Isolated hearts from 11-, 12- and 13-day rat embryos were incubated in a simple defined salt solution to which was added a variety of single substrates. Utilization of the added substrate was determined by comparing the contractile rates of the hearts in the presence and absence of the compound being tested. Of all the compounds tested only those involved in the Embden-Meyerhof glycolytic pathway were capable of maintaining cardiac contraction at a maximum rate in the 11-day heart. This was accomplished under both aerobic and anaerobic conditions. Although glycolysis remained important, the 12- and 13-day hearts exhibited a shift in dependence towards other metabolic pathways. This conclusion was based on the observations that anaerobic glycolysis could no longer maintain maximum heart rates and that a variety of non-glycolytic compounds could be utilized for contractile activity by the 12- and 13-day organs.

A survey of the literature reveals that little information is available concerning mammalian cardiac metabolism in the early stages of functional activity. Biochemical and histochemical analysis of the early embryonic heart has led to some understanding of the changing metabolic patterns in this organ during development, but these experimental approaches are limited by the small amount of tissue available for study and by factors intrinsic to the techniques themselves.

Spratt (1949, 1950a) has successfully utilized chick embryos cultured on defined media in his studies of the nutritional requirements of the developing chick embryo, which indirectly yielded information concerning cardiac metabolism. The addition of metabolic inhibitors to the defined media (Spratt, 1950 b; Duffey & Ebert, 1957) further elucidated the pathways involved in energy metabolism in the heart and heart-forming areas of the chick embryo. Unfortunately this technique has not been applicable to mammalian embryos of the beating-heart stages since a more complex media is required to promote growth in these organisms (New, 1967).

Hall (1957) and Roberts (1966) used isolated embryonic hearts from rat and chick, incubated for short periods in Krebs-Ringer’s bicarbonate plus glucose, in their investigation of the effects of neurohumoral agents and anaerobiosis on heart rate. Preliminary experiments begun in these laboratories indicated that cardiac contraction rate could be maintained at an in vivo level when isolated embryonic rat hearts were incubated in a simple buffered salt solution containing glucose, but not other metabolites with the possible exception of mannose. It appeared, however, that this investigator’s experimental system did not provide an adequate mixture of oxygen and media, so that substrates were tested under essentially anaerobic conditions.

In order to extend and clarify these preliminary observations a more satisfactory incubation chamber was designed and constructed which allowed rapid equilibration between the media and gas phase. The following investigations involve a study of energy metabolism in the developing embryonic rat heart as determined by substrate utilization.

Sprague-Dawley rats were housed in an inverted light-cycle room and the female’s estrous cycles followed by vaginal smears according to the method of Blandau, Boling & Young (1941). The female in estrous was placed with a male for 2 h and checked for vaginal sperm at the end of the breeding period. Copulation was assumed to have taken place at the midpoint of the 2 h breeding period.

Embryos were obtained by laparotomy and uterine section 264 ± 1 h (11 days), 288 ± 1 h (12 days) or 312 ± 1 h (13 days) after copulation. This careful timing of gestation produced litters that were remarkably uniform in development. Eleven-day embryos possessed an average of 16 ±2 somites and the 12-day embryos 28 ±2 somites. Other external features were used to ascertain the stage of development of the 13-day embryos which were comparable to the stage 23 described by Christie (1964).

Incubation techniques

The embryos were removed from the uterus in their decidual capsules and transferred to a dish of sterile normal saline. Extra-embryonic membranes were removed in a fashion similar to that described by New & Stein (1964) and the hearts then dissected free by severing the truncus arteriosus and sinus venosus at the sites of attachment to the pericardial sac. The hearts separated from the pericardial sac were transferred by pipette to perforated glass inserts, a part of the incubation chamber described below. The organs were bathed by 500 ml of incubating media, maintained at 37·5°C by a circulating water bath which consisted of a standard Krebs-Ringers (K-R) bicarbonate solution to which was added 50 mg/ml of streptomycin and 100 i.u./ml of penicillin. Substrates to be tested were added as isosmotic (300 m-osm) solutions (Table 1).

