Department of Pathology, VU University Medical Center, De Boelelaan 1117, 1007 MB Amsterdam, The Netherlands.
Mol Cell Biochem. 2011 Dec;358(1-2):229-39. doi: 10.1007/s11010-011-0973-4. Epub 2011 Jul 8.
We previously found that homocysteine (Hcy) induced plasma membrane flip-flop, apoptosis, and necrosis in cardiomyocytes. Inactivation of flippase by Hcy induced membrane flip-flop, while apoptosis was induced via a NOX2-dependent mechanism. It has been suggested that S-adenosylhomocysteine (SAH) is the main causative factor in hyperhomocysteinemia (HHC)-induced pathogenesis of cardiovascular disease. Therefore, we evaluated whether the observed cytotoxic effect of Hcy in cardiomyocytes is SAH dependent. Rat cardiomyoblasts (H9c2 cells) were treated under different conditions: (1) non-treated control (1.5 nM intracellular SAH with 2.8 μM extracellular L -Hcy), (2) incubation with 50 μM adenosine-2,3-dialdehyde (ADA resulting in 83.5 nM intracellular SAH, and 1.6 μM extracellular L -Hcy), (3) incubation with 2.5 mM D, L -Hcy (resulting in 68 nM intracellular SAH and 1513 μM extracellular L -Hcy) with or without 10 μM reactive oxygen species (ROS)-inhibitor apocynin, and (4) incubation with 100 nM, 10 μM, and 100 μM SAH. We then determined the effect on annexin V/propodium iodide positivity, flippase activity, caspase-3 activity, intracellular NOX2 and p47(phox) expression and localization, and nuclear ROS production. In contrast to Hcy, ADA did not induce apoptosis, necrosis, or membrane flip-flop. Remarkably, both ADA and Hcy induced a significant increase in nuclear NOX2 expression. However, in contrast to ADA, Hcy additionally induced nuclear p47(phox) expression, increased nuclear ROS production, and inactivated flippase. Incubation with SAH did not have an effect on cell viability, nor on flippase activity, nor on nuclear NOX2-, p47phox expression or nuclear ROS production. HHC-induced membrane flip-flop and apoptosis in cardiomyocytes is due to increased Hcy levels and not primarily related to increased intracellular SAH, which plays a crucial role in nuclear p47(phox) translocation and subsequent ROS production.
我们之前发现同型半胱氨酸（Hcy）诱导心肌细胞的质膜翻转、凋亡和坏死。Hcy 对翻转酶的失活诱导了质膜翻转，而凋亡则是通过 NOX2 依赖性机制诱导的。有人认为 S-腺苷同型半胱氨酸（SAH）是高同型半胱氨酸血症（HHC）引起心血管疾病发病机制的主要致病因素。因此，我们评估了 Hcy 在心肌细胞中观察到的细胞毒性作用是否依赖于 SAH。用不同条件处理大鼠心肌细胞（H9c2 细胞）：（1）非处理对照（1.5 nM 细胞内 SAH 和 2.8 μM 细胞外 L-Hcy），（2）用 50 μM 腺苷-2,3-二醛（ADA 导致 83.5 nM 细胞内 SAH 和 1.6 μM 细胞外 L-Hcy）孵育，（3）用 2.5 mM D, L-Hcy（导致 68 nM 细胞内 SAH 和 1513 μM 细胞外 L-Hcy）孵育，并用 10 μM 活性氧（ROS）抑制剂 apocynin 处理，或（4）用 100 nM、10 μM 和 100 μM SAH 孵育。然后，我们测定了对膜联蛋白 V/碘化丙啶阳性、翻转酶活性、半胱天冬酶-3 活性、细胞内 NOX2 和 p47(phox)表达和定位以及核 ROS 产生的影响。与 Hcy 相反，ADA 不会诱导细胞凋亡、坏死或质膜翻转。值得注意的是，ADA 和 Hcy 均显著增加核 NOX2 的表达。然而，与 ADA 不同，Hcy 还诱导核 p47(phox)表达、增加核 ROS 产生和失活翻转酶。SAH 孵育对细胞活力、翻转酶活性、核 NOX2-、p47phox 表达或核 ROS 产生均无影响。HHC 诱导的心肌细胞质膜翻转和凋亡是由于 Hcy 水平升高，而不是主要与细胞内 SAH 升高有关，SAH 在核 p47(phox)易位和随后的 ROS 产生中起关键作用。
