Hearts isolated from 11-and 12-day rat embryos were incubated in a simple salt solution to which was added the metabolic inhibitors iodoacetate, malonate, 2,4-dinitrophenol or trypan blue. Comparisons were made between the two age-groups and it was observed that both 11-and 12-day hearts exhibited depressed contractile activity with the glycolytic inhibitor iodoacetate. Malonate did not depress contraction rate in the younger hearts but significantly depressed the rate in the older hearts. A greater inhibitory action on the older hearts as compared with the younger hearts was also produced by 2,4-dinitrophenol. These results were interpreted as further evidence of a shift in dependence on pathways other than glycolysis between days 11 and 12 of development.
The teratogenic agent trypan blue was shown to depress contraction rate when added to the incubation medium. This effect could be reversed by adding glycolytic intermediates such as fructose-l,6-diphosphate or alpha glycerophosphate. These results were suggestive of a direct inhibitory effect of trypan blue either on glucose uptake or its complete catabolism.
The inclusion of metabolic inhibitors in culture media has been one of the techniques employed in the study of metabolic pathways utilized during embryonic development. Spratt (1950) examined the effects of inhibitors acting on the Embden-Meyerhof pathway, the tricarboxylic acid cycle (TCA) and the terminal respiratory chain as part of his investigations of the development of cultured chick embryos. Thompson (1967) employed metabolic inhibitors to block the same pathways in her studies of energy metabolism in preimplantation mouse embryos and Harary & Slater (1965) reported the effects produced by uncouplers of oxidative phosphorylation on the contractile activity of single beating rat heart cells in culture.
Results from investigations of substrate utilization (Cox & Gunberg, 1972) indicated that in isolated embryonic rat heart preparations an increase in the number of metabolites capable of maintaining contractile activity occurred between the 11th and 12th days of development. The series of experiments reported here tested the effects on in vitro contractile activity of embryonic rat hearts induced by inhibitors of various pathways involved in metabolic energy production. These experiments were undertaken to further elucidate the apparent shift in energy producing pathways utilized for maintenance of contraction rates during the early development of the embryonic rat heart.
MATERIALS AND METHODS
The breeding technique, preparation of isolated hearts, incubation apparatus and the general procedures employed were the same as those reported previously (Cox & Gunberg, 1972). The basic incubation media consisted of a Krebs-Ringer bicarbonate solution. Inhibitors and substrates, when indicated below, were added to the incubation medium in isosmotic concentrations. Gas mixtures for oxygenated conditions included 65% O2, 35% N2, 5% CO2 and for anaerobic conditions included 95% N2 and 5% CO2.
Several known metabolic inhibitors were tested for an effect on the contraction rate of isolated embryonic rat hearts. The inhibitors tested and the concentrations employed were as follows: (1) an inhibitor of glycolysis, 3×10−5M iodoacetate (IAA); (2) an inhibitor of the TCA cycle, 1·5×10−2M malonate; (3) an uncoupler of oxidative phosphorylation, 1 ×10−4 M 2,4-dinitrophenol (2,4-DNP); and (4) a suspected inhibitor with an uncertain site of action, 1 ×10−5 M trypan blue. Another inhibitor of the terminal respiratory chain, an anaerobic gas phase, has been reported elsewhere (Cox & Gunberg, 1972) and the results will not be presented here.
A comparison was made between heart contraction rates in 11-and 12-day hearts after the addition to the incubation medium of either iodoacetate or malonate. Three groups of hearts obtained from the same litter were used each time this experiment was conducted. After 1 h of incubation in the oxygenated Krebs-Ringer bicarbonate solution the inhibitors were added separately to two of the three chambers. The third group of hearts served as the control and received neither inhibitor nor substrate.
It was noted that in both age-groups examined the addition of iodoacetate resulted in complete depression of heart activity after 2 h. This effect could be reversed by the addition of pyruvate (1 ×10−2M) to the medium. The presence of malonate had no apparent effect on heart rates observed in organs obtained from 11-day embryos (Fig. 1) whereas malonate significantly depressed contractile activity in the hearts from 12-day embryos (Fig. 2).
A comparison was made between 11-and 12-day embryonic rat heart contraction rates after the addition of 2,4-DNP. Two groups of hearts obtained from litter-mates were allowed to equilibrate for 1 h in oxygenated Krebs-Ringer bicarbonate solution which contained 5 × 10−3M glucose. The inhibitor was added to the medium after one hour of equilibration. One chamber was supplied with oxygen and the other an anaerobic gas phase consisting of 95% N and 5% CO2. After 2 h of anaerobiosis these hearts were supplied with fructose-1,6-diphosphate (5x 103M).
