Effects of physical training on several glycolytic enzymes in rat heart.

In response to a program of daily swimming for 16 weeks, the activities of pyruvate kinase and lactate dehydrogenase increased significantly in the hearts of young male rats. The isozyme composition in M of cardial lactate dehydrogenase increased from 28.5 to 32.7% in the trained animals. Phosphofructokinase activity and glycogen content were unchanged. The hearts of the exercising animals were 28% heavier than those of sedentary paired weight controls.     

The myocardium and its vasculature: a histochemical comparison of the normal and chronically sympathectomized dog heart

Summary Using histochemical techniques, the reactivities of selected enzymes and other metabolic components were examined in the myocardium, coronary arteries, and coronary arterioles of normal, two-week-sympathectomized, and sham-operated canine hearts. There were no differences in the histochemistry of coronary arteries in any of the hearts, but important differences were noted in the myocardium and especially in the arterioles. The reactivities of the enzyme glucose-6-phosphate dehydrogenase and the nucleic acids were increased in arterioles of the sympathectomized heart, possibly indicating an increased protein synthesis. The reactivities of succinate dehydrogenase, NAD-isocitrate dehydrogenase, and cytochrome oxidase were reduced in myocardium and arterioles of sympathectomized hearts as well as in arterioles of sham-operated hearts; the changes were greater in the sympathectomized arterioles where there was also observed an increase in reactivity of lactate dehydrogenase. These findings suggest a depression in aerobic metabolic capacity and, in the case of the sympathectomized arteriole, imply a possible shift in adaptation from aerobic to anaerobic metabolism.     

Changes in the activity of selected enzymes during starvation of Physarum polycephalum

The levels of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, lactate dehydrogenase, and cyclic phosphodiesterase activities were examined in growing and starving plasmodia of Physarum polycephalum. The activities of lactate dehydrogenase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase decreased whereas that of cyclic phosphodiesterase increased. The change in activity of lactate dehydrogenase was the result of the variation of the activity of a single enzyme quite similar to the lactate dehydrogenases of higher animals.     

Oxfenicine diverts rat muscle metabolism from fatty acid to carbohydrate oxidation and protects the ischaemic rat heart

In isolated diaphragms from rats fed on a high-fat diet, oxfenicine (S-4-hydroxyphenylglycine) stimulated the depressed rates of pyruvate decarboxylation (2-fold) and glucose oxidation (5-fold). In diaphragms from normal-fed rats, oxfenicine had no effect on pyruvate decarboxylation but doubled the rate of glucose oxidation and inhibited the oxidation of palmitate. Treatment of fat-fed rats with oxfenicine restored the proportion of myocardial pyruvate dehydrogenase in the active form to that observed in normal-fed rats. In rat hearts perfused in the presence of glucose, insulin and palmitate, oxfenicine increased carbohydrate oxidation and stimulated cardiac performance with no increase in oxygen consumption - i.e. improved myocardial efficiency. Working rat hearts perfused with glucose, insulin and palmitate and subjected to 10 min global ischaemia recovered to 81% of their pre-ischaemic cardiac output after 30 min reperfusion, and released large amounts of lactate dehydrogenase into the perfusate. Hearts perfused with oxfenicine had slightly higher pre-ischaemic cardiac outputs and, on reperfusion, recovered more completely (to 96% of the pre-ischaemic value). Oxfenicine reduced the amount of lactate dehydrogenase released by 73%. We conclude that, in rat hearts with high rates of fatty acid oxidation, a relative increase in carbohydrate oxidation will improve myocardial efficiency, and preserve mechanical function and cellular integrity during acute ischaemia.     