Table 1

A summary of the substrates tested, the concentrations employed and the cardiac contractile rates observed

A summary of the substrates tested, the concentrations employed and the cardiac contractile rates observed
A summary of the substrates tested, the concentrations employed and the cardiac contractile rates observed

The chamber (Fig. 1) was made of heavy Pyrex glass with a flanged top which supported the lid. The lower portion of the chamber contained a ground-glass female fitting designed to receive a tapered air-stone apparatus (labelled ‘Gas inflow’ in Fig. 1). Humidified gas supplied to the chamber through the air-stone consisted of 35% nitrogen, 65% oxygen and 5% carbon dioxide for aerobic conditions or 95% nitrogen and 5% carbon dioxide for anaerobic conditions. The gas was allowed to escape from the chamber through an exhaust port, thus keeping the barometric pressure at ground level. Condensation on the inner surface of the optical-glass lid was prevented by maintaining a lid temperature slightly greater than that of the incubation media. This was accomplished by passing a low-voltage current through a thin ring of tin oxide baked onto the surface of the lid.

Fig. 1

A schematic drawing of the incubation chamber. The chamber was immersed in a constant-temperature water bath to the level of the exhaust port and filled with the medium being tested to the level of the suspending plate which supported the inserts containing the hearts under observation.

Fig. 1

A schematic drawing of the incubation chamber. The chamber was immersed in a constant-temperature water bath to the level of the exhaust port and filled with the medium being tested to the level of the suspending plate which supported the inserts containing the hearts under observation.

The inserts in which the embryonic hearts were placed (one heart/insert) contained 20 0·5-mm diameter holes which permitted free exchange between the 1 ml of media in the insert and the 500 ml of media contained in the chamber. This large volume of media ensured that the concentration of the substrate was not significantly altered during the experiment and that any metabolic by-products which might prove toxic to the function of developing heart were diluted to presumably non-toxic levels. The chamber was designed so that the gas phase could be changed and compounds could be added to the media with-out disturbing the hearts under observation.

Heart rate was measured directly by examining the organs with a stereoscopic microscope through the chamber lid. Ten beats of each heart were timed with an electric timer and the data converted to the number of beats per minute. Duplicate counts were routinely made to check on the accuracy of the initial observation, and only synchronized functional heart beats were counted. Comparisons were made between the mean heart rates of the various groups only when each group contained samples from the same females. The litters from each animal, usually containing 12–15 offspring, were evenly divided into three groups, one serving as the control and the other two as the experimental groups. This was repeated for each of the three or more pregnant animals used in each series.

Comparison of average heart rates between groups was made after calculating the variance and standard deviation about the mean. Application of Student’s ‘t’-test and reference to P values contained in the table of distribution of by Fisher & Yates (1963) was used to determine the level of significance of any inter-group differences. The value P < 0·05 was considered indicative of a statistically significant difference.

Glucose with and without oxygen

The mean contractile rates of hearts obtained from 11-, 12- and 13-day embryos, incubated in medium containing glucose were determined in the presence and absence of oxygen (Fig. 2). It was observed that the lack of oxygen exerted no effect on the contractile rates of the 11-day hearts. The anaerobic gas phase produced a significant depression of the contractile rate in both the 12- and 13-day hearts, with the 13-day hearts exhibiting the greatest retardation in rate (Fig. 2).

Fig. 2

A comparison of the average contractile rates (beats per min) observed in the isolated heart preparations obtained from 11-, 12- and 13-day rat litter-mates when incubated in a medium containing glucose and subjected to aerobic or anaerobic gas phases.

Fig. 2

A comparison of the average contractile rates (beats per min) observed in the isolated heart preparations obtained from 11-, 12- and 13-day rat litter-mates when incubated in a medium containing glucose and subjected to aerobic or anaerobic gas phases.

Substrates other than glucose

Embden-Meyerhof pathway intermediates

The ability of embryonic hearts to utilize several substrates other than glucose was tested in organs obtained from 11- and 12-day embryos. In these experiments each litter was divided into three groups. The first group was incubated in the presence of glucose to establish the optimal contractile rate. A second group was incubated in the buffered salt solution alone to establish a base-line of contractile activity supported by intrinsic energy stores. The third group was furnished the metabolite being tested and the contractile activity observed in these organs could then be compared to the optimal and intrinsic rates established by the hearts obtained from litter-mates. In each experiment the first hour of incubation was conducted under anaerobic conditions and the remaining 3 h in the presence of oxygen.

A graphic example of the type of data obtained from such experiments is presented in Fig. 3. In this experiment which tested the ability of fructose to maintain cardiac contraction in 12-day hearts it will be noted that under anaerobic conditions fructose did not support cardiac function at a level different than the established intrinsic rate. In the presence of oxygen, however, fructose maintained cardiac contractile rates in 12-day hearts at a level comparable to that established with glucose.

