nieuw Amerongen, G.P.; Stehouwer, C.D.A.; Rauwerda, J.A.; van Hinsbergh, V.W.M.; Niessen, H.W.M.; Krijnen, P.A.J.

In: Cellular Physiology and Biochemistry, Vol. 28, No. 1, 2011, p. 53-62.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Homocysteine induces phosphatidylserine exposure in cardiomyocytes through inhibition of Rho kinase and flippase activity

AU - Sipkens, J.A.

AU - Hahn, N.E.

AU - van Nieuw Amerongen, G.P.

AU - Stehouwer, C.D.A.

AU - Rauwerda, J.A.

AU - van Hinsbergh, V.W.M.

AU - Niessen, H.W.M.

AU - Krijnen, P.A.J.

N1 - Copyright © 2011 S. Karger AG, Basel.

PY - 2011

Y1 - 2011

N2 - AIMS: 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.CONCLUSION: 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.

AB - AIMS: 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.CONCLUSION: 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.

KW - Adenosine Triphosphate

KW - Amides

KW - Animals

KW - Cells, Cultured

KW - Guanosine Triphosphate

KW - Homocysteine

KW - Myocytes, Cardiac

KW - Phosphatidylserines

KW - Phospholipid Transfer Proteins

KW - Protein Kinase Inhibitors

KW - Pyridines

KW - Rats

KW - rho-Associated Kinases

KW - rhoA GTP-Binding Protein

KW - Journal Article

U2 - 10.1159/000331713

DO - 10.1159/000331713

M3 - Article

VL - 28

SP - 53

EP - 62

JO - Cellular Physiology and Biochemistry

JF - Cellular Physiology and Biochemistry

SN - 1015-8987

IS - 1

ER -