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.

Fatty acid-membrane interactions in isolated cardiac mitochondria and erythrocytes.

The effects of long-chain fatty acids on mitochondrial functions and red cell stability were studied. In albumin-containing incubation media, fatty acid distribution between the albumin-bound and the unbound fraction was estimated by calculation. When fatty acids are compared to one another on the basis of identical unbound concentrations, their effectiveness differs by orders of magnitude. Fatty acids stimulate mitochondrial basic oxygen consumption, thus lowering the respiratory control index, without changing the ATP/O ratio at lower concentrations. Lower concentrations increase Ca2+ uptake velocity, but decrease maximal Ca2+ storage capacity. The order of effectiveness of different fatty acids is the same for both oxidative phosphorylation and Ca2+ uptake. The influence of fatty acids on red cell stability in hypotonic media is similar to these effects both in concentration range and in order of effectiveness. The influence of fatty acids on red cell stability and their critical micellar concentrations were investigated because these are general characteristics of 'detergent-like' compounds. Critical micellar concentrations of the fatty acids in physiological salt buffers are, in general, at least 10-fold higher than the concentrations exhibiting membrane effects in vitro. Based on these findings it is suggested that, of the various concentrations reported in literature for myocardial non-esterified fatty acids, only the lowest values are physiologically possible.

Action of synthetic atrial natriuretic factor on contractility and coronary perfusion in isolated working rat hearts.

The effects of atrial natriuretic factor were studied in isolated working rat hearts. Increasing concentrations, from 0.1 to 100 nM, of a synthetic rat-ANF (atrial natriuretic factor) were added to the perfusate. ANF had no effect on heart rate, contraction cycle or coronary resistance in concentrations up to 3 nM. At concentrations above 10 nM, peak dP/dt and oxygen consumption showed a slight dose-dependent increase. The greater decrease in coronary resistance, and the lowering of coronary oxygen extraction, suggest that ANF has a small coronary dilating effect. No arrhythmias were observed at any concentration of ANF.

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.

A central venous catheter for long-term studies on drug effects and pharmacokinetics in Munich minipigs.

We have developed an implantable venous catheter for repeated blood sampling in the Munich minipig. The catheter consists of silicone tubing, with a polyesterfibre net for fixation in the subcutaneous tissue of the neck. It was introduced into the right jugular vein, and its tip was positioned in the right atrium. The extravascular part of the catheter was tunnelled subcutaneously to the exit point in the subscapular region. In order to avoid contamination of the silicone tubing by drugs, intravenous administrations were performed via a second polyethylene catheter inserted into the implanted catheter. In 9 minipigs, the catheter remained patent for an average of 200 days (maximum 515 days). The animals did not show any signs of discomfort or impaired health.

A new method for determination of drug effects on the arterial distensibility on conscious dogs.

We developed a method for recording drug effects on the distensibility of the arterial system on conscious catheterized beagle dogs. Assuming a pressure-dependent volumic flow from the large arteries into the peripheral vessels, the volume-elasticity of the arterial windkessel is computed by the time-course of the diastolic pressure decline and the total peripheral resistance. A continuous recording of this parameter shows a rapid decrease during each diastolic relaxation, which was in agreement with studies on isolated arteries. An elevation of the mean pressure by an infusion of angiotensin I causes an increase of the elasticity. However, nitroglycerin administration (5 micrograms/kg/min) reduced this parameter significantly. Simultaneous recording of the pulse-wave velocity, which is a measure of the elastic modulus of the arterial wall suggests that nitroglycerin-induced enhancement of the arterial elasticity is caused by a reduced stiffness of the arterial walls as well as by an increased volume of the windkessel. The effects of the nitroglycerin infusion on the venous system and the pulmonary artery showed a faster onset than the decrease of the arterial elasticity. However, the venous effect showed a significant drug tolerance, whereas enhancement of the arterial distensibility remained constant.

Role of fatty acid metabolites in the development of myocardial ischemic damage.

1. The review deals with possible mechanisms by which fatty acids amplify ischemic damage in myocardium. 2. The accumulation of free fatty acids, long chain acyl CoA and carnitine esters during hypoxia and their effects on various enzymatic systems are discussed. 3. Findings on the influence of exogenous fatty acids as well as observations concerning an inhibition of fatty acid degradation are also considered. 4. Finally the role of an oxygen steal effect, as an indirect mechanism for the fatty acid induced amplification of ischemic damage, is discussed.