Impairment of glucose metabolism and energy transfer in the hypertriglyceridemic rat heart

The metabolic pathways involved in ATP production in hypertriglyceridemic rat hearts were evaluated. Hearts from male Wistar rats with sugar-induced hypertriglyceridemia were perfused in an isolated organ system. Mechanical performance, oxygen uptake and beat rate were evaluated under perfusion with different oxidizable substrates. Age- and weight-matched animals were used as control. The hypertriglyceridemic (HTG) hearts showed a decrease in the mechanical work and slight diminution in the oxygen uptake when perfused with glucose, pyruvate or lactate. No differences were found when perfused with palmitate, octanoate or -hydroxybutyrate. The glycolytic flux in HTG hearts was 2.4 times lower than in control hearts. Phosphofructokinase-I (PFK-I) was 16% decreased in HTG hearts, whereas pyruvate kinase activity did not change. The increased levels of glucose-6hyphen;phosphate in HTG heart, suggested a flux limitation by the PFK-I. Pyruvate dehydrogenase in its active form (PDHa) diminished as well. The PDHa level in the HTG hearts was restored to control values by dichloroacetate; however, this addition did not significantly improve the mechanical performance. Levels of ATP and phosphocreatine as well as total creatine kinase activity and the MB fraction were significant lower in the HTG hearts perfused with glucose. The data suggested that supply of ATP by glucose oxidation did not suffice to support cardiac work in the HTG hearts; this impairment was exacerbated by the diminution of the creatine kinase system output.     

Effects of cold acclimation on the activity levels of creatine kinase, lactate dehydrogenase and lactate dehydrogenase isoenzymes in various tissues of the rat.

The effects of cold acclimation on the activity levels of creatine kinase, lactate dehydrogenase and lactate dehydrogenase isoenzymes in various tissues/ organs of the rat (Rattus norvegicus) were investigated. Male Sprague-Dawley rats were divided into two groups. One group was housed at 4+/-1 degrees C (experimental group) and the other at 24+/-1 degrees C (control group) for six months. The rats were housed in single cages and had access to food and water ad libitum. The tissues/organs investigated were heart, liver, lung, kidney, gastrocnemius muscle and interscapular brown adipose tissue as well as serum. With the exception of lung, (which showed a decrease of 24%) total creatine kinase activity levels were significantly increased (P< 0.05) in all the tissues/organs investigated (17-51%) as well as serum (34%), in cold acclimated animals. Cold acclimation also resulted in significantly increased (P< 0.05) activity levels of lactate dehydrogenase in all the tissues/organs investigated (14-24%) as well as serum (35%). Cold exposure resulted in an increase of the activity levels of all the detectable isoenzymes of lactate dehydrogenase, although not always significant, in all the tissues/organs investigated as well as serum. The M(4)tetramer of lactate dehydrogenase was the only detectable isoenzyme in serum.     

Proteomic analyses of transgenic LQT1 and LQT2 rabbit hearts elucidate an increase in expression and activity of energy producing enzymes

Various biochemical and genomic mechanisms are considered to be a hallmark of metabolic remodeling in the stressed heart, including the hypertrophied and failing heart. In this study, we used quantitative proteomic 2-D Fluorescence Difference In-Gel Electrophoresis (2-D DIGE) in conjunction with mass spectrometry to demonstrate differential protein expression in the hearts of transgenic rabbit models of Long QT Syndrome 1 (LQT1) and Long QT Syndrome 2 (LQT2) as compared to littermate controls (LMC). The results of our proteomic analysis revealed upregulation of key metabolic enzymes involved in all pathways associated with ATP generation, including creatine kinase in both LQT1 and LQT2 rabbit hearts. Additionally, the expression of lamin-A protein was increased in both LQT1 and LQT2 rabbit hearts as was the expression of mitochondrial aldehyde dehydrogenase and desmoplakin in LQT1 and LQT 2 rabbit hearts, respectively. Results of the proteomic analysis also demonstrated down regulation in the expression of protein disulfide-isomerase A3 precuorsor and dynamin-like 120 kDa protein (mitochondrial) in LQT1, and of alpha-actinin 2 in LQT2 rabbit hearts. Up regulation of the expression of the enzymes associated with ATP generation was substantiated by the results of selective enzyme assays in LQT1 and LQT2 hearts, as compared to LMC, which revealed increases in the activities of glycogen phosphorylase (+50%, +65%, respectively), lactate dehydrogenase (+25%, +25%) pyruvate dehydrogenase (+31%, +22%), and succinate dehydrogenase (+32%, +60%). The activity of cytochrome c-oxidase, a marker for the mitochondrial function was also found to be significantly elevated (+80%) in LQT1 rabbit hearts as compared with LMC. Western blot analysis in LQT1 and LQT2 hearts compared to LMC revealed an increase in the expression of very-long chain-specific acyl-CoA dehydrogenase (+35%, +33%), a rate-limiting enzymes in β-oxidation of fatty acids. Collectively, our results demonstrate similar increases in the expression and activities of key ATP-generating enzymes in LQT1 and LQT2 rabbit hearts, suggesting an increased demand, and in turn, increased energy supply across the entire metabolic pathway by virtue of the upregulation of enzymes involved in energy generation.     