The 11-day hearts exhibited a moderate depression of heart rate after the addition of 2,4-DNP (Fig. 3). No difference in performance could be demonstrated between the aerobic and anaerobic groups. Neither group contained an organ in which the contraction rate declined to zero during the 4 h of observation. In contrast, the 12-day hearts exhibited a complete cessation of contractile activity in less than 30 min after the addition of 2,4-DNP (Fig. 4). The anaerobic group remained at zero for the full course of the experiment and the addition of fructose-1,6-diphosphate did not restore contractile activity in these quiescent organs. The aerobic group after 1 h of exposure to the inhibitor showed a spontaneous partial recovery of function. Heart contraction rates in these organs returned to approximately 50% of the rate established before the addition of the inhibitor.
Trypan blue as a metabolic inhibitor
Two groups of 11-day embryonic hearts obtained from litter-mates were allowed to equilibrate for 1 h in an oxygenated Krebs-Ringer bicarbonate solution which contained 5 × 10−3M glucose. Semi-purified and desalted trypan blue was added to one of the two incubation chambers. The quantity of dye used was adjusted to bring the media concentration to 1 × 10−5M. After the initial hour in oxygen the gas phase was changed to provide anaerobic conditions for both groups for the duration of the experiment.
It was observed that after 1 h in oxygen no difference in the mean contraction rates existed between the two groups of hearts. After changing to an anaerobic gas phase, however, a marked separation between the two groups was observed, with the hearts exposed to trypan blue exhibiting a significantly lower contraction rate (Fig. 5). After 2 h of exposure to anaerobic conditions fructose-1,6-diphosphate (5×10−3M) was added to both chambers. One hour after the addition of the phosphorylated hexose the average contraction rates in hearts exposed to trypan blue had returned to the control level. It should be noted that alpha-glycerophosphate in a concentration of 3 × 10−2M had the same effect as fructose-1,6-diphosphate in reversing the trypan blue block. The same pattern, with higher heart rates, was observed for 12-day hearts under similar conditions.
Glycolysis and the TCA cycle
The observation that inhibition of the TCA cycle with malonate resulted in a depression of the contraction rate in 12-but not 11-day embryonic rat hearts illustrates the rapid metabolic shift that takes place during the 24 h period of development under study. It is of interest to note that on the 12-day of development the allantoic placenta begins to replace the visceral yolk sac as the site of maternal-embryonic gas exchange. Since the allantoic placenta possesses a greater surface area for maternal-embryonic gas exchange and a better circulation than the visceral yolk sac, it appears that the embryo in vivo resides in a better oxygenated environment at the time the isolated hearts exhibit an increasing dependence on extraglycolytic metabolism.
The reduction in the importance of the Embden-Myerhof pathway for the maintenance of cardiac contractile activity in the 12-day hearts, as suggested by the substrate utilization investigations previously reported (Cox & Gunberg, 1972), was demonstrated here to be of a relative nature. The low concentrations of iodoacetate employed here plus reversal of the block by pyruvate are highly suggestive of a specific action on glycolysis (Webb, 1963). It will be recalled that iodoacetate poisoning of this metabolic pathway resulted in stoppage of heart contraction in both the 11-and 12-day hearts. In light of this observation it appears that although the TCA cycle gains in importance as an energy producing pathway, the Embden-Myerhof pathway is probably responsible for providing the majority of the three carbon intermediates destined for oxidation aerobically. While it has been demonstrated that the 12-day embryonic rat heart can utilize some amino acids and ketone bodies to maintain contractile activity (Cox & Gunberg, 1972) it seems unlikely that these compounds would contribute significantly to the energy required by these young organs during a phase of rapid development and protein synthesis.
It is also of interest to note that in the experiments which tested the effects of iodoacetate and malonate on the function of the isolated embryonic rat hearts no substrate was added to the incubation medium. The adverse effects of iodoacetate on both the 11-and 12-day organs suggests that the intrinsic stores of metabolite utilized by the embryonic hearts enters the glycolytic pathway at a point above the blockade induced by this metabolic poison. Presumably the intrinsic metabolite is stored in the form of glycogen which can be demonstrated histochemically in the 12-day heart and ultrastructurally in the 11-day organ (Chacko, 1971).
