Influence of plating density on individual cell growth, cell division and differentiation of neonatal rat heart primary cultures.

The influence of plating cell density of an originally enriched myocardial cell population has been studied in neonatal rat heart cells in culture. Low density (LDM) is defined as a density (24 h after plating) of 209 +/- 44 cells/mm2 (mean +/- SEM) and is compared with high density (HDM), 419 +/- 67 cells/mm2. Cell growth is evaluated by the total cell number, the percentage of myocardial cells (M) in culture (PAS method) and the protein content per cell. Some differentiation parameters such as beating rates, glycogen concentration, enzymatic activities (cytochrome C oxidase and glycogen phosphorylase) are studied with time in culture (48, 96 and 192 hr). High density was designed to yield a complete confluency of the cells within 24 hr after plating and to minimize cell division of the non-muscle cells (F). At high density, cell division of F cells is effectively limited, thus leading to a more stable model regarding the cell density per plate and the percentage of M cells: 85.7 +/- 4% and 33.4 +/- 6% in LDM cultures compared with 86.5 +/- 4.7% and 51.7 +/- 9.8% in HDM cultures at 24 and 192 hr (mean +/- SEM). Heart cells increase similarly in size with age in culture in both groups. In HDM cultures the spontaneous contractions begin sooner (24 hr) than in LDM cultures and are more rapidly synchronized. The beating rate is higher in HDM cultures between 48 and 96 hr; however, after this time it falls in HDM and does not fall in LDM. Thus the overgrowth of muscle cells by non-muscle cells is not responsible for loss of beating with time in culture but more likely high density could be a limiting factor for isotonic contraction. There is more glycogen per myocyte in LDM than in HDM cultures. The cell density influences the enzymatic activities of cytochrome C oxidase and glycogen phosphorylase. The cytochrome oxidase activity is higher in HDM cultures than in LDM cultures at 96 hr whereas glycogen phosphorylase activity is higher in LDM cultures at time 96 and 192 hr. In LDM cultures, the ratio cytochrome C oxidase/glycogen phosphorylase decreases with time in culture from 1.685 +/- 0.680 at 48 hr to 0.780 +/- 0.290 at 192 hr but not in HDM cultures (2.13 +/- 0.36 and 1.64 +/- 0.34 respectively). Thus plating density influences properties of heart cell cultures with regard to the overgrowth of the F-cell population and the differentiated state of M cells.

Effects of adriamycin on enzymes of adenine nucleotide metabolism in heart cell cultures.

Effects of adriamycin (ADM) treatment on rat heart cells in culture were examined. ADM treatment (lug/ml) for 3 hr, 48 to 51hr of culture age, had no effect on oxygen consumption, myosin content or myosin ATPase activity. In contrast, the activities of creatine kinase, adenylate kinase and adenosine deaminase were significantly lower in ADM-treated cells than in corresponding controls at 72 and 96 hr of culture age. Fall in the activities of these key enzymes of adenine nucleotide metabolism would lead to disturbances in energy metabolism, and results in the functional impairment of heart cells.

Oxygen consumption of mammalian myocardial cells in culture: measurements in beating cells attached to the substrate of the culture dish.

A Lucite attachment which permitted the measurement of oxygen consumption in cells in culture without manipulating the cells was constructed. The attachment fit over commercially available dishes for cell culture and had an oxygen electrode built into it. Oxygen uptake of cells in culture was thus measured. Cells were attached to the substrate of the culture dish during the measurements and could be observed in an inverted phase microscope. Cells did not show any morphological changes, e.g., cell shapes or beating rate in case of myocardial cells, before and after the measurements of oxygen consumption. Using this method the rate of oxygen consumption was determined in rat myocardial and heart non-muscle cells in culture and also in HeLa and L6 cell lines. Myocardial cells in culture had an approximately four times higher rate of oxygen uptake compared with heart non-muscle, HeLa, and L6 cells. The oxygen uptake of beating myocardial cells was higher by about 50% compared with quiescent myocardial cells.

Isozymes of creatine kinase in mammalian cell cultures.