Combined glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase in catecholamine-stimulated guinea-pig cardiac muscle. Comparison with mass-action ratio of creatine kinase.

The steady-state reactant levels of triose-phosphate isomerase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system were examined in guinea-pig cardiac muscle. Key glycolytic intermediates, including glyceraldehyde 3-phosphate were directly measured and compared with those of creatine kinase. Non-working Langendorff hearts as well as isolated working hearts were perfused with 5 mM glucose (plus insulin) under normoxia conditions to maintain lactate dehydrogenase near-equilibrium. The cytosolic phosphorylation potential ([ATP]/([ADP].[Pi])) was derived from creatine kinase and the free [NAD+]/([NADH].[H+]) ratio from lactate dehydrogenase. In Langendorff hearts glycolysis was varied from near-zero flux (hyperkalemic cardiac arrest) to higher than normal flux (normal and maximum catecholamine stimulation). The triose-phosphate isomerase was near-equilibrium only in control or potassium-arrested Langendorff hearts as well as in postischemic 'stunned' hearts. However, when glycolytic flux increased due to norepinephrine or due to physiological pressure-volume work the enzyme was displaced from equilibrium. The alternative phosphorylation ratio [ATP]'/([ADP]).[Pi]) was derived from the magnesium-dependent glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system assigning free magnesium different values in the physiological range (0.1-2.0 mM). As predicted, [ATP]/([ADP].[Pi]) and [ATP]'/([ADP]'.[Pi]') were in excellent agreement when glycolysis was virtually halted by hyperkalemic arrest (flux approximately 0.2 mumol C3.min-1.g dry mass-1). However, the equality between the two phosphorylation ratios was not abolished upon resumption of spontaneous beating and also not during adrenergic stimulation (flux approximately 5-14 mumol C3.min-1.g dry mass-1). In contrast, when flux increased due to transition from no-work to physiological pressure-volume work (rate increase from approximately 3 to 11 mumol C3.min-1.g dry mass-1), the two ratios were markedly different indicating disequilibrium of the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase. Only during adrenergic stimulation or postischemic myocardial 'stunning', not due to hydraulic work load per se, glyceraldehyde-3-phosphate levels increased from about 4 microM to greater than or equal to 16 microM. Thus the guinea-pig cardiac glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system can realize the potential for near-equilibrium catalysis at significant flux provided glyceraldehyde-3-phosphate levels rise, e.g., due to 'stunning' or adrenergic hormones.     

Lipid Peroxidation, Tissue Necrosis, and Metabolic and Mechanical Recovery of Isolated Reperfused Rat Heart as a Function of Increasing Ischemia

Isolated Langendorff-perfused rat hearts, after 30 min of preperfusion, were submitted to increasing times of global normothermic ischemia (1, 2, 5, 10, 20 and 30 min) or to the same times of ischemia followed by 30 min of reperfusion. Analysis of malondialdehyde, ascorbic acid, oxypurines, nucleosides, nicotinic coen-zymes and high-energy phosphates was carried out by HPLC on neutralized perchloric acid extracts of freeze-clamped tissues. In addition, maximum rate of intra-ventricular pressure development and cardiac output of malondialdehyde, lactate dehydrogenase, oxypurines and nucleosides were monitored during both preperfusion and reperfusion. Besides decreasing energy metabolites and nicotinic coenzyme pool, prolonged ischemia produced oxidation of significant amounts of hypoxanthine and xanthine to uric acid and generation of detectable levels of malondialdehyde (0.002 μmollg dry weight). After oxygen and substrate readmission, tissue and perfusate malondialdehyde increased only if previous ischemia was longer than 5 min, while lactate dehydrogenase was detected in perfusate of reperfused hearts following 10, 20, and 30 min of ischemia. Highest values of tissue malondialdehyde and total malondialdehyde output were recorded in reperfused hearts subjected to 30 min of ischemia (0.043 μmol/g dry weight and 0.069 μmol/ 30 min/g dry weight, respectively). Since tissue malondialdehyde was observed without detectable lactate dehydrogenase release in perfusate, it might be stated that malondialdehyde generation (i.e., lipid peroxidation) temporally preceded lactate dehydrogenase release (i.e., tissue necrosis). In reperfused hearts, evaluation of myocardial energy state and of mechanical recovery allowed us to determine times of ischemia beyond which reperfusion did not positively affect these metabolic and functional parameters. Main findings are that, under these experimental conditions, lipid peroxidation might be the cause and not the consequence of tissue necrosis and that duration of ischemia might be the factor deciding effectiveness of reperfusion.     