Temperature dependence of nifedipine action.

Recently, the use of calcium antagonists has been proposed as a new cardioplegic principle. At high doses (e.g. 10(-6)M nifedipine [3]) these drugs can be used for induction of reversible cardiac arrest. Apart from their effect on coronary flow, calcium antagonists seem to be beneficial to ischemic tissue because of their negative inotropic effect at high doses [6, 9]. Ionic cardioplegic solutions are commonly used in an advantageous combination with hypothermia. In the case of calcium antagonists, there is an indication that the specific cardioprotective effect is lessened at low temperatures [3]. Nifedipine is known to reduce contraction force without abolishing the generation of action potentials even at excessive doses [2]. To quantify the suppressing effect of nifedipine on the generation of contractions, we determined the maximal possible contraction frequency (Fm) under electrical stimulation at different temperatures. In isolated myocardial cells, Fm can be determined from the cell contour movements even with an almost complete force reduction and therefore it represents a measure for effective contractile refractoriness.

Hemodynamic effects of new angiotensin converting enzyme inhibitors during continuous angiotensin I infusion on conscious dogs.

The effects of a 6 h infusion of 50 ng/kg.min angiotensin I on hemodynamic parameters were recorded in conscious male beagle dogs. Mean arterial pressure was elevated from 107 +/- 3 mmHg to 152 +/- 5 mmHg (mean +/- SEM, n = 5) within the first hour, followed by a slow decrease to 145 +/- 5 mmHg after 6 h. Cardiac output fell from 2.3 +/- 0.2 l/min to 1.6 +/- 0.2 l/min followed by an increase to 2.5 +/- 0.3 l/min. The peripheral resistance showed an exceeding increase from 2.9 +/- 0.2 mmHg.s/ml to 5.7 +/- 0.7 mmHg.s/ml and a slow decrease to 3.5 +/- 0.5 mmHg.s/ml. Heart rate was found to be decreased from 62 +/- 7 min-1 to 58 +/- 7 min-1 after 1 h. The angiotensin converting enzyme (ACE)-inhibitor enalapril caused a complete reduction of the angiotensin induced hypertension. Three new non-sulfhydryl containing ACE-inhibitors ((S)-N-[N-[1-[ethoxycarbonyl]-3-phenylpropyl]-L-alanyl]-N- [1-pyrrolidinyl]-glycine (REV 6207), (S)-N-[N-[1-[ethoxycarbonyl]-3-phenylpropyl]-L-alanyl]-N- [2-ethoxyethoxy]-glycine (REV 6134), (S)-N-[N-[1-[ethoxycarbonyl]-3-phenylporpyl]-L-alanyl]-N- [phenylmethyl]-glycine (REV 5975] were found to show a partial reduction of the angiotensin I effect. The duration of action which was found to be similar after oral and intravenous administration ranged from 83 +/- 33 min (n = 3) for REV 5975 to more than 360 min for enalapril. A correlation of the arterial pressure lowering and the percentage of the ACE-inhibition suggests that a considerable blood pressure effect can only be observed when more than about 80% of the ACE-activity is inhibited.

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.

A new method for continuous measuring of respiratory quotient in a computer-assisted working heart preparation.

A method was developed for continuous monitoring of the respiratory quotient (RQ) and the ratio of O2-consumption to O2-demand (EQ) in an isolated working rat heart preparation. The RQ allows to get informations about substrates actually oxidized in the myocardium. The EQ is a parameter which behaves reciprocally to the ATP/O of the oxidized substrates during steady state and additionally it allows monitoring of a transitory oxygen debt. For registration of RQ the arterio-venous O2-difference and the CO2-release are measured. As the CO2-production is very small compared with arterial CO2- and HCO-3 -concentrations when using a bicarbonate buffer for perfusion, a bicarbonate-free perfusion-fluid is used. In a portion of coronary effluent the pH is lowered to shift the CO2/HCO-3 -equilibrium to the side of dissolved CO2. Then the pCO2 is measured with a CO2-Nelectrode. Additionally, ventricular pressure, cardiac input and coronary flow are continuously registrated. All data are fed via an AD-converter into an LSI 11-computer. Heart rate, maximum ventricular pressure, dP/dtmax, oxygen demand after Bretschneider's formula, RQ and EQ are calculated. The advantages of the system described are demonstrated with two examples.

