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Excessive right heart hypertrophy was investigated under additional acute hypoxic stress to find out a possible contribution of mitochondrial dysfunction to sudden heart failure. Severe right heart hypertrophy in rats was induced by exposure to hypobaric pressure (46,663 Pa) for 4 weeks. Heart rate, isovolumic pressure and coronary flow were determined in the Langendorff mode of perfusion. After normoxia, the hearts were subdued to acute hypoxia/reoxygenation. Mitochondrial membrane potential was measured at the heart surface by fluorometry using 2-(dimethylaminostyryl)-l-ethylpyridinium iodide (DASPEI). At the end of each experiment mitochondria were isolated and ATP synthesis, ATPase, as well as creatine kinase activity were determined. Compared to normal hearts the heart rate is decreased in the hypertrophied group whereas right ventricular systolic and (end)diastolic pressure (adjusted to isovolumetric maxima) are increased. Coronary flow is decreased. Cytosolic creatine phosphate ATP levels and ATP/ADP ratios are significantly (p < 0.01) decreased. Furthermore, ATP synthesis and creatine kinase activities are diminished. At high ADP, respiration is loosely coupled or partially uncoupled. Acute hypoxia is particularly deleterious to hypertrophied hearts: Mitochondrial membrane potential as measured by heart surface fluorometry decreases extensively and is only very incompletely restored during reoxygenation. Rate-pressure product decreases precipitously and is restored during reoxygenation only to a very low extent. The results indicate an insufficient energy metabolism of mitochondria during acute hypoxia/reoxygenation which adds to the earlier described shifted isozyme pattern of myosin and decreased activities of myosin and sarcoreticular Ca2+ ATPase, leading to myocardial failure in right heart hypertrophy.

Metabolism in cultured cells strongly depends on cell density, thus cell density may be an important factor in biotechnological maintenance of cell cultures. Therefore the energy metabolism of three related cell types with different proliferation activity has been characterised: density controlled primary or secondary fibroblasts [pmf], immortalized, but still density controlled 3T3 cells, and SV40-transformed 3T3 lacking growth control. The investigations revealed the decrease of oxygen consumption, net lactate production, ATP-content, NAD-content and NAD-redox potential, and F-actin content with increasing culture density in pmfs, less distinct in 3T3 cells and in SV40-3T3 cells. The main decrease of these factors is related to cell-cell contacts rather than to proliferation which ceases at least two division cycles after most cells contacted a neighbouring cell. SV40-3T3 cells also at preconfluent densities exhibit relatively low metabolic activity as revealed by the above mentioned factors. Supply with metabolites for catalytic processes seems to be the rate limiting factor as deduced from a decreasing NADH/total NAD ratio. SV40-3T3 cells lack a contact mediated reduction of energy metabolism which is in accord with the missing contact inhibition of motility and proliferation in SV40-3T3 cells. Because of a possible association of glycolytic enzymes with actin, F-actin content has been determined. No correlation, however, was revealed by the F-actin-/lactate ratio. This may be due to the fact that about 50% of the lactate released into the culture medium originated to from glutaminolysis rather than glycolysis. Only in SV40-3T3 cells were respiration and lactate production insensitive to glutamine deprivation and in these cells both these parameters did not change significantly, therefore they did not allow testing as to whether f-actin content and glycolytic activity are correlated. Fractionated release of adeninnucleotides revealed that energy charge in the cytosolic fraction is the only factor which strictly correlates with the cessation of proliferation. Thus energy charge of the cytosol seems to be the only factor of these studied here, which can be used as an indicator of a culture's commitment for further proliferation. To compare energy metabolic parameters among different cell types one always has to take into account the density dependence of these factors.

In the present study we investigated the direct effects of potential uremic toxins on the energy metabolism of cultured cardiac myocytes. High-energy phosphates were extracted with perchloric acid and determined by high performance liquid chromatography. Energy charge (calculated from the ratio of [ATP], [ADP] and [AMP] was significantly reduced by 20 mM urea and the combination of creatinine (5 mM) plus urea (200 mM). On the other hand, perfusion with culture media containing clinically relevant amounts of urea (20 mM) or creatinine (1 mM) increased the PCr/ATP ratio. This effect was more pronounced after application of an artificial uremic medium (consisting of uremic serum, urea, creatinine and cytokines) or high amounts of creatinine (5 mM) plus urea (200 mM). As contractility of myocytes is reduced due to application of uremic compounds or uremic serum, we attribute changes in contraction frequency or inotropy to dysregulation of calcium availability within the cell. In fact, the cardiodepressive action of uremic serum (2.5%) could be completely reversed by the calcium agonist, Bay K 8644, thus indicating disturbances in myocardial calcium homeostasis in uremia. Altered calcium regulation by uremic toxins might therefore be responsible for the observed changes in myocardial energy metabolism. These results might contribute to the understanding of the pathogenesis of cardiac damage in end-stage renal disease.