Fatty acid oxidation and its impact on response of spontaneously hypertensive rat hearts to an adrenergic stress: benefits of a medium-chain fatty acid

The spontaneously hypertensive rat (SHR) is a model of cardiomyopathy characterized by a restricted use of exogenous long-chain fatty acid (LCFA) for energy production. The aims of the present study were to document the functional and metabolic response of the SHR heart under conditions of increased energy demand and the effects of a medium-chain fatty acid (MCFA; octanoate) supplementation in this situation. Hearts were perfused ex vivo in a working mode with physiological concentrations of substrates and hormones and subjected to an adrenergic stimulation (epinephrine, 10 microM). (13)C-labeled substrates were used to assess substrate selection for energy production. Compared with control Wistar rat hearts, SHR hearts showed an impaired response to the adrenergic stimulation as reflected by 1) a smaller increase in contractility and developed pressure, 2) a faster decline in the aortic flow, and 3) greater cardiac tissue damage (lactate dehydrogenase release: 1,577 +/- 118 vs. 825 +/- 44 mU/min, P < 0.01). At the metabolic level, SHR hearts presented 1) a reduced exogenous LCFA contribution to the citric acid cycle flux (16 +/- 1 vs. 44 +/- 4%, P < 0.001) and an enhanced contribution of endogenous substrates (20 +/- 4 vs. 1 +/- 4%, P < 0.01); and 2) an increased lactate production from glycolysis, with a greater lactate-to-pyruvate production ratio. Addition of 0.2 mM octanoate reduced lactate dehydrogenase release (1,145 +/- 155 vs. 1,890 +/- 89 mU/min, P < 0.001) and increased exogenous fatty acid contribution to energy metabolism (23.7 +/- 1.3 vs. 15.8 +/- 0.8%, P < 0.01), which was accompanied by an equivalent decrease in unlabeled endogenous substrate contribution, possibly triglycerides (11.6 +/- 1.5 vs. 19.0 +/- 1.2%, P < 0.01). Taken altogether, these results demonstrate that the SHR heart shows an impaired capacity to withstand an acute adrenergic stress, which can be improved by increasing the contribution of exogenous fatty acid oxidation to energy production by MCFA supplementation.     

Comparative effects of experimental lead poisoning on enzymatic activities of kidney and liver in rat.

Rats were fed for 6 months with 0.1% lead acetate diet. At the end of the intoxication period urinary levels of ALA and lead were comparable with the usual urinary levels of chronically lead exposed humans. In kidney homogenates of poisoned rats succinate dehydrogenase, cytochrome oxidase and lactate dehydrogenase were unmodified, malate dehydrogenase and glutamate dehydrogenase were decreased, while Dt diaphorase and glucose-6-phosphate dehydrogenase were greatly increased compared with controls. In liver homogenates of the same poisoned animals no alteration of the enzymatic activities was observed.     

