P.G. Spieckermann

Culturing of calcium stable adult cardiac myocytes.

Abstract Cardiac myocytes isolated from adult rats can be kept in culture with physiological calcium concentrations up to four days. Over this time cardiocytes preserve their normal ultrastructure. At intercalated disc area they round up and develop pseudopodia. With exogenous creatine energy rich metabolites are maintained at levels known from intact tissue. Isolated cells are mechanically at rest, their O 2 consumption is comparable with values measured in resting hearts. With 50 μ m 2,4-DNP they can be stimulated to values comparable with maximal O 2 consumption of working hearts. Freshly isolated cells are either morphologically and metabolically intact and electromechanically coupled or rounded up and metabolically inactive. After 3h in culture a preparation of almost 100% intact cells can be obtained, since only intact cells attach to the dish.

Regulation of glycolysis in the ischemic and the anoxic myocardium

Abstract Experiments were done in dogs with various durations of myocardial anoxia in the following conditions: (1) so-called ischemic cardiac arrest in normo- or hypothermia; (2) anoxic perfusion of the heart with and without added glucose; (3) ischemia with cardioplegia in normo- or hypothermia. Even after a pronounced reduction of the myocardial energy demands following cardioplegia and/or hypothermia, anaerobic glycolytic energy production is insufficient to cover the myocardial energy demands during ischemia. When the oxygen tension becomes critical in the ischemic myocardium ( p O 2 in vitro can be utilized in vivo for lactate production under anaerobic conditions. An exception is the activity of hexokinase which in vivo can be about 20% of its maximal in vitro activity. Enzymes of tricarboxylic acid cycle and of the respiratory chain operate in vivo nearer their maximal activity determined in vitro (20 to 60%). The glycolytic flux in the ischemic myocardium is mainly limited by the phosphofructokinase reaction. Only during the earliest stages of ischemia does activation of phosphorylase determine lactate production. When the myocardial ATP content has fallen to about 3.5 μmol/g wet weight (i.e. almost half of normal) the phosphoglucomutase reaction becomes the rate-limiting step of glycolysis; the ATP values of 3.5 μmol/g correlate with the limit of tolerated myocardial ischemia under clinical conditions. The cessation of lactate production is due to lack of ATP for the phosphorylation of fructose-6-phosphate to fructose diphosphate. This happens at a myocardial ATP-content

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.

Testosterone inhibits expression of inducible nitric oxide synthase in murine macrophages.

We investigated the effect of testosterone, the main sexual steroid hormone in men, upon inducible nitric oxide synthesis in murine macrophages. Incubation of murine macrophages (RAW 264.7 cells) stimulated by bacterial lipopolysaccharide (2 microg/ml) with increasing amounts of testosterone (0.1-40 microM) showed a dose dependent inhibition of inducible nitric oxide synthesis. Inducible nitric oxide synthase protein expression was reduced in a dose dependent manner as revealed by immunoblotting when cells were incubated with increasing amounts of testosterone. This was associated with a decline in iNOS mRNA-levels as determined by competitive semiquantitative PCR. As nitric oxide plays an important role in immune defense and atherosclerosis prevention, testosterone-induced iNOS inhibition could lead to an elevated risk of infection as well as to the development of atherosclerotic lesions.

Influence of dipyridamol (Persantin) on myocardial adenosine metabolism

Abstract The degradation of adenine nucleotides to inosine in the ischemic myocardium of the dog is inhibited by about 80% by dipyridamol, which hinders the deamination of adenosine. The K i -values of dipyridamol for the intramyocardial deamination of adenosine in vivo are about 1000 times smaller (corresponding to a 1000 times stronger inhibition) than the inhibitory constants of dipyridamol for the enzymic deamination in vitro . The K i -values of dipyridamol for the penetration of adenosine through the membranes of different blood cells and for the degradation of adenosine in the ischemic myocardium are of the same order of magnitude. From the kinetic studies it appears that dipyridamol inhibits the degradation of adenosine within the myocardial cell by hindering its penetration through intracellular membranes prior to its enzymic deamination. Dipyridamol also increases the resistance of the myocardial cell membrane to the penetration of adenosine out of the cell. Thus it can be concluded that the diffusion of adenosine from the intracellular to the extracellular space cannot be essential for the coronary dilating action of dipyridamol and, therefore, not for the local metabolic regulation of coronary blood flow. These findings suggest the existence of alternate or additional mechanisms to the postulated adenosine mechanism of local metabolic regulation of coronary blood flow.

Stimulation of nitric oxide synthesis by the aqueous extract of Panax ginseng root in RAW 264.7 cells

In this study, we investigated the effect of Panax ginseng root aqueous extracts upon inducible nitric oxide synthesis in RAW 264.7 cells. Panax ginseng root extract has been used in the Asian world for centuries as a traditional herb to enhance physical strength and resistance and is becoming more and more popular in Europe and North America. Incubation of murine macrophages (RAW 264.7 cells) with increasing amounts of aqueous extracts of Panax ginseng (0.05 – 0.8 μg μl−1) showed a dose dependent stimulation of inducible nitric oxide synthesis. Polysaccharides isolated from Panax ginseng showed strong stimulation of inducible nitric oxide synthesis, whereas a triterpene‐enriched fraction from an aqueous extract of Panax ginseng did not show any stimulation. Inducible nitric oxide synthase protein expression was enhanced in a dose dependent manner as revealed by immunoblotting when cells were incubated with increasing amounts of Panax ginseng extract. This was associated with an incline in inducible nitric oxide synthase mRNA‐levels as determined by semiquantitative polymerase chain reaction and electromobility shift assay studies indicated enhanced nuclear factor‐κB DNA binding activity. As nitric oxide plays an important role in immune function, Panax ginseng treatment could modulate several aspects of host defense mechanisms due to stimulation of the inducible nitric oxide synthase.

Enzyme release resulting from total ischemia and reperfusion in the isolated, perfused guinea pig heart

Abstract A study of enzyme release induced by total myocardial ischemia was undertaken using isolated guinea pig hearts perfused at constant flow (4 ml/min). Mechanical activity (perfusion pressure and heart rate) and perfusate enzyme activity (malate dehydrogenase, lactate dehydrogenase and creatine phosphokinase) were examined throughout experiments in which the duration of total ischemia was varied (5, 10 and 20 min). The enzyme releases occurred generally in a biphasic pattern; a marked release just after reperfusion (first phase) and later a moderate release (second phase). The level of enzyme activity returned close to the control level within 7 min. The first phase of release was not accompanied by resumption of contraction. The rate of enzyme release correlated closely with the duration of total ischemia. The present results suggest that enzyme release during/after the tested durations of ischemia may not necessarily reflect serious myocardial cell damage, but rather alterations of the membrane permeability of myocardial cells.

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.