Uncoupling of oxidative phosphorylation
The observation that 2,4-DNP caused a slight depression of heart activity uniformly in 11-day hearts under both aerobic and anaerobic conditions suggests that some interference in energy production occurs at the glycolytic substrate phosphorylation level. Harary & Slater (1965) proposed that 2,4-DNP might inhibit extramitochondrial ATP levels as well as uncouple oxidative phosphorylation. In their studies utilizing single beating neonatal-rat-heart cells, they found that 2,4-dinitrophenol completely inhibited contractile activity, as seen here for the 12-day isolated hearts. The difference in response to 2,4-DNP by the 12-day as compared to the 11-day preparations could be due to one or more of several possibilities: (1) greater inhibition of extramitochondrial ATP levels by 2,4-DNP in the older hearts, (2) increased permeability of the cells to the inhibitor in the older hearts, and (3) a greater dependence on mitochondrial oxidative processes with a relatively less active glycolytic cycle in the 12-day hearts. The last explanation gains favour when considering the partial recovery in activity of the inhibited hearts in oxygen since it is known that uncoupling of oxidative phosphorylation stimulates glycolysis (Mueller, 1962). This explanation is not entirely satisfactory, however, and further investigation in this area is necessary.
The failure of the 12-day hearts subjected to an anaerobic gas phase to recover their contractile activity when furnished fructose-1,6-diphosphate has significance only when compared with the following discussion.
Trypan blue as a metabolic inhibitor
The mechanism of action of this teratogenic agent has been the subject of much investigation and speculation since it was originally observed to produce malformations in the offspring of treated pregnant rats (Gillman, Gilbert, Gillman & Spence, 1948). The most popular theory of the mode of action of this teratogen was proposed by Beck and his associates (1967) and implicates yolk-sac dysfunction. These investigators propose that since the dye was concentrated in the visceral yolk-sac epithelium and since this extraembryonic membrane was of importance in providing nutrients to the early embryo, any disruption of its function may lead to maldevelopment of the embryo it supports. This hypothesis was strengthened by the observation that trypan blue inhibits, in vitro, several hydrolytic enzymes found in lysosomal fractions from disrupted rat visceral yolk-sac (Lloyd, Beck, Griffiths & Parry, 1968).
Another mechanism of action for trypan blue has been proposed by Kaplan & Johnson (1968). These investigators reported that oxygen consumption by dye-treated chick embryos increased over control values and suggested that trypan blue might act in a manner similar to 2,4-DNP in uncoupling oxidative phosphorylation. A comparison of the results presented here indicate that trypan blue and 2,4-DNP elicit markedly different responses from the embryonic rat heart in vitro. This observation plus the ability of fructose-1,6-diphosphate to alleviate the effect of trypan blue on the function of the embryonic rat heart but not that of 2,4-DNP, suggests that these two agents act in a dissimilar fashion.
The possibility that trypan blue injected into a pregnant rat might reach the embryo and have a direct effect on embryonic tissues was proposed by Adams-Smith (1963). This investigator reported an accumulation of glycogen in the hearts of embryos from dye-treated rats not observed in the controls. It was suggested in this report that trypan blue might act within the myoepicardial cells to cause a premature shift in metabolism to that of a more mature form. The concept of direct action of trypan blue on embryonic tissues has not been widely accepted because of the difficulty in visualizing the dye in the embryo. Davis & Gunberg (1968), however, have recently reported that dye deposits could be observed in the entoderm of embryos obtained from a treated rat and Schmidt (1971) has described dye-like particles observed with the electron microscope in the neuroectoderm of such animals.
The results reported here indicated that trypan blue did have a direct effect on the embryonic rat heart when it was exposed to very low concentrations of the dye. The effective concentration of the dye employed in these experiments (1 × 10−5M) was approximately 4% of the peak concentration reported in maternal circulation following a teratogenic dose of trypan blue (Beck & Lloyd, 1966). The depression of the contraction rate in embryonic hearts induced by trypan blue when these organs are presumably deriving energy for function primarily from anaerobic glycolysis, suggests that the dye acts to interfere with glucose uptake or catabolism. The reversal of the trypan blue block with the addition of fructose-1,6-diphosphate or alpha glycerophosphate further localizes the site of interference. It appeared that trypan blue interfered with the process of glycolysis at some point between the transport of glucose across the cell membrane and the enzyme phosphofructokinase. Investigations have been initiated which hopefully will more clearly define the metabolic site of action of this teratogenic agent on the isolated embryonic rat heart.