The effect of dichloroacetate on the isolated no flow arrested rat heart

Ischemic dysfunction, including contracture, has been attributed to lack of ATP, although previous work has not been consistent with this concept. We describe here a model of no flow ischemic arrest, characterized by depressed levels of mechanical function upon reperfusion and high energy phosphate stores within normal limits. The decreased mechanical function bears an inverse relationship to myocardial lactate levels after twenty-minutes of reperfusion in the absence or presence of dichloroacetic acid (DCA). Post-ischemic non-DCA treated hearts attained peak work of only 25% of that of controls, while those treated with DCA following ischemia performed almost as well as controls. ATP and CP levels remained high in both DCA treated and non-DCA treated hearts. Lactate levels were high in hearts immediately following ischemia, but were reduced to control levels in post-ischemic hearts perfused with DCA within twenty minutes, whereas those not treated with DCA had lactate levels two to three times that of controls within the same time period. Pyruvate dehydrogenase (PDH) activity was reduced in non-DCA treated post ischemic hearts after twenty minutes reperfusion but was elevated above controls in hearts reperfused with DCA. The data indicates that DCA increases mechanical performance of the isolated post-ischemic rat heart and the proposed mechanism for this increase is the oxidative removal of lactate resulting from an increase in PDH activity.     

Pyruvate carboxylation as an anaplerotic mechanism in the isolated perfused rat heart.

1. The role of pyruvate carboxylation in the net synthesis of tricarboxylic acid-cycle intermediates during acetate metabolism was studied in isolated rat hearts perfused with [1-14C]pyruvate. 2. The incorporation of the 14C label from [1-14C]pyruvate into the tricarboxylic acid-cycle intermediates points to a carbon input from pyruvate via enzymes in addition to pyruvate dehydrogenase and citrate synthase. 3. On addition of acetate, the specific radioactivity of citrate showed an initial maximum at 2 min, with a subsequent decline in labelling. The C-6 of citrate (which is removed in the isocitrate dehydrogenase reaction) and the remainder of the molecule showed differential labelling kinetics, the specific radioactivity of C-6 declining more rapidly. Since this carbon is lost in the isocitrate dehydrogenase reaction, the results are consistent with a rapid inactivation of pyruvate dehydrogenase after the addition of acetate, which was confirmed by measuring the 14CO2 production from [1-14C]pyruvate. 4. The results can be interpreted to show that carboxylation of pyruvate to the C4 compounds of the tricarboxylic acid cycle occurs under conditions necessitating anaplerosis in rat myocardium, although the results do not identify the enzyme involved. 5. The specific radioactivity of tissue lactate was too low to allow it to be used as an indicator of the specific radioactivity of the intracellular pyruvate pool. The specific radioactivity of alanine was three times that of lactate. When the hearts were perfused with [1-14C]lactate, the specific radioactivity of alanine was 70% of that of pyruvate. The results suggest that a subcompartmentation of lactate and pyruvate occurs in the cytosol.     

Effects of increased mechanical work by isolated perfused rat heart during production or uptake of ketone bodies. Assessment of mitochondrial oxidized to reduced free nicotinamide-adenine dinucleotide ratios and oxaloacetate concentrations.

Metabolic effects of increased mechanical work were studied by comparing isolated pumping rat hearts perfused by the atrial-filling technique with aortic-perfused non-pumping hearts perfused by the technique of Langendorff. The initial medium usually contained glucose (11 mm) and palmitate (0.6 mm bound to 0.1 mm albumin). During increased heart work (comparing pumping with non-pumping hearts) the uptake of oxygen and glucose increased threefold, but that of free fatty acids was unchanged. Tissue contents of alpha-oxoglutarate, NH4+, malate, lactate, pyruvate and Pi rose with increased heart work, but contents of ATP, phosphocreatine and citrate fell. Ketone bodies were produced with a ratio of beta-hydroxybutyrate/acetoacetate of about 3:1 in both pumping and non-pumping hearts but with higher net production rates in non-pumping hearts. When ketone bodies were added in relatively high concentrations (total 4 mm) to a glucose (11 mm) medium the medium, ratios of beta-hydroxybutyrate/acetoacetate were not steady even after 60 min of perfusion. The validity of calculating mitochondrial free NAD+/NADH ratios from the tissue contents of the reactants of the glutamate dehydrogenase system or the beta-hydroxybutyrate dehydrogenase system is assessed. The activities of these enzymes are considerably less in the rat heart than in the rat liver, introducing reservations into the application to the heart of the principles used by Williamson et al. (1967) for calculation of mitochondrial free NAD+/NADH ratios of liver mitochondria...     

