Cultured Rat Heart Cells Research Articles

Electrical properties of cultured heart cell reaggregates from newborn rat ventricles: Comparison with intact non-cultured ventricles

The electrophysiological properties of cultured rat heart cells were investigated and compared with those of cells in intact hearts of the same age rats. Reaggregates (100 to 200 μm diameter) from newborn rat hearts (ventricles) were prepared from collagenase-hyaluronidase-dissociated cells and cultured for periods up to 7 days. Intracellular micro-electrode recordings were made using standard techniques. Ventricular reaggregates cultured under standard conditions had a low resting potential (−67 ± 1.5 mV) and the action potentials had a slow rate of rise (18 ± 1.4 V/s). Tetrodotoxin (5 × 10 −6 m) decreased only slightly the rate of rise and verapamil (10 −6 m) inhibited completely the action potentials. In contrast the ventricular action potentials recorded from isolated ventricles were characterized by a high resting potential (−77 ± 0.7 mV) and a fast rate of rise (80 to 193 V/s). Tetrodotoxin (5 × 10 −6 m) decreased the rate of rise and verapamil (10 −6 m) only depressed the plateau level of the action potentials. In cultured reaggregates hyperpolarizing current pulses increased the rate of rise. This effect was prevented by tetrodotoxin. Reducing the amount of fibroblasts in the reaggregates by using the differential attachment technique, caused the AP parameters to become similar to those found in non-cultured cells. We conclude that, under certain conditions (e.g. removal of fibroblasts) fully differentiated properties can be maintained in reaggregate cell cultures. Thus, as in the case of cultured chick heart cells, cultured rat heart cells can be maintained in vitro in a highly differentiated state.

The regulation of protein turnover in newborn rat heart cell cultures.

The regulation of myocardial protein turnover was studied using newborn rat heart cells in monolayer cultures. Protein accumulation in this model system was regulated by the presence of serum in the culture medium. The influences of serum on the rates of protein synthesis and degradation were investigated. Serum deprivation or arginine deprivation did not modify protein synthesis. In contrast serum strongly inhibited the rates of [3H]leucine release from prelabeled long-lived myocardial proteins. The inhibiting serum factor was found heat-stable, displayed slight species specificity, was of plasmatic origin, and its activity was decreased in the plasma of hypophysectomized rats. Platelet growth factor, which promotes DNA synthesis in heart cell cultures, did not inhibit protein degradation. The influence of known regulators of myocardial protein balance was investigated. Leucine had no influence on protein synthesis and degradation. Supraphysiological concentrations of insulin and growth hormone inhibited protein degradation but only in the absence of serum. It is suggested that these compounds may not be the natural regulators of myocardial protein balance.

The use of 5-bromodeoxyuridine and irradiation for the estimation of the myoblast and myocyte content of primary rat heart cell cultures.

A method for killing dividing cells (Puck and Kao, '67) was adapted for the elimination of dividing heart muscle cells (myoblasts) in cultures. We have used this method to demonstrate their presence and to estimate their number as well as the number of nondividing heart muscle cells (myocytes) in the neo-natal rat heart. Cells were cultivated in BUdR (5-bromodeoxyuridine) 10(-4) M for 3 days and then irradiated with long UV light. The selective elimination of dividing cells led to a loss of myosin Ca2+-activated ATPase in the cultures. This indicates the presence of dividing cells which contain myosin. The percent of ATPase left after irradiation was 32% of the control in cultures derived from 1-day postnatal rats and 48% in cultures from 4-day postnatal rats. This reflects an in vivo shift of myoblasts to myocytes in the muscle cell population as the rat ages.

