Homocysteine affects cardiomyocyte viability: concentration-dependent effects on reversible flip-flop, apoptosis and necrosis.
BACKGROUND
Hyperhomocysteinaemia (HHC) is thought to be a risk factor for cardiovascular disease including heart failure. While numerous studies have analyzed the role of homocysteine (Hcy) in the vasculature, only a few studies investigated the role of Hcy in the heart. Therefore we have analyzed the effects of Hcy on isolated cardiomyocytes.


METHODS
H9c2 cells (rat cardiomyoblast cells) and adult rat cardiomyocytes were incubated with Hcy and were analyzed for cell viability. Furthermore, we determined the effects of Hcy on intracellular mediators related to cell viability in cardiomyocytes, namely NOX2, reactive oxygen species (ROS), mitochondrial membrane potential (DeltaPsi (m)) and ATP concentrations.


RESULTS
We found that incubation of H9c2 cells with 0.1 mM D,L-Hcy (= 60 microM L-Hcy) resulted in an increase of DeltaPsi (m) as well as ATP concentrations. 1.1 mM D,L-Hcy (= 460 microM L-Hcy) induced reversible flip-flop of the plasma membrane phospholipids, but not apoptosis. Incubation with 2.73 mM D,L-Hcy (= 1.18 mM L-Hcy) induced apoptosis and necrosis. This loss of cell viability was accompanied by a thread-to-grain transition of the mitochondrial reticulum, ATP depletion and nuclear NOX2 expression coinciding with ROS production as evident from the presence of nitrotyrosin residues. Notably, only at this concentration we found a significant increase in S-adenosylhomocysteine which is considered the primary culprit in HHC.


CONCLUSIONS
We found concentration-dependent effects of Hcy in cardiomyocytes, varying from induction of reversible flip-flop of the plasma membrane phospholipids, to apoptosis and necrosis.
Sipkens
Jessica A
JA
Department of Pathology, VU University Medical Centre, Room 0E46, De Boelelaan 1117, Amsterdam, 1081 HV, The Netherlands. j.sipkens@vumc.nl
Krijnen
Paul A J
PA
Meischl
Christof
C
Cillessen
Saskia A G M
SA
Smulders
Yvo M
YM
Smith
Desirée E C
DE
Giroth
Cindy P E
CP
Spreeuwenberg
Marieke D
MD
Musters
René J P
RJ
Muller
Alice
A
Jakobs
Cornelis
C
Roos
Dirk
D
Stehouwer
Coen D A
CD
Rauwerda
Jan A
JA
van Hinsbergh
Victor W M
VW
Niessen
Hans W M
HW
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; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects; D006710:Homocysteine / Q000032:analysis / Q000494:pharmacology*; D008560:Membrane Fluidity / Q000187:drug effects*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008929:Mitochondria, Heart / Q000187:drug effects; D008954:Models, Biological; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects* / Q000473:pathology; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D009336:Necrosis / Q000139:chemically induced; D010743:Phospholipids / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D012435:S-Adenosylhomocysteine / Q000032:analysis; D012436:S-Adenosylmethionine / Q000032:analysis
D008562:Membrane Glycoproteins; D010743:Phospholipids; D006710:Homocysteine; D012436:S-Adenosylmethionine; D000255:Adenosine Triphosphate; D012435:S-Adenosylhomocysteine; C500593:Cybb protein, rat; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D053148:Caspase 3