Diurnal and Seasonal Variations of Serum Enzyme Activity in Cattle and Sheep

In order to test the variation of enzyme activity in serum of cattle and sheep during the day, blood samples were taken at three hrs. interval from 6 a.m. to 9 p.m. The following enzymes were assayed: Aspartate aminotransferase (AspAT = GOT), alanine aminotransferase (AlAT = GPT), total lactate dehydrogenase (LDH), and a-hydroxybutyrate dehydrogenase (HBD). The variation between animals contributed by far to the greatest part of the total variation in clinical healthy animals. The time-of-day-dependant variation was less than 3 %, except for alanine aminotransferase. During the first two weeks of spring pasture serum aspartate and alanine aminotransferase levels were significantly raised in both cows and ewes, compared with serum levels of the same animals on indoor feeding. No such increase occurred in total lactate dehydrogenase.     

Proteomic analysis of cardiac metabolic enzymes in asphyxiated newborn piglets

Hypoxia/reoxygenation (H/R) creates an energetic deficiency in the heart, which may contribute to myocardial dysfunction. We hypothesized that H/R-induced impairment of cardioenergetic enzymes occurs in asphyxiated newborn animals. After hypoxia for 2 h (10-15% oxygen), newborn piglets were resuscitated with 100% oxygen for 1 h, followed by 21% oxygen for 3 h. Sham-operated control piglets had no H/R. Hemodynamic parameters in the piglets were continuously measured. At the end of experiment, hearts were isolated for proteomic analysis. In asphyxiated hearts, the level of isocitrate dehydrogenase and malate dehydrogenase was reduced compared to controls. Inverse correlations between the level of myocardial malate dehydrogenase and cardiac function were observed in the control, but not the H/R hearts. We conclude that reoxygenation of asphyxiated newborn piglets reduces the level of myocardial isocitrate dehydrogenase and malate dehydrogenase. While the cause is not clear, it may be related to the impaired tricarboxylic acid cycle pathway and energy production in the heart.     

Oxygen Radical Injury and Loss of High-Energy Compounds in Anoxic and Reperfused Rat Heart: Prevention By Exogenous Fructose-1, 6-Bisphosphate

Isolated Langendorff-perfused rat hearts after 10 minutes preperfusion, were subjected to a substrate-free anoxic perfusion (20 minutes) followed by 20 minutes reperfusion with a glucose-containing oxygen-balanced medium. Under the same perfusion conditions, the effect of exogenous 5mM fructose-1, 6-bisphosphate has been investigated. The xanthine dehydrogenase to xanthine oxidase ratio, concentrations of high-energy phosphates and the TBA-reactive material (TBARS) were determined at the end of each perfusion period in both control and fructose-1, 6-bisphosphate-treated hearts. Results indicate that anoxia induces the irreversible transformation of xanthine dehydrogenase into oxidase as a consequence of the sharp decrease of the myocardial energy metabolism. This finding is supported by the protective effect exerted by exogenous fructose-1, 6-bisphosphate which is able to maintain the correct xanthine dehydrogenase/oxidase ratio by preventing the depletion of phosphorylated compounds during anoxia. Moreover, in control hearts, the release oflactate dehydrogenase during reperfusion, is paralleled by a 50% increase in the concentration of tissue TBARS. On the contrary, in fructose-1, 6-bisphosphate-treated hearts this concentration does not significantly change after reoxygenation, while a slight but significant increase of lactate dehydrogenase activity in the perfusates is observed.

On the whole these data indicate a direct contribution of oxygen-derived free radicals to the worsening of post-anoxic hearts. A hypothesis on the mechanism of action of fructose-1, 6-bisphosphate in anoxic and reperfused rat heart and its possible application in the clinical therapy of myocardial infarction are presented.     

Tumor necrosis factor-alpha pretreatment is protective in a rat model of myocardial ischemia-reperfusion injury.

We have demonstrated that tumor necrosis factor-alpha (TNF-alpha) pretreatment protected the rat heart from ischemia-reperfusion injury. This effect was monitored by assaying for lactate dehydrogenase (LDH), an enzyme whose release correlates with loss of cell membrane integrity. Intact hearts removed from rats pretreated with TNF-released significantly lower amounts of LDH compared to control hearts after 20 min. of total global ischemia followed by reperfusion. Hearts from TNF-alpha-pretreated animals contained higher levels of manganous superoxide dismutase (MnSOD) mRNA than hearts from untreated rats. Because oxygen free radicals have been implicated as a major cause of reperfusion damage and the function of MnSOD is to detoxify superoxide anions in the mitochondria, a possible protective mechanism for TNF-alpha may be to induce expression of MnSOD in the heart and thus confer resistance to oxygen free radicals generated during reperfusion.     