Interaction between parathyroid hormone and the β-adrenoceptor system in cultured rat myocardial cells

The effect of parathyroid hormone (PTH) alone and in combination with dl-sotalol, dl-propranolol, isoprenaline and calcium chloride has been investigated on cultured rat heart cells beating. PTH induced a dose-dependent positive chronotropic effect which was antagonized by sotalol non-competitively. PTH decreased the positive chronotropic effect of isoprenaline by a non-competitive antagonism. PTH reduced the negative chronotropic effect of propranolol by a competitive antagonism and the positive chronotropic effect of calcium chloride by a non-competitive antagonism. Such interactions suggest that PTH may act in vivo not entirely by stimulating a proper receptor.

The effects of incubation conditions on enzyme release from anoxic rat heart cell cultures.

The effects of incubation conditions on enzyme release from anoxic rat heart cell cultures.

The effect of extracellular calcium concentration and Ca-antagonist drugs on enzyme release and lactate production by anoxic heart cell cultures

Tissue cultures of neonate rat heart cells were subjected to anoxia for 16 h in the presence of 5 m m glucose and increasing concentrations of calcium in the incubation medium. When the calcium content of the culture medium was increased, release of intracellular enzyme was observed. Increased extracellular calcium concentration appeared to have no effect on normoxic tissue preparations. The time course of this anoxia-related change was examined. Increasing the calcium concentration of the medium caused an acceleration of the anoxia-induced rate of enzyme leakage when compared with anoxic cultures exposed to a low medium calicum concentration. The calcium-induced increase in anoxic enzyme release could be partially reversed by various concentrations of the calcium antagonist drugs verapamil and nifedipine. Verapamil and nifedipine both appear to reduce the extent to which an elevated medium calcium concentration is able to reduce myocyte ATP content under anoxic conditions. These drugs appear to be without effect on ATP content of anoxic cells exposed to a low calcium concentration on the medium. Calcium-induced increases in enzyme leakage cannot be modified by the provision of high medium glucose concentrations, and hypertonic solutions of mannitol exacerbate damage to myocytes under anoxic and hypercalcaemic conditions. These data suggest that high extracellular concentrations of calcium exacerbate damage to heart cells subjected to anoxia although the mechanism underlying this exacerbation is uncertain.

Flow of "injury" current and patterns of excitation during early ventricular arrhythmias in acute regional myocardial ischemia in isolated porcine and canine hearts. Evidence for two different arrhythmogenic mechanisms.

SUMMARY We recorded 60 DC-extracellular electrograms simultaneously from epicardial and intramural sites of the left ventricle of isolated perfused porcine and canine hearts during the first 15 minutes after occlusion of the left anterior descending coronary artery. During coronary occlusion, maximal current flow across the ischemic border occurred when normal cells had repolarized and ischemic cells had not. At that moment, maximal current sources at the normal side of the ischemic border were in the order of 2 /iA/mm 3 and maximal current sinks were — 5 /tiA/mm 3 . During propagation of a broad wavefront in nonischemic myocardium, current sources in the wake of the wavefront were about twice as large. Ventricular premature beats usually followed deep negative T waves in ischemic myocardium, when "injury" currents were maximal. Earliest activity always occurred at the normal side of the ischemic border, and whenever Purkinje activity was recorded it preceded myocardial activity in both single premature beats and the initial beats of ventricular tachycardia (VT) or ventricular fibrillation (VF). For later beats of VT, circus movements with a diameter of 1-2 cm were responsible for continuation of the arrhythmia. Dimension and position of the reentrant circuit changed from beat to beat. In VF, fragmentation of wavefronts occurred, and multiple wavelets followed tortuous paths. Circus movements were seldom completed; when they were, their diameter was 0.5 cm. It is concluded that two mechanisms are responsible for the very early ischemic arrhythmias: one, a "focal" mechanism located at the normal side of the ischemic border, possibly induced by injury currents in normal Purkinje fibers and, two, macro- and micro-reentry in ischemic myocardium. Circ Res 47:151-165, 1980

Effects of adrenaline-tablets on rat heart cells in tissue culture at normal and raised temperature

Primary cultures of rat heart cells were incubated at 37 degrees C or 39.5 degrees C. For permanent treatment with adrenaline recently developed tablets were added to the cultures. After 10 hours the cell counts, glucose, lactate, LDH, alpha-HBDH and GOT were determined. Permanent adrenaline-application led to a decrease of cell counts, an increase of lactate, LDH, alpha-HBDH and GOT. The results indicate an injured membrane function of rat heart cells. Raised temperature sensitized the cells for adrenaline-treatment.

