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Homocysteine-induced cardiomyocyte apoptosis and plasma membrane flip-flop are independent of S-adenosylhomocysteine: a crucial role for nuclear p47phox

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Abstract

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 p47phox 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 p47phox 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 p47phox translocation and subsequent ROS production.

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Acknowledgments

This project was financed by the ICaR-VU (nr. 200370).

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Authors and Affiliations

  1. Department of Pathology, VU University Medical Center, Room nr. 0E46, De Boelelaan 1117, 1007 MB, Amsterdam, The Netherlands

    Jessica A. Sipkens, Paul A. J. Krijnen, Nynke E. Hahn, Melissa Wassink, Christof Meischl & Hans W. M. Niessen

  2. Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands

    Desirée E. C. Smith

  3. Department of Physiology, VU University Medical Center, Amsterdam, The Netherlands

    René J. P. Musters & Victor W. M. van Hinsbergh

  4. Department of Internal Medicine, Academic Hospital Maastricht, Maastricht, The Netherlands

    Coen D. A. Stehouwer

  5. Department of Vascular Surgery, VU University Medical Center, Amsterdam, The Netherlands

    Jan A. Rauwerda

  6. Department of Cardiac Surgery, VU University Medical Center, Amsterdam, The Netherlands

    Hans W. M. Niessen

  7. ICaR-VU, Institute of Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands

    Jessica A. Sipkens, Paul A. J. Krijnen, Nynke E. Hahn, Christof Meischl, René J. P. Musters, Jan A. Rauwerda, Victor W. M. van Hinsbergh & Hans W. M. Niessen

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  1. Jessica A. Sipkens
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Correspondence to Paul A. J. Krijnen.

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Jessica A. Sipkens and Paul A. J. Krijnen contributed equally to this manuscript.

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Sipkens, J.A., Krijnen, P.A.J., Hahn, N.E. et al. Homocysteine-induced cardiomyocyte apoptosis and plasma membrane flip-flop are independent of S-adenosylhomocysteine: a crucial role for nuclear p47phox . Mol Cell Biochem 358, 229–239 (2011). https://doi.org/10.1007/s11010-011-0973-4

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