Previous studies on the energy metabolism of rat myocardial cells in culture supported the hypothesis that the creatine-phosphocreatine-creatine kinase system plays an important role in the intracellular transport of energy from the mitochondria to the myofibrils and in the regulation of energy production coupled to energy utilization in this model system. Effective functional compartmentation of ATP could result from the binding of creatine kinase to cellular organelles (e.g., myofibrils and mitochondria) such that high energy charge at the myofibrils is maintained by the reverse creatine kinase reaction, while phosphocreatine is synthesized mainly at the mitochondria in the forward creatine kinase reaction. It was, therefore, essential to demonstrate the presence of mitochondrial creatine kinase in the cultured myocardial cells to support this hypothesis, particularly since the mitochondrial creatine kinase was reportedly absent in fetal hearts. Using electrophoresis on cellulose acetate strips, the mitochondrial creatine kinase isozyme, as well as MM, MB, and BB isozymes, have now been demonstrated in myocardial cultures derived from neonatal rats. The mitochondrial creatine kinase increased with age in culture and with age of animal from which the culture is derived. Furthermore, the addition of creatine to culture media stimulates its synthesis. The mitochondrial creatine kinase isozyme was not detected in nonmuscle cells in culture derived from the neonatal rat hearts, nor in L6 muscle cell line. Phosphocreatine was present in all cells, but the regulation of energy metabolism and energy shuttle by creatine-phosphocreatine-creatine kinase could be operative only in the cells where the mitochondrial creatine kinase is present. This regulatory mechanism provides for an efficient system concomitant with the continuous energy demand of the myocardium; it is not ubiquitous and its development in myocardial cells seems to be triggered postnatally.

Mitochondrial creatine kinase in mammalian myocardial cells in culture.

Previous studies on the energy metabolism of rat myocardial cells in culture supported the hypothesis that the creatine-phosphorylcreatine-creatine kinase system is essential for intracellular transport of energy from the mitochondria to the myofibrils and in the regulation of energy production to meet energy utilization. Effective functional compartmentation of ATP could result from the binding of creatine kinase to cellular organelles (e.g., myofibrils and mitochondria) so that the high-energy charge at the myofibrils is maintained by the reverse creatine kinase reaction, whereas phosphorylcreatine is synthesized mainly at the mitochondria in the forward creatine kinase reaction. It was essential to demonstrate the presence of mitochondrial creatine kinase to support the hypothesis. Using polyacrylamide gel electrophoresis and electrophoresis on cellulose acetate strips, the mitochondrial creatine kinase isozyme, as well as MM, MB, and BB isozymes, has now been demonstrated in myocardial cells in culture. Nonmuscle cells in culture also derived from neonatal rat hearts lack the mitochondrial creatine kinase isozyme. Total creatine kinase in myocardial cells is greatly decreased by treatment of the cells with adriamycin, a cardiotoxic chemotherapeutic agent, and the relative amounts of the isozymes are altered. The mitochondrial creatine kinase seems to be reduced less than either the BB or MM isozymes.

Metabolic involvement in adriamycin cardiotoxicity.

The cardiotoxic effects of adriamycin were studied in mammalian myocardial cells in culture as a model system. Adriamycin inhibited cell growth and the rhythmic contractions characteristic of myocardial cells in culture. A possible involvement of energy metabolism was suggested previously, and in this study the adenylate energy charge and phosphorylcreatine mole fraction were determined in the adriamycin-treated cells. The adenylate energy charge was found to be significantly decreased, while the phophorylcreatine mole fraction was unchanged. Such disparity suggests an inhibition of creatine phosphokinase. The addition of 1 mM adenosine to the myocardial cell cultures markedly increases the ATP concentration through a pathway reportedly leading to a compartmentalized ATP pool. In the adriamycin-treated cells, the addition of adenosine increased the adenylate charge and, concomitant with this inrcease, the cells' functional integrity, in terms of percentage of beating cells and rate of contractions, was maintained.

Replicative and unscheduled DNA synthesis in adriamycin-treated myocardial cells.

The effect of the potent antitumor antibiotic adriamycin (ADM) on chromosome integrity, DNA replication, and unscheduled DNA synthesis was investigated in cultured rat cardiac cells. Chromosome distribution, autoradiography, and [3H]thymidine (dThd) incorporation studies were carried out on separate cultures. A 3-hr pulse of ADM at a concentration of 1 microgram/ml was sufficient to cause chromosomal aberrations that were evident for up to 3 days post-ADM treatment. At the same ADM dosage, DNA replication was depressed for up to 6 days by as much as 90%, yet the ability of the cardiac cells to repair UV-damaged DNA was not impaired. However, cells exposed to higher concentrations of ADM failed to undergo significant UV-induced repair.

Modification by adenosine of the effect of adriamycin on myocardial cells in culture.