Chronotropic effect of histamine on cultured neonatal rat heart cells

The positive chronotropic effect (PCE) of histamine in cultured neonatal rat heart cells was monitored using a microscopic method as well as an electro-optically recording device. The action potential frequency was also measured (by means of microelectrodes). An increase in PCE was noted when histamine (from 1 × 10 −6 M to 1 × 10 −5 M) was added to the cells. However, higher concentrations (from 1 × 10 −5 M to 1 × 10 −4 M) were less effective. The PCE of histamine was reduced by pretreating the cells with antihistaminic drugs. H 1-blocking agents (promethazine and mepyramine) were more potent than H 2-blocking drugs (metiamide and cimetidine). In addition, the PCE of histamine was abolished when the cells were in presence of high K + medium (26 mEq) but contraction and action potential amplitudes were increased. Our results demonstrate that these cultures respond to histamine and that this response is abolished by antihistaminic drugs thus suggesting that H 1 and/or H 2 receptors may be present in the neonatal rat heart cell cultures.

Release of alpha hydroxybutyrate from neonatal rat heart cell cultures exposed to anoxia and reoxygenation: comparison with impairment of structure and function of damaged cardiac cells.

Spontaneously beating monolayer cultures of neonatal rat heart cells first exposed to depletion of oxygen and metabolic substrates for 1 to 7 h (anoxia). Subsequently the cultures were resupplied with oxygen and substrates (reoxygenation). The release of alpha-hydroxybutyrate dehydrogenase (HBDH) from the cells, the extent of necrosis, and the changes in spontaneous contractile activity were measured. HBDH release was observed to start after 1 h of anoxia and to increase to 84% of intracellular HBDH activity after 7 h of anoxia. Reoxygenation of anoxic heart cells is associated with accelerated HBDH release (oxygen paradox). The activity of HBDH released by cardiac cells, the number of necrotic cells and the impairment of beating capacity of the cells depend on the duration of the anoxic period in a similar way. This study demonstrates that cardiac cell death can be assessed quantitatively and reliably by the measurement of the activity of HBDH released by the cells.

Lipoprotein lipase of cultured mesenchymal rat heart cells: IV. Modulation of enzyme activity by VLDL added to the culture medium

Lipoprotein lipase activity was studied in rat heart cell cultures grown in the presence of 20% fetal calf and horse serum and a medium concentration of triacylglycerol of 0.03 mg/ml. After 6–8 days, when the enzyme activity had reached high levels, the cells were incubated for 24 h in a medium containing 20% serum derived from fasted or fed rats. No change in enzyme activity occurred in the presence of fasted rat serum, but a 50% fall was observed with fed rat serum. When the complete culture medium was supplemented with rat plasma VLDL (0.075–0.75 mg triacylglycerol) a pronounced decrease in lipoprotein lipase activity occurred after 3–5 h of incubation. Similar extent of enzyme fall was observed also in the presence of triacylglycerol-rich lipoproteins isolated from rat plasma after feeding of safflower oil or lard, even though the fatty acid composition of the triacylglycerol varied markedly. As the addition of VLDL to the culture medium resulted in a lesser fall of heparin releasable than residual activity it seems that there was no direct inhibition of surface bound enzyme activity and that the transport of the enzyme to the cell surface was not affected. These data indicate that addition of VLDL to the culture medium resulted in a fall in enzyme synthesis, while total protein synthesis as determined by incorporation of [ 3H]leucine, remained unchanged. This inhibition could be reproduced by increasing free fatty acid concentration of the-medium, however addition of excess albumin to VLDL containing medium did not prevent the fall in enzyme activity. The present results obtained with cultured rat hearts cells suggest that in vivo plasma levels of triacylglycerol-rich lipoproteins could modulate the lipoprotein lipase activity of the heart.

