J.F. Hütter

Early enzyme release from myocardial cells is not due to irreversible cell damage.

It is often assumed that the release of enzymes from oxygen deficient heart tissue is due to the irreversible damage of myocardial cells. However, because of diffusion barriers and inhomogeneity of oxygen-deficient tissue this hypothesis cannot be proven in heart tissue. The question whether enzyme release may already occur during reversible injury is of considerable relevance in clinical medicine: first, because the amount of released enzyme activity has been used to estimate the mass of damaged tissue in cardiac infarction and, second, because the stress of some diagnostic interventions may lead to cardiac enzyme release, which according to the irreversibility hypothesis would indicate the death of cells in a cell constant organ.

Development of ischemia-induced damage in defined mitochondrial subpopulations

Two mitochondrial subpopulations were isolated from guinea-pig heart by density gradient centrifugation. Under control conditions, both contain functionally intact mitochondria in which ischemic damage develops similarly. However, in one subpopulation adenine nucleotide content, adenine nucleotide translocase activity, oxidative phosphorylation and Ca2+ uptake are a quarter lower than in the other one when related to mitochondrial protein mass. Cytochrome contents and uncoupled electron flux are the same. Changes develop most evidently at the very beginning of ischemia for NAD-linked respiration. When ischemia progresses, cytochromes and the translocator protein are gradually lost or inactivated. Thereupon only partial recovery of mitochondrial function can be obtained after 20 min of reperfusion.

Energy metabolism and enzyme release of cultured adult rat heart muscle cells during anoxia

An intact preparation of adult ventricular muscle cells was incubated in substrate-free, pH-constant, anoxic Tyrode solution. The time course of metabolic changes was found to depend on the relation of cell number to incubation volume: the smaller the volume, the faster anoxic damage develops. Energy needs decline rapidly during anoxia. Yet glycolytic energy production remains insufficient, since it also declines. Glycogenolysis stops after degradation of only half the glycogen present initially. Release of cytosolic enzymes (LDH, MDH) starts with the initial decrease in high-energy phosphates and proceeds correlated to the actual ATP content (r = -0.98) during the stage of reversible cell injury. An ATP content of 2 mumol/g wet wt. marks a critical threshold, below which more and more cells become irreversibly damaged. In the cell culture system, the anoxic process develops similarly to that of the oxygen deficient organ, however prolonged as in arrested hearts.

Myocardial fatty acid oxidation: evidence for an albumin-receptor-mediated membrane transfer of fatty acids

SummaryUsing a computer-assisted working rat heart preparation, which allows continuous registration of the respiratory quotient, it was tested which parameters determine fatty acid oxidation in the myocardium.Supplying albumin and palmitate in different concentrations the rate of fatty acid oxidation was measured. The UFA concentrations were calculated using stepwise equilibrium constants. When keeping constant the NEFA/albumin ratio and raising total NEFA concentration, an increase in fatty acid oxidation was found showing a saturation curve. Increasing NEFA at constant albumin concentration, however, results in a linear increase in fatty acid oxidation. Keeping constant the total NEFA concentration elevation of albumin shows an inhibitory effect. These results suggest the existence of a receptor for albumin on heart cell surface, which mediates uptake of albumin-bound NEFA.An additional supply of glucose and lactate does not show any effect on these relations. Acetate and dichloroacetate, an activator of the pyruvate dehydrogenase, are found to be competitive inhibitors of fatty acid oxidation.

Kinetic analysis of myocardial fatty acid oxidation suggesting an albumin receptor mediated uptake process

The relationship between extracellular albumin and non-esterified fatty acid (NEFA) concentrations and the rate of fatty acid oxidation was studied. The data were obtained from tests performed on a working rat heart. When NEFA concentration was increased the rate of fatty acid oxidation showed a saturation curve at a constant NEFA/albumin ratio. Keeping constant the albumin concentration, a rise in NEFA concentration resulted in a linear increase of fatty acid oxidation. No correlation, however, was found between fatty acid oxidation and the unbound fraction of fatty acids. These results suggest an albumin receptor mediated NEFA uptake. With this assumption the following rate law of the NEFA uptake was derived: UPT = UPT0 X [FA]/(Km + [ALB] ) where [FA] and [ALB] are the total NEFA- and albumin concentrations, UPT0 and Km are constants. The rates of oxidation computed with this equation show a very good congruence to the values obtained experimentally. The validity of the rate law is confirmed by the fact that it is shown to be in agreement to the results of other investigators.

