R. Spahr

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.

Substrate oxidation by adult cardiomyocytes in long-term primary culture.

In medium 199 plus 20% fetal calf serum adult rat cardiomyocytes establish a long-term culture (25 days). During the first 10 days they change their gross morphology from the typical elongated in vivo shape (day 1), to a smooth spherical intermediate form (days 2 to 5), to a spread cell type beating spontaneously (days 10 to 15). During the first 10 days in culture, protein content per cell increases and the cell population decreases. By the tenth day, protein content has doubled, and about half of the cells originally plated remain. Thereafter both the protein content and the number of cells are essentially constant for the remainder of the 25-day period investigated. On days 1, 15 and 25 adenine nucleotide contents (213, 216 and 225 nmol/10(6) cells) and values of adenylate energy charge (0.91, 0.87 and 0.88) were similar. At all times in culture, palmitate (0.1 mM) is oxidized at higher rates than lactate (1 mM) and glucose (5 mM). At all times in culture glycolytic flux is sensitive to insulin with half maximal effect seen around 10(-9) M. Oxidation rates for all exogenous substrates are maximal at 15 days in culture, indicating maximal energy demand at this time. The conversion of glucose to lactate, however, progressively increases, so that at 25 days in culture, 70% of ATP derived from degradation of exogenous glucose is glycolytic. The results of this study demonstrate that oxidative metabolism of cardiomyocytes in long-term culture resembles, in its basic characteristics, that of the intact heart. In their increased glycolytic activity, however, they are clearly different.

Glycine fermentation via a glycine reductase in peptococcus glycinophilus and peptococcus magnus

Peptococcus glycinophilus and P. magnus (P. variabilis) utilized only glycine-containing compounds for growth and required selenium compounds for the fermentation of glycine in an optimized medium. Under these conditions an active glycine reductase was expressed in vivo as demonstrated for both species by tracer experiments and additionally in vitro for P. glycinophilus in cell extracts. It is concluded that the availability of selenium determines the fermentation pathway of glycine. In the presence of selenite glycine is converted via the glycine reductase rather than the hitherto known glycine-serine-pyruvate interconversion. The molar growth yield of P. magnus was determined to be 8.9 g of dry weight per mol of glycine. The significance of the low vitamin B12 content and a low carbon monoxide dehydrogenase activity in P. glycinophilus for the reduction of CO2 to acetate is discussed.

Substrate utilization of adult cardiac myocytes

Cultured adult myocytes are in a state of basal metabolism. When glucose is the only exogenous substrate, they produce lactate over CO2 at a constant rate of 2.7 from this substrate. Increase of oxygen tension does not change this behaviour. Insulin preferentially increases lactate formation, dichloroacetate only CO2 production. The fact that the lactate/CO2 ratio can be varied from 0.5 to 16 indicates that there is no close coupling between glycolytic flux and pyruvate oxidation. Both exogenous lactate and fatty acids are used preferentially over glucose. But increase of fatty acid oxidation and inhibition of glucose oxidation are not complementary. Glycolytic flux is only slightly decreased when fatty acid oxidation is already saturated. The results indicate that fatty acids interact with glucose oxidation primarily by inactivation of the pyruvate dehydrogenase. Neither insulin nor dichloroacetate in the presence of glucose inhibit fatty acid oxidation.

Morphological dedifferentiation of adult cardiac myocytes in coculture with hepatocytes

When adult heart cells are plated on a dish covered with a monolayer of hepatocytes gradual morphological changes are observed. While during the first day the myofibrils are still organized in rod-like shape, later the cells become flat and spread on top of underlying hepatocytes. After two days most cells have a flat, polygonal appearance with spread myofibrillar bundles. At this stage they start spontaneous rhythmic contractions which are characteristic for embryonic myocytes, but not for isolated adult ventricular cells. In this culture myocytes form specific contact structures to adjacent myocytes as well as to hepatocytes. These results demonstrate the phenotypical plasticity of adult heart muscle cells which are believed to be terminally differentiated.

Importance of endogenous substrates for cultured adult rat cardiac myocytes

In Ca-tolerant adult cardiomyocytes the contribution of endogenous substrates (glycogen, tri- and diacylglycerol) to oxidative substrate metabolism was investigated. After 4 h in culture medium (M 199 plus 4% fetal calf serum) the cellular triacylglycerol content is 3.6-fold higher than in fresh myocardium and reflects the free fatty acid composition of the medium. When triacylglycerol is degraded, all long-chain fatty acids are hydrolysed at equal rates. In these quiescent cells, the activity of pyruvate dehydrogenase is low (10% of full activity, in Tyrode solution with 5 mM glucose). Up to 30% of full pyruvate dehydrogenase activity, the contribution of non-lipid substrates (glycogen, glucose, lactate and pyruvate) to oxidative energy production is correlated to pyruvate dehydrogenase activity. At 5 mM medium concentration, glucose, lactate and pyruvate share in energy production the proportions of 15, 36 and 50%, whereas endogenous lipolysis accounts for 78, 61 and 46%. It is concluded that these quiescent cardiomyocytes represent cardiac metabolism in a basal state in which the preference for fatty acids, especially from endogenous lipids, is very pronounced. The utilization of endogenous substrates therefore has to be considered in all studies investigating the oxidative metabolism of these isolated cells.

Enzyme release and glycolytic energy production

In substrate-free anoxia, activities of released cytosolic enzymes (LDH, MDH) correlate inversely with the actual ATP level (for both: r = -0.98). At the same time there is a close correlation between lactate production from glycogen and the ATP content (r = 0.98). With external glucose present enzyme release is greatly delayed, but this could be due to the stimulation of glycolysis as well as to the maintenance of high ATP levels. When glycolysis is blocked by iodoacetate under aerobic conditions, the cells also become depleted of high-energy phosphates. This depletion is delayed in the presence of pyruvate. Cytosolic enzyme release again is correlated with total ATP contents, by the same relation in the presence or absence of pyruvate. Glycolytic energy production is negligible in both cases and does not seem to determine enzyme release directly.