The effects of 0.1 nmol/l ouabain (CAS 630-60-4) and digitoxin (CAS 71-63-6) on oxygen consumption rate (OCR) and the contractility of cultured chick embryo cardiomyocytes were investigated using a perfusion system which allowed microscopic observation during the experiment. contractility was assessed by a novel image analysis procedure. During substrate free perfusion the OCR was increased on application of 0.1 nmol/l ouabain or digitoxin for 60 min, whilst contractility was not affected. Digitoxin (0.1 nmol/l) elicited no change in the OCR in the presence of glucose, pyruvic acid, lactic acid and caprylic acid. Ouabain (0.1 nmol/l) did not affect the OCR or contractility in the presence of glucose, pyruvic acid and lactic acid, however together with caprylic acid the OCR was increased significantly. These results demonstrate a hitherto unknown stimulation of fatty acid utilization by low concentrations of ouabain.

The spreading of trypsinized XTH-2 cells (a line derived from Xenopus laevis tadpole heart endothelia) on glass was investigated. Three phases can be distinguished: (1) blebbing of rounded cells, first attachment to a solid substratum and formation of a broad smooth contact area; (2) organization of a peripheral zone of actin fibrils and reinforcement of the basal cytoplasm by a stress fibre-like pattern; (3) extension of lamellae. The first phase seems to be independent of a supply of metabolic energy, while the others clearly depend on it. This is concluded from the close relationship between cellular projection area and energization of mitochondria as revealed by (a) the fluorescence intensity of cells vitally stained with the mitochondria-specific fluorochrome DASPMI (2-4-(dimethylamino)-styryl-1-methylpyridinium-iodine); (b) the degree of spreading in the presence of inhibitors of respiration; (c) effective amelioration of spreading (phases (2) and (3] under conditions of high ATP content. In phase (2) the extension of the central part of the cells becomes stabilized, the cell body settles on the basal cytoplasmic layer and further expansion of the projection area is achieved by lamella formation (phase (3]; motile and stabile regions of the cells become separated. This sequence of events is interpreted as a self-organizing process based on the development of internal hydraulic pressure, actin polymerization and contraction of the newly developed actomyosin network. During trypsinization, depolymerization of actin does not occur but rather on addition of Ca2(+)-containing media. Cellular ATP content drops as well on trypsinization, as on addition of Ca2+. Manganese promotes spreading by decreasing F-actin disassembly and maintaining a high level of cytosolic ATP, most probably because it is not accepted by the calcium pumps. Regarding the association of glycolytic enzymes with F-actin and their influence on actin assembly, lactate dehydrogenase has been inhibited with oxamic acid. This treatment improves the correlation between F-actin content and the degree of spreading; however, the total amount of F-actin remains smaller and the cells spread more.

The physiological significance of the association of glycolytic enzymes with actin fibrils was investigated in cell culture. Cytochalasin D (CD) was used to induce the known actin-based sequence of events in a culture of an endothelial-cell line (XTH-2) derived from hearts from tadpoles of Xenopus laevis. 1 min following addition of CD, ruptures in the cortical fibrillar meshwork and in stress fibres are seen. At the same time the cellular ATP level decreases by ca. 25%. This and the following reactions resulting in a kind of arborization depend on a continuous supply with metabolic energy. As shown by measurements of oxygen consumption, cells with intact energy metabolism provide the ATP needed from glycolysis; ATP produced by oxidative phosphorylation is not utilized as long as lactate dehydrogenase (LDH) reoxidizes NADH2. After inhibition of LDH, respiration in XTH-2 cells doubles. CD treatment induces a transient increase in oxygen consumption, indicating an increased energy supply by respiration. From these results we conclude: The energy needed by the actomyosin system is - under normal metabolic conditions -supplied from ATP phosphorylated in glycolysis. The processes of energy metabolism seem to be highly compartmentalized; ATP is not a parameter that is kept constant in time intervals of minutes up to one hour.