Fig. 3

A comparison of the average contractile rates (beats per min) observed in the isolated heart preparations obtained from 12-day rat litter-mates and incubated in media containing glucose, fructose or no substrate.

Fig. 3

A comparison of the average contractile rates (beats per min) observed in the isolated heart preparations obtained from 12-day rat litter-mates and incubated in media containing glucose, fructose or no substrate.

Similar experiments were undertaken to test each of the following compounds: galactose, mannose, glucose-l-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-l,6-diphosphate, alpha-glycerophosphate and pyruvate. A summary of the results obtained from these experiments is presented in Table 1, where heart rates from hours 1 and 3, representative of anaerobic and aerobic conditions, respectively, were compared statistically for evidence of substrate utilization. In general the following was observed:

  1. Of the non-phosphorylated hexoses, glucose and mannose alone were able to maintain contractile activity at rates significantly above the base-line values under anaerobic conditions. Mannose, however, was utilized less effectively than glucose.

  2. In the presence of oxygen the effectiveness of the non-phosphorylated hexoses in maintaining contractile rates above base-line values in the isolated embryonic rat heart can be ranked as follows: glucose = mannose = fructose > galactose.

  3. Singly phosphorylated hexoses were not utilized by the preparations to support cardiac contraction while doubly phosphorylated fructose and alpha-glycerophosphate were effective.

  4. Pyruvate, under aerobic conditions, provided energy for contraction in both the 11-and 12-day preparations under the experimental conditions employed.

Amino acids, ketone bodies and tricarboxylic acid cycle intermediates

Embryonic hearts obtained from litter-mates were divided into two groups for this series of experiments. The control group was incubated in the buffered salt solution and the experimental group in the same solution to which was added the substrate to be tested. The design of these experiments is illustrated by the graphic presentation of data obtained from hearts provided OH-L-proline as a substrate (Fig. 4). It will be noted that the gas phase during the first hour was anaerobic, as in the hexose experiments, and was changed to aerobic for the remainder of the incubation period. Anaerobically, OH-L-proline could not maintain cardiac contraction at rates significantly different than the control functioning in the absence of substrate. In the presence of oxygen, however, this substrate supported cardiac contraction in 12-day hearts at a level comparable to that observed previously in hearts of the same age incubated in the presence of glucose.

Fig. 4

A comparison of the average contractile rates (beats per min) observed in the isolated heart preparations obtained from 12-day rat litter-mates and incubated in media containing OH-L-proline or no substrate.

Fig. 4

A comparison of the average contractile rates (beats per min) observed in the isolated heart preparations obtained from 12-day rat litter-mates and incubated in media containing OH-L-proline or no substrate.

Similar experiments were conducted on 11-, 12- and in some instances 13-day hearts testing the following substrates: acetoacetate, beta-hydroxybutyrate, L-valine, L-alanine, succinate, isocitrate, and malate. The results of these experiments are summarized in Table 1. Unlike the data from the Embden-Meyerhof pathway experiments, in which the same pattern of substrate utilization pertained to both 11- and 12-day embryo hearts, this series of experiments demonstrated several changes in pattern associated with age.

  1. The 11-day embryonic rat heart under the experimental conditions employed was unable to utilize the amino acids, ketone bodies or tricarboxylic acid cycle intermediates tested to maintain contractile rates above the intrinsic level.

  2. Cardiac contraction in the 12-day hearts was supported by the ketone bodies acetoacetate and beta-hydroxybutyrate, and by two of the three amino acids tested.

  3. By the thirteenth day of development, succinate could be added to the list of substrates which were capable of maintaining contractile activity in these isolated preparations.

Differences in the utilization of hexoses by developing chick brain were noted by Spratt (1950a) in his investigations of the nutritional requirements of the developing chick embryo. He noted no differences, however, in the functional development of the heart when the embryos were cultured on defined medium containing one of the four hexoses, glucose, mannose, fructose or galactose. Based on the observations presented above it would appear that the embryonic rat heart is different in this regard. Of the four simple hexoses investigated, only glucose was able to maintain a maximum heart rate in nitrogen; mannose was next best, with fructose and galactose being essentially incapable of maintaining contractile activity under anaerobic conditions. Aerobically, glucose utilization appeared to be equalled by mannose and fructose, while galactose was found to be less effective as a source of energy. The differences in hexose utilization observed could be related to either differences in cell membrane transport mechanisms associated with each compound or the enzyme levels necessary for the degradation of the respective hexoses.