Overexpression of CuZnSOD in coronary vascular cells attenuates myocardial ischemia/reperfusion injury

Superoxide dismutase scavenges oxygen radicals, which have been implicated in ischemia/reperfusion (I/R) injury in the heart. Our experiments were designed to study the effect of a moderate increase of copper/zinc superoxide dismutase (CuZnSOD) on myocardial I/R injury in TgN(SOD1)3Cje transgenic mice. A species of 0.8 kb human CuZnSOD mRNA was expressed, and a 273% increase in CuZnSOD activity was detected in the hearts of transgenic mice with no changes in the activities of other antioxidant enzymes. Furthermore, immunoblot analysis revealed no changes in the levels of HSP-70 or HSP-25 levels. Immunocytochemical study indicated that there was increased labeling of CuZnSOD in the cytosolic fractions of both endothelial cells and smooth muscle cells, but not in the myocytes of the hearts from transgenic mice. When these hearts were perfused as Langendorff preparations for 45 min after 35 min of global ischemia, the functional recovery of the hearts, expressed as heart rate x LVDP, was 48 +/- 3% in the transgenic hearts as compared to 30 +/- 5% in the nontransgenic hearts (p <.05). The improved cardiac function was accompanied by a significant reduction in lactate dehydrogenase release from the transgenic hearts. Our results demonstrate that overexpression of CuZnSOD in coronary vascular cells renders the heart more resistant to I/R injury.     

Regulation of pyruvate dehydrogenase in rat heart. Mechanism of regulation of proportions of dephosphorylated and phosphorylated enzyme by oxidation of fatty acids and ketone bodies and of effects of diabetes: role of coenzyme A, acetyl-coenzyme A and reduced and oxidized nicotinamide-adenine dinucleotide.

The proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart was decreased by alloxan-diabetes or by perfusion with media containing acetate, n-octanoate or palmitate. The total activity of the dehydrogenase was unchanged. 2. Pyruvate (5 or 25mM) or dichloroacetate (1mM) increased the proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart, presumably by inhibiting the pyruvate dehydrogenase kinase reaction. Alloxan-diabetes markedly decreased the proportion of active dehydrogenase in hearts perfused with pyruvate or dichloroacetate. 3. The total activity of pyruvate dehydrogenase in mitochondria prepared from rat heart was unchanged by diabetes. Incubation of mitochondria with 2-oxo-glutarate plus malate increased ATP and NADH concentrations and decreased the proportion of active pyruvate dehydrogenase. The decrease in active dehydrogenase was somewhat greater in mitochondria prepared from hearts of diabetic rats than in those from hearts of non-diabetic rats. Pyruvate (0.1-10 mM) or dichloroacetate (4-50 muM) increased the proportion of active dehydrogenase in isolated mitochondria presumably by inhibition of the pyruvate dehydrogenase kinase reaction. They were much less effective in mitochondria from the hearts of diabetic rats than in those of non-diabetic rats. 4. The matrix water space was increased in preparations of mitochondria from hearts of diabetic rats. Dichloroacetate was concentrated in the matrix water of mitochondria of non-diabetic rats (approx. 16-fold at 10 muM); mitochondria from hearts of diabetic rats concentrated dichloroacetate less effectively. 5. The pyruvate dehydrogenase phosphate phosphatase activity of rat hearts and of rat heart mitochondria (approx. 1-2 munit/unit of pyruvate dehydrogenase) was not affected by diabetes. 6. The rate of oxidation of [1-14C]pyruvate by rat heart mitochondria (6.85 nmol/min per mg of protein with 50 muM-pyruvate) was approx. 46% of the Vmax. value of extracted pyruvate dehydrogenase (active form). Palmitoyl-L-carnitine, which increased the ratio of [acetyl-CoA]/[CoA] 16-fold, inhibited oxidation of pyruvate by about 90% without changing the proportion of active pyruvate dehydrogenase.