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

Using 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.

Effect of fatty acid oxidation on efficiency of energy production in rat heart.

Myocardial fatty acid oxidation has been reported to be accompanied by an elevated O2 consumption compared with carbohydrate oxidation. The exact amount of this additional O2 consumption is controversial. Different investigators have observed an O2 wasting effect that is too large to be explained by the different ATP-to-O2 ratios of these substrates. With the use of isolated perfused rat hearts, O2 consumption and hemodynamic measurements were computer analyzed to provide on-line estimates of the ratio between O2 consumption and demand (EQ). Increasing palmitate or octanoate concentrations decreased the respiratory quotient, which was accompanied by a disproportionate increase of EQ. Inhibition of fatty acid oxidation by an inhibitor of acylcarnitine transferase or a blockade of mitochondrial thiolase caused a drastic reduction of fatty acid oxidation. The fatty acid-induced enhancement of O2 consumption was decreased to a much smaller extent, indicating that there are two different mechanisms responsible for the O2-wasting effect, one that depends on mitochondrial fatty acid oxidation and another that is not affected by an inhibition of this pathway.

An index for estimation of oxygen consumption in rat heart by hemodynamic parameters.

To find a suitable index for the estimation of O2 consumption of rat hearts by use of hemodynamic parameters, isolated hearts were perfused under different working conditions. Coronary flow, arteriovenous O2 difference, cardiac output, and ventricular pressure curve were recorded and continuously fed into a computer. O2 consumption and different hemodynamic parameters such as dP/dtmax, maximum ventricular pressure, pressure-time integral, and heart rate were computed on-line. With the use of these data of 64 different steady states, numerous formulas containing hemodynamic parameters were tested with respect to their ability to predict O2 consumption. The best fit of the data was obtained by a linear combination of the products of rate times dP/dtmax and rate times pressure-time integral of one beat (r = 0.993). Indexes containing approximations of wall stress were not found to improve the predictive ability.

Temperature dependence of calcium antagonist action.

Calcium-tolerant ventricular myocytes from adult rats were electrically stimulated. The maximal contraction frequency (fm) was determined at different temperatures. In drug-free Tyrode solution, fm follows the Arrhenius equation from 7 to 39.5 degrees C. However, all investigated calcium antagonist drugs (verapamil, nifedipine, diltiazem, fendiline) introduce a discontinuity between 27 and 30 degrees C into the Arrhenius plots of fm. Above this transition temperature calcium antagonists lower fm more pronouncedly than below. Below, 10- or 100 fold higher concentrations are needed for the same relative effect as at 37 degrees C. It is argued that these findings might be important in cardiac surgery when calcium antagonists are used for cardioplegia at deep hypothermia.

A new method for studying the incorporation of nonesterified fatty acids into cardiac lipids by using deuterium-labelled palmitate.

A new method for measuring the incorporation of exogenous fatty acids into myocardial lipids of working rat hearts using deuterium-labelled palmitate has been developed. After perfusing isolated hearts, the fatty acid composition of triglycerides, diglycerides, monoglycerides, cholesterol esters, and nonesterified fatty acids (NEFA) was measured by a mass fragmentographic method. Hearts perfused only with 5 mM glucose and 1 mM lactate perfusion as basic substrates showed a significant decrease of triglyceride content, while the other lipids were not found to be significantly reduced. The fatty acid composition of all lipids were not affected. An addition of D31-palmitate complexed to albumin at a molar ratio of 5:1, caused a dose-dependent incorporation into triglycerides and diglycerides which suggested saturation kinetics. The tissue content of nonesterified D31-palmitate was found to be linearly related to its concentration in the perfusate. It may be of note that the concentration of the other NEFA was significantly affected neither by a fatty-acid-free perfusion nor by a perfusion with D31-palmitate. A significant incorporation of the supplied fatty acid into cholesterol esters and monoglycerides could not be detected.

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.

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.

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.

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 mumol 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.