Sipkens Jessica A, Hahn Nynke E, van Nieuw-Amerongen Geerten P, Stehouwer Coen D A, Rauwerda Jan A, van Hinsbergh Victor W M, Niessen Hans W M, Krijnen Paul A J
Department of Pathology, VU University Medical Centre, Amsterdam, The Netherlands.
Cell Physiol Biochem. 2011;28(1):53-62. doi: 10.1159/000331713. Epub 2011 Aug 16.
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.
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.
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.
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.
同型半胱氨酸（Hcy）水平升高是心血管疾病的一个独立危险因素。在之前的一项研究中，我们已表明Hcy可诱导心肌细胞中磷脂酰丝氨酸（PS）暴露于质膜外层小叶，从而诱导促炎表型。在本研究中，我们分析了Hcy诱导PS暴露所涉及的机制。
将H9c2大鼠心肌母细胞置于2.5 mM D，L-Hcy中，分析RhoA转位和活性、Rho激酶（ROCK）活性及表达以及翻转酶活性。此外，分析了用Y27632抑制ROCK对Hcy诱导的PS暴露和翻转酶活性的影响。此外，还测定了GTP和ATP水平。
用2.5 mM D，L-Hcy孵育H9c2细胞不会抑制RhoA转位至质膜。即便GTP水平显著降低，它也不会抑制RhoA的激活。Hcy确实显著抑制ROCK激活，但不抑制其表达，并且在ATP水平显著降低之前就抑制了翻转酶活性。通过Y27632抑制ROCK对此没有显著的附加作用。
Hcy通过抑制ROCK和翻转酶活性诱导心肌细胞质膜外层小叶中的PS暴露。因此，Hcy可能会使高同型半胱氨酸血症患者体内的心肌细胞易于发生炎症。