Inhibition of fatty acid oxidation and decrease of oxygen consumption of working rat heart by 4-bromocrotonic acid

Nonesterified fatty acids (NEFA), glucose and lactate are major fuels for myocardial energy production. The ratio of energy produced and oxygen consumed, which can be expressed as ATP/O ratio, is different for each substrate: e.g. 3.17 for glucose and 2.83 for palmitate. Direct measurements, however, have shown that the difference of oxygen consumption is about twice as great as theoretically expected. This difference is of little significance under aerobic conditions, but may be important when oxygen supply is restricted. Numerous attempts have been made to reduce oxygen consumption by activating carbohydrate oxidation or inhibiting fatty acid metabolism. As the rate of fatty acid oxidation has been shown to depend on arterial concentrations of NEFA and albumin, this may be one point of control. Further approaches such as increasing the arterial levels of glucose, insulin and potassium, have been controversially discussed. As 4-bromocrotonic acid has been found to inhibit the fatty acid oxidation in isolated rat heart mitochondria [8], this might be an effective agent to save oxygen by reducing the rate of fatty acid oxidation in intact hearts.

Absence of reoxygenation damage in isolated heart cells after anoxic injury

Cultured adult cardiac myocytes were exposed to anoxia under substrate-free conditions and then reoxygenated. When comparing the oxygen deficient organ to the anoxic cell culture, we see that metabolic changes in the latter system proceed in a similar, yet prolonged manner, as in arrested hearts. Release of cytosolic enzymes starts with minor energetic disturbances and proceeds closely correlated to the actual ATP level. Below 2 μmol ATP/gww, an increasing number of cells becomes irreversibly damaged, above this level, 30 min reoxygenation leads to extensive recovery of the whole preparation. The results indicate that leakage of cytosolic enzymes during the early stage of anoxia is due to a gradual protein release from the individual cells and is related to reversible membrane alterations. Reoxygenation does not induce changes considered typical of the ‘oxygen paradox’. Since mechanical cell-cell interactions are absent in this model, it is suggested that aggravation of tissue damage in heart tissue reoxygenated late is mainly caused by mechanical forces.

Acyl-carnitine effects on isolated cardiac mitochondria and erythrocytes.

SummaryThe effects of various long-chain acyl-carnitines (AC) on mitochondrial functions and red cell membrane stability were studied. Lower concentrations slightly stimulate respiration-dependent functions such as phosphorylation rate and Ca++ uptake velocity, whereas higher concentrations inhibit these functions with concomitant depression of the ATP/O ratio. The order of effectiveness among the AC is very similar for different mitochondrial function. The differences among AC in their actions on red cell stability in hypotonic media and their differences in influence on mitochondiral functions exhibit less resemblance. The relative order of erythrolytic concentrations of AC follows the order of their critical micellar concentration. Model calculations indicate that the concentrations of AC found in ischemic hearts are below those which exhibit inhibitory effects in vitro. Ultrastructural changes in mitochondria incubated with AC are different from those found in ischemic tissue. From this, it seems questionable whether the elevated AC levels in ischemic hearts are indeed as importan for the development of membrane damage as is often supposed.

Effects of hypoxia and fatty acids on the distribution of metabolites in rat heart.

The effects of exogenous fatty acids and hypoxia on cardiac energy metabolism were studied by measuring mitochondrial and cytosolic adenine nucleotides as well as CoA and carnitine esters using a tissue fractionation technique in non-aqueous solvents. During normoxia, the administration of 0.5 mM palmitate caused a considerable increase in acyl-CoA and acylcarnitine, particularly in mitochondria. High-energy phosphates, however, were only slightly altered. A 90 min low-flow hypoxia caused a dramatic increase in mitochondrial acyl esters. The mitochondrial ATP content decreased significantly, while the cytosolic concentration was only slightly diminished, suggesting an inhibition of mitochondrial adenine nucleotide translocation by long-chain acyl-CoA. Addition of palmitate during hypoxia amplified hypoxic damage and reduced adenine nucleotides in both compartments considerably, while fatty acid metabolites were only slightly affected. In presence of an inhibitor of fatty acid oxidation (BM 42.304), the fatty-acid-induced acceleration of cardiac injury was prevented. Since BM 42.304 decreased mitochondrial acylcarnitine and increased the cytosolic concentration significantly, BM 42.304 was presumed to inhibit mitochondrial acylcarnitine translocase. However, a causal relationship between lipid metabolites and ischemic damage seemed unlikely.

Relation between enzyme release and metabolic changes in reversible anoxic injury of myocardial cells.

Cultured adult cardiac myocytes were exposed to anoxia under substrate-free conditions. When compared to the metabolic changes in the oxygen deficient organ, those in the anoxic cell culture proceed in a similar, yet prolonged manner. Release of cytosolic enzymes starts with minor energetic disturbances and proceeds in close correlation to the actual ATP decay. Below 2 mumol ATP/gww, an increasing number of cells becomes irreversibly damaged, but above, 30 min reoxygenation leads to extensive recovery of the whole preparation. The results indicate that leakage of cytosolic enzymes during the early stage of anoxia is due to a gradual protein release from the individual cells, related to reversible membrane alterations.