The cardiotoxic effects of Adriamycin (ADM) were studied utilizing mammalian myocardial cells in culture as a model system. ADM inhibited cell growth and the rhythmic contractions characteristic of these cells. Because a possible involvement of energy metabolism in the action of ADM was suggested previously, the adenylate energy charge and phosphorylcreatine mol fraction were determined in the ADM-treated cells. The adenylate energy charge was found to be significantly decreased, while the phosphorylcreatine mol fraction was not. Such disparity suggests an inhibition of creatine phosphokinase. The addition of 1 mM adenosine to the myocardial cell cultures markedly increased the adenosine triphosphate concentration but not the adenylate charge. In the ADM-treated cells, the addition of adenosine increased both the adenosine triphosphate concentration and the adenylate charge, and, concomitant with this increase, the functional integrity of the cells in terms of percentage of beating cells and rate of contractions was maintained.

Differential effect of adriamycin on DNA replicative and repair synthesis in cultured neonatal rat cardiac cells.

The effect of the potent antitumor antiobiotic Adriamycin (ADM) on DNA replication and unscheduled DNA synthesis in cultured rat cardiac cells was investigated. Autoradiography and [3H]thymidine incorporation studies were carried out on parallel cultures. DNA replication was depressed for up to 6 days following a 3-hr pulse of ADM administration. An ADM concentration of 1 microgram/ml which was effective in reducing replicative DNA synthesis by as much as 75% did not reduce the ability of cardiac cells to repair UV-damaged DNA. However, cells exposed to higher ADM concentrations failed to undergo significant UV-induced repair. In the absence of UV treatment, ADM did not stimulate unscheduled DNA synthesis. To account for the differential response of the cardiac cell cultures to replicate and repair DNA, we propose that ADM exerts a localized effect on DNA synthesis covering a region proximal to its primary intercalation site.

Cardiotoxic effects of adriamycin in mammalian cardiac cells in culture.

Cardiotoxicity of unknown etiology may preclude the use of adriamycin, a cancer chemotherapeutic agent. Mammalian cardiac cells in culture were used as a model system in the study of the mechanisms involved. Adriamycin inhibited cell growth, particularly of the fast-dividing nonmuscle cells. This inhibition might be a contributory factor to cardiomyopathy, but it does not explain the cessation of the rhythmic contractions characteristic of myocardial cells in culture. The concentrations of ATP and phosphorylcreatine (PC) were decreased in the adriamycin-treated cells, but the addition of creatine resulted in a several-fold increase of PC. Therefore, the regulation of energy production and the potential to maintain a high, steady-state concentration of PC were not impaired.

Studies on the control of energy metabolism in mammalian cardiac muscle cells in culture.

Myocardial cells in a monolayer culture are a myogenic model system wihch shows functional differentiation and in which the intracellular metabolism and energy utilization do not differ markedly from those of the fresh tissue. In the myocardial cells, as in numerous other muscle tissues, the concentration of high energy phosphate compounds, primarily phosphorylcreatine, correlates well with the functional integrity of the cells. The decline of adenosine triphosphate (ATP) below a critical level leads to the cessation of rhythmic contractions of the cells in culture, and, conversely, an increased steady state level of ATP correlates with an increased rate of excitability. When creatine, in the concentration range known to be present in the cardiac tissue, is added to the growth medium of the cultured myocardial cells, the intracellular concentration of phosphorylcreatine increases up to 100%. Since the only metabolic path known for phosphorylcreatine synthesis is via creatine phosphokinase, the rate of transphosphorylation and ATP synthesis must have been increased. This stimulation of energy production is due to the regeneration of mitochondrial ADP brought about by the phosphosphorylation of creatine to phosphorylcreatine and possibly, also, to an enhanced rate of glycolysis. No net transfer of approximately P from ATP to creatine was observed under any of the experimental conditions. The high steady state level of phosphorylcreatine was not maintained upon the addition of either oligomycin or 2-deoxyglucose, and the addition of both metabolic inhibitors simultaneously resulted in a depletion of phosphorylcreatine and ATP and in cessation of rhythmic contractions. The excitability was also inhibited upon the addition of 1-fluoro-2,4-dinitrobenzene, a creatine phosphokinase inhibitor, and the accompanying depletion of approximately P was primarily reflected in a decrease of ATP concentration. These findings support the following conclusions: (1) phosphorylcreatine serves as a source of approximately P for the resynthesis of ATP at the site of utilization (i.e., myofibrils, membrane) and thus maintains the optimal energy charge; (2) production site; (3) the regeneration of phosphorylcreatine is coupled to oxidative phosphorylation and possibly to glycolysis. Creatine-phosphorylcreatine system operates as a undirectional shuttle for approximately P and as a control system regulating energy production according to demand. Reports of studies on intact mitochondria and on isoenzymes of creatine phosphokinase give further support to the above conditions.