Mitochondrial dysfunction accompanies diastolic dysfunction in diabetic rat heart.

The objective of this study was to determine whether a defect in mitochondrial respiratory function accompanies the development of diabetic cardiomyopathy. The hypothesis tested in this study is that a decrease in Ca2+ uptake into mitochondria may prevent the stimulation of Ca(2+)-sensitive matrix dehydrogenases and the rate of ATP synthesis. Streptozotocin (55 mg/kg)-induced diabetic rats were used as a model of insulin-dependent diabetes mellitus. Hearts from 4-wk diabetic rats had basal heart rates and rates of contraction and relaxation similar to control. Isoproterenol caused a similar increase in the rate of contraction in diabetic and control hearts, whereas the peak rate of relaxation was reduced in diabetic hearts. Mitochondrial Ca2+ uptake was reduced in mitochondria from diabetic hearts after 2 wk of diabetes. Na(+)-induced Ca2+ release was unchanged. State 3 respiration rate was depressed in mitochondria from diabetic rats only when the respiration was supported by the substrate of a Ca(2+)-regulated matrix enzyme. The pyruvate dehydrogenase activity was reduced in diabetic mitochondria compared with that of control. It was concluded that mitochondria from diabetic hearts had a decreased capacity to upregulate ATP synthesis via stimulation of Ca(2+)-sensitive matrix dehydrogenases. The impairment in the augmentation of ATP synthesis rate accompanies a decreased rate of relaxation during increased work load.

Expression of rat alpha 1 Na,K-ATPase containing substitutions of "essential" amino acids in the catalytic center.

Previous chemical modification studies have suggested that Cys-369, Cys-658, Lys-482, Asp-712, and Asp-716 are essential for Na,K-ATPase function. To determine if the side chains of these amino acid residues are required for enzyme activity, rat alpha 1 cDNAs containing the mutations Cys-369-->Ser, Cys-658-->Ala, Lys-482-->Ala, Asp-712-->Asn, and Asp-716-->Asn were prepared and stably expressed in HeLa cells, which normally cannot survive in medium containing microM concentrations of ouabain. Expression of rat alpha 1 wild-type and the mutants Cys-369-->Ser, Cys-658-->Ala, and Lys-482-->Ala causes the appearance of HeLa cells that survive in medium containing 0.5 microM ouabain. Membranes isolated from the rat alpha 1 mutant clones exhibit Na,K-ATPase activities, ratios of Na,K-ATPase:phosphoenzyme, and apparent affinities for ouabain and ATP which are comparable to those of the rat alpha 1 wild-type enzyme. HeLa cells expressing mRNA and protein for Asp-712-->Asn and Asp-716-->Asn rat alpha 1 mutants cannot survive in 0.1 microM ouabain, and membranes isolated from these clones exhibit very little or no ouabain-insensitive rat alpha 1 Na,K-ATPase activity. The Asp-712-->Asn, but not Asp-716-->Asn, mutant rat alpha 1 can be phosphorylated by ATP. We conclude that Cys-369, Cys-658, and Lys-482 are not essential for Na,K-ATPase function but that the Asp residues at 712 and 716 are both essential.

Swelling, reductive stress, and cell death during chemical hypoxia in hepatocytes.

In rat hepatocytes, we examined the relationship between cell volume, bleb formation, and loss of cell viability during chemical hypoxia with KCN plus iodoacetic acid. In hypotonic media (150-200 mosmol/kgH2O), cells swelled to a greater extent during chemical hypoxia than in isotonic media, but rates of cell killing were identical. Sucrose (300 mM) added to isotonic media prevented early cell swelling but actually accelerated cell killing. In contrast, mannitol (300 mM) improved cell survival but did not prevent cell swelling. Bleb formation occurred regardless of buffer tonicity. The antioxidants desferrioxamine and cyanidanol but not superoxide dismutase +/- catalase delayed lethal cell injury. Cell killing was greater during aerobic compared with anaerobic chemical hypoxia. Hydroperoxide formation was measured using a dichlorofluorescin assay and was accelerated during aerobic but not anaerobic chemical hypoxia. The results indicate that cell swelling is not the driving force for bleb formation or lethal cell injury. We conclude that "reductive stress" caused by respiratory inhibition favors formation of toxic oxygen species and may contribute to lethal cell injury during intermittent or incomplete oxygen deprivation.