Some phosphorylated compounds were able to provide energy for cardiac contraction while others proved ineffective. The monophosphorylated hexoses did not support function of the isolated embryonic hearts. Fructose-1,6-diphosphate and alpha-glycerophosphate, on the other hand, were both effective metabolites. The observation that phosphorylated compounds can cross the cardiac cell membrane in an in vitro system has been previously demonstrated and it has been reported that molecules as large as adenosinetriphosphate can cross the cell membrane in cultured heart cells (Bloom, 1970). The results reported above probably represent a difference in cardiac cell permeability to molecules of varying configuration rather than representing a selective permeability based on molecular size.

The observation that 11-day hearts can maintain an optimal contractile rate in the absence of oxygen, if furnished glucose as a substrate, suggests that the energy requirements for contraction in these young organs can be provided by glycolysis. Extraglycolytic energy production under anaerobic conditions has been postulated by Penney & Cascarano (1970) from observations on the adult rat heart and cannot be ruled out for the embryonic heart on the basis of data presented here. The ability of the 11-day heart to utilize pyruvate in the presence of oxygen for the maintenance of contractile activity suggests that this organ has an effective aerobic metabolic pathway but it is not certain as to how important it is in vivo during this stage of development. Tricarboxylic acid cycle activity has been demonstrated in the early undifferentiated preimplantation embryo by Daniel (1967) and Fridhandler, Wastila & Palmer (1967).

It should be pointed out that of all the metabolites tested only those entering the Embden-Meyerhof glycolytic pathway were effective in maintaining contractile activity in the 11-day heart. These observations, coupled with Tanimura & Shephard’s (1970) report of large quantities of lactate produced by the 11-day embryo, strongly suggests that the early rat embryo is quite dependent on glycolysis. The importance of glucose metabolism in developing heart has been emphasized by other investigators of early mammalian cardiac metabolism (Breuer, Barta, Pappoua & Zlatos, 1967; Clark, 1971; Wildenthal, 1971).

By the twelfth day of development optimal contraction rates could not be maintained by anaerobic glucose utilization alone. The greater dependence of the 12-day hearts on other metabolic pathways was also indicated by changes seen in the pattern of substrate utilization. Contraction of the heart at this stage of development could be maintained by the four additional compounds, acetoacetate, beta-hydroxybutyrate, L-alanine and hydroxy-L-proline. It will be noted that all these compounds can be metabolized by entering into the tricarbocylic acid cycle. The 13-day heart appears to be even more dependent on aerobic metabolism for maintenance of contractions. The pattern observed in the 12- and 13-day embryonic rat heart is similar to that reported by Wildenthal (1971) for late fetal mouse heart. This investigator reported that pyruvate, lactate, octanoate, acetoacetate and beta-hydroxybutyrate prolonged the life of isolated hearts cultured in complex media, but not to the same extent as glucose under like conditions. Williamson & Krebs (1961) observed that both acetoacetate and beta-hydroxybutyrate were oxidized by the adult rat heart and that by this stage of development acetoacetate was oxidized preferentially over glucose. The increasing importance of extraglycolytic metabolism during the stages of development under study here was stressed by Mackler, Grace & Duncan (1971). They found an increased activity of the enzymes concerned with terminal oxidation and phosphorylation in homogenates of 10-through 14-day rat embryos. Paralleling the enzyme changes was an increased complexity in the ultrastructure of mitochondria in the developing hearts of these animals.

The observations reported in this paper suggested that during the 48 h of development which were investigated there was a shift from dependence on glycolysis to a metabolic pattern which utilized extraglycolytic energy sources as well. This was characterized by the inability of the older hearts to function optimally in the absence of oxygen and the apparent development or activation of enzyme systems capable of metabolizing substrates along extraglycolytic pathways. It is of interest that this occurs at the same time developmentally that an effective gas-exchange organ, the allantoic placenta, is becoming functional. It can be postulated, therefore, that compounds interfering with the normal functioning of the glycolytic cycle would have a more harmful effect on the postimplantation embryo prior to day 12 than after the twelfth day of development. This question is further explored in another report which deals with the effect of metabolic inhibitors on the isolated heart preparations (Cox & Gunberg, 1972).

This work was supported in part by the National Institutes of Health Training Grant GM 445.

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