Thiol depletion induces lethal cell injury in cultured cardiomyocytes.
Treatment of cultured neonatal cardiomyocytes with ethacrynic acid (EA) induced a rapid depletion of glutathione (GSH) that preceded a gradual elevation of cytosolic Ca2+ (monitored by phosphorylase a activation), a loss of protein thiols, and a marked inactivation of the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PD). A subsequent decline of mitochondrial transmembrane potential (delta psi) and ATP occurred prior to the onset of lipid peroxidation which closely paralleled a loss of cardiomyocyte viability. The antioxidant N,N'-diphenyl-p-phenylenediamine prevented lipid peroxidation and cell death but had no effect on elevated cytosolic Ca2+, delta psi loss, GSH depletion, or G3PD inactivation. Pretreatment with the iron chelator, deferoxamine, decreased both lipid peroxidation and cell death. EA-induced lipid peroxidation and cell damage were also diminished by preincubation with acetoxymethyl esters of the Ca2+ chelators Quin-2 and ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid, even though cytosolic Ca2+ remained elevated. The extent of GSH depletion was unaltered by either chelator; however, Quin-2 did protect G3PD from inactivation by EA. An inhibitor of the mitochondrial respiratory chain, antimycin A, decreased EA-induced lipid peroxidation and cell death but had no effect on thiol depletion or elevated cytosolic Ca2+. These data suggest that cardiomyocyte thiol status may be linked to intracellular Ca2+ homeostasis and that peroxidative damage originating in the mitochondria is a major event in the onset of cell death in this cardiomyocyte model of thiol depletion.
Dhanbhoora
C M
CM
Department of Pharmacology and Toxicology, College of Pharmacy, University of Rhode Island, Kingston 02881.
Babson
J R
JR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004734:Energy Metabolism / Q000187:drug effects; D004976:Ethacrynic Acid / Q000633:toxicity*; D005978:Glutathione / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006706:Homeostasis; D008054:Lipid Peroxides / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D008929:Mitochondria, Heart / Q000502:physiology; D009206:Myocardium / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D002614:Chelating Agents; D008054:Lipid Peroxides; D013438:Sulfhydryl Compounds; D000968:Antimycin A; D000255:Adenosine Triphosphate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005978:Glutathione; D004976:Ethacrynic Acid; D002118:Calcium