The activity of cardio-specific isoenzymes of creatine phosphokinase and lactate dehydrogenase in monolayer cultures of neonatal rat heart cells

Monolayer cultures of neonatal rat heart cells, prepared according to Harary and Farley [ 12], contain, besides muscle cells (myoblasts, myocytes), a number of non-muscle cells (fibroblasts, endothelial cells) proliferating at a faster rate than the muscle cells. In ageing cultures of this type changes in enzyme and isoenzyme activities have been reported. These activity shifts have been interpreted either as an adaptation to hypoxic conditions, or as inherent to ageing of muscle cells. In this study we determined the changes in time of the activity of creatine phosphokinase including its isoenymes MM, MB and BB, and the activity of lactate dehydrogenase (LDH) including the isoenzyme combinations LDH 1+2 and LDH 3+4+5. It is shown that for creatine phosphokinase and its isoenzymes as well as for lactate dehydrogenase and its isoenzymes the activity changes in time are compatible with the hypothesis, that the isoenzyme shifts are the result of a change in the proportion of muscle cells in the culture, rather than an adaptation to hypoxia or ageing of the muscle cells. It is also demonstrated that inhibition of cell proliferation, by using a nutrient medium without serum, prevents the occurrence of these activity shifts.

Inhibition of cell-substratum attachment of cultured rat heart cells by protein synthesis inhibitors

Addition of cycloheximide to growth medium of neonatal rat heart cell cultures prevented cell-substratum attachment. Even concentrations of cycloheximide which inhibited only 50% of normal protein synthesis prevented some cells from attaching. Cells which required the longest time to attach were most dependent on protein synthesis. The kinetics of cell-substratum adhesion in the presence of various concentrations of cycloheximide supported the hypothesis that repair of damaged cell membranes was required prior to attachment. An alternate hypothesis that protein synthesis was required for substratum attachment either to synthesize new unique proteins or higher concentrations of existing proteins not damaged by enzymes was not supported by experimentally obtained data. If the second hypothesis were true, no cells would have attached when protein synthesis was completely inhibited (greater than 95%) and all cells should have been equally affected by protein synthesis inhibition; such was not the case. Inhibition of mRNA formation by actinomycin D also should have inhibited attachement completely and this was not observed. Since attachment was minimally affected by actinomycin D, protein synthesis on long-lived mRNA was apparently sufficient for cell-substratum adhesion.

Cation exchange and glycoside binding in cultured rat heart cells

The Na/K-exchange characteristics, ouabain-binding kinetics, and Na pump turnover rates of synchronously contracting monolayers of neonatal rat myocardial cells were studied. The cells exchange Na rapidly (T1/2 = 35 s) with a mean Na flux of approximately 25 (pmol/cm2)/s. The half time (T1/2) of K exchange is much longer (12 min); the mean K flux is 13 (pmol/cm2)/s. Active Na/K transport, as measured by K influx, is relatively ouabain sensitive, and 10(-6) M ouabain produces half-maximal inhibition. Ouabain (10(-2)M) inhibits 60% of the Na efflux and 75% of the K influx. The cells bind [3H]ouabain rapidly (T1/2 = 8 min), but release it very slowly (T1/2 = 11 h), and both the amount bound and the rate of binding were inversely proportional to extracellular K. Specific [3H]ouabain binding demonstrates saturation reaching a maximum of 1.6 x 10(6) molecules per cell at 2 x 10(-7) M [3H]ouabain. From cell surface area and ouabain-sensitive flux measurements, the Na pump density was calculated at 720/micrometer2 with an individual pump turnover rate of 50/s. Thus the studies indicate that despite their neonatal origin, the behavior of the Na pump in these cells is very similar to that in other mammalian tissues.

