title: Homocysteine-induced cardiomyocyte apoptosis and plasma membrane flip-flop are independent of S-adenosylhomocysteine: a crucial role for nuclear p47(phox).
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 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.
authors: Department of Pathology, VU University Medical Center, De Boelelaan 1117, 1007 MB Amsterdam, The Netherlands.
authors: Jessica A
authors: JA
authors: Sipkens
authors: Paul A J
authors: PA
authors: Krijnen
authors: Nynke E
authors: NE
authors: Hahn
authors: Melissa
authors: M
authors: Wassink
authors: Christof
authors: C
authors: Meischl
authors: Desirée E C
authors: DE
authors: Smith
authors: René J P
authors: RJ
authors: Musters
authors: Coen D A
authors: CD
authors: Stehouwer
authors: Jan A
authors: JA
authors: Rauwerda
authors: Victor W M
authors: VW
authors: van Hinsbergh
authors: Hans W M
authors: HW
authors: Niessen
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology*; D002470:Cell Survival / Q000187:drug effects; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D006710:Homocysteine / Q000378:metabolism / Q000494:pharmacology*; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D012435:S-Adenosylhomocysteine / Q000494:pharmacology*; D012436:S-Adenosylmethionine / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D008562:Membrane Glycoproteins; D049294:Phospholipid Transfer Proteins; D017382:Reactive Oxygen Species; D006710:Homocysteine; C027579:periodate-oxidized adenosine; C002744:3-nitrotyrosine; D014443:Tyrosine; D012436:S-Adenosylmethionine; D000255:Adenosine Triphosphate; D012435:S-Adenosylhomocysteine; C500593:Cybb protein, rat; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D053148:Caspase 3; D000241:Adenosine