Mechanism of peroxide-induced cellular injury in cultured adult cardiac myocytes.
Reactive oxygen species contribute to the tissue injury seen after reperfusion of ischemic myocardium. We propose that toxicity originates from the effect that mitochondrial peroxide metabolism has on substrate entry into oxidative pathways. To support our contention, cultured adult rat cardiomyocytes were incubated with physiological concentrations of peroxide. The cellular extract and incubation medium were analyzed for adenine nucleotides and purines by reverse-phase high-pressure liquid chromatography. Cellular glutathione efflux was determined by enzymatic analysis of the incubation medium. Pyruvate dehydrogenase (PDH) activity was determined in the cultured myocytes as well as in freshly isolated cardiac mitochondria using [1-C14]pyruvate. Extracellular glutathione rose 3.3-fold in response to small doses of peroxide (approximately 108 nmol/mg protein). Likewise, small quantities of peroxide reduced total cellular adenine nucleotides to 50-60% of control values with only a modest (0.95-0.91) reduction in energy charge [ATP + 1/2 ADP)/(ATP + ADP + AMP]. Peroxide-treated myocytes selectively release inosine and adenosine, as only these two purine degradation products were detected in the incubation medium. The most dramatic response was a peroxide dose-dependent inhibition of PDH activity in cultured myocytes as well as freshly isolated mitochondria; just 65 and 30 nmol peroxide/mg protein induced a 50% reduction in cellular and mitochondrial PDH activity, respectively. In conclusion, physiological quantities of peroxide potently inhibit PDH in cultured cardiomyocytes and isolated cardiac mitochondria. PDH inhibition blocks the aerobic oxidation of glucose and inhibits the oxidative phosphorylation of ADP, which in turn leads to cellular adenine nucleotide degradation.
Vlessis
A A
AA
Department of Surgery, Oregon Health Sciences University, Portland 97201.
Muller
P
P
Bartos
D
D
Trunkey
D
D
D000227:Adenine Nucleotides / Q000378:metabolism; D000241:Adenosine / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000818:Animals; D015687:Cell Hypoxia; D002851:Chromatography, High Pressure Liquid; D005978:Glutathione / Q000378:metabolism; D006321:Heart / Q000187:drug effects*; D007288:Inosine / Q000378:metabolism; D008297:Male; D008929:Mitochondria, Heart / Q000187:drug effects / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology / Q000473:pathology*; D010084:Oxidation-Reduction; D010085:Oxidative Phosphorylation; D010545:Peroxides / Q000633:toxicity*; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D000227:Adenine Nucleotides; D010545:Peroxides; D011768:Pyruvate Dehydrogenase Complex; D007288:Inosine; D000255:Adenosine Triphosphate; D005978:Glutathione; D000241:Adenosine