Release of hypoxanthine from and enzyme depletion in rat heart cell cultures deprived of oxygen and metabolic substrates

In monolayer cultures of neonatal rat heart cells deprived of oxygen and metabolic substrates we measured (1) the release of hypoxanthine, a product of ATP catabolism, and (2) enzyme depletion with creatine phosphokinase (CPK) and α-hydroxybutyrate dehydrogenase (α-HBDH). Taking samples each hour up to 7 h of anoxia we were able to demonstrate that hypoxanthine release precedes cellular enzyme depletion. The release rate of hypoxanthine is maximal in the second hour of anoxia and the depletion rate of CPK and α-HBDH is maximal in the fourth hour. These results suggest that for early diagnosis of damage to heart cells due to deprivation of oxygen and metabolic substrates the measurement of hypoxanthine release is preferred to that of enzyme release.

Temperature-control system for biological preparations

A temperature-control system was developed in order to maintain cultured rat heart cells at a constant temperature within the range of 20–40 deg C during microscopy. A linearised thermistor bridge was used in conjunction with a 3-term controller and negative feedback to achieve a temperature stability of ±0·05 deg C. Cooling fluid passing through the heating unit improved transient response and acted as an antagonist to the heating coil. The interaction between the heating and cooling elements helped to maintain better temperature regulation and allowed for operation below room temperature.

Uptake and utilization of 1-14C palmitic acid by heart cells treated with fresh or thermally oxidized fats.

The effects of fractions isolated from thermally oxidized corn oil or olive oil on the metabolic activity of heart endothelial and muscle cells were studied. Rat heart cells in culture, exposed to thermally oxidized fat components, took up more exogenous 1-14C-palmitic acid and incorporated more of it into the cell triacylglycerol fraction than when the cells were treated with fresh fats. Particularly with the heated corn oil compared to fresh corn oil, much less of the radioactivity from the labeled palmitic acid was deposited in the phospholipid fraction. Also, with heated corn oil when the incubation period was extended beyond 12 hr, there was a decline in the radioactivity retained in the triacylglycerol fraction of the heart muscle cells. When the fresh fats were compared for 14C-radioactivity incorporation into the heart cells, the olive oil gave much higher values, indicating a distinct difference in response to the proportion of fatty acids supplied.

Effect of dibutyryl cyclic AMP and analogs on the rate of contractions of myocytes in culture

2O, 6N-butyryl, 3', 5'-cyclic monophosphate (dibu cAMP) when added to fetal rat heart cells in culture inhibits myocyte contraction. This inhibition is 100, 84 and 51% complete when the dibu cAMP concentration used is 2, 0.2 and 0.02 mM, respectively. The potency of dibu cAMP derivatives in myocyte contraction inhibition follows the order, dibu cAMP greater than 6N-bu cAMP greater than 2O-bu cAMP = AMP greater than butyrate. The inhibition caused by the first three chemicals is greater than 70%.

A quantitative method for measurement of lysosomal acid phosphatase latency in cultured rat heart cells with 210Pb.

A method is described for measuring the latency of lysosomal acid phosphatase in cultured rat heart endotheloid cells. 210Pb was added to a medium used to demonstrate acid phosphatase activity by the Gomori lead method, and the amount of lead deposited was measured with a liquid scintillation counter. Deposition rates were measured after enzyme activation pretreatments with acetate buffer (pH 5.0) at various osmolalities, and after formaldehyde fixation. Formaldehyde, alloxan, or fluoride in the Gomori medium were evaluated for their differential effects on lysosomal and non-lysosomal acid phosphatase. The method was found to provide a sensitive, rapid and quantitative evaluation of acid phosphatase latency and should be useful for studying the integrity of lysosomes within cells.

Progesterone metabolism by new born rat heart cells in culture

Progesterone metabolism by new born rat heart cells in culture