/. Embryol. exp. Morph. Vol. 28, 2, pp. 235-245, 1972
Printed in Great Britain
235
Metabolite utilization by isolated embryonic
rat hearts in vitro
By STEVEN J. COX1 AND DAVID L. GUNBERG 2
From the Department of Anatomy, University of Oregon Medical School
Portland, Oregon
SUMMARY
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.
INTRODUCTION
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, 1950^) 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, 1950Z?;
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).
1
Author's address: Sacred Heart Medical Center, West 101 Eighth Avenue, Spokane,
Washington
99204, U.S.A.
2
Author's address: Anatomy Department, Faculty of Medicine, University of Malaya,
Kuala Lumpur, Malaysia.
l6
.,
EMB 28
236
S. J. COX AND D. L. GUNBERG
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.
MATERIALS AND METHODS
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. Elevenday 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).
The chamber (Fig. 1) was made of heavy Pyrex glass with a flanged top which
Embryonic rat heart metabolism
237
Table 1. A summary of the substrates tested, the concentrations employed and the
cardiac contractile rates observed
Average heart rate beats per minute ±S.D.
Age
...
11-day
13-day
Hour 1
Hour 3
Hour 1
Hour 3
Hour 1 Hour 3
(anaerobic) (aerobic) (anaerobic) (aerobic) (anaerobic) (aerobic)
149±11
145±12
162±12
162±17
131 ±12
192±10
31 ±12
69±10
84±7
119+15
90+17
127±16
Substrate
Glucose (1 x 10~2M)
No substrate
Mannose (1 x 10~2M)
Fructose (1 x 10- 3 M to 5 X 10- 2 M)
Galactose(lxlO- 3 M to 5X10- 2 M)
Glucose-l-PO 4 (lxl0- 2 M to 5X10- 2 M)
Glucose-6-PO4(lxl0-2M to 5 X 1 0 - 2 M )
2
2
Fructose-6-PO4 ( 1 X 1 0 - M to 5 X 1 0 - M )
Fructose-l,6-diPO4(5xl0-3M)
Alpha glycero-PO4 (3x1 0- 2 M)
Pyruvate (1 x 10~2M)
Acetoacetate (1 x 10~2M)
Beta-Hydroxybutyrate (1 x 10~2M)
L-Valine(5xlO-3M to 5X10- 2 M)
L-Alanine (5x 10~3M)3
OH-L-Proline(5xl0M)
2
Succinate (1 x 10~ M)
12-day
3
Isocitrate(5xlOM to 5X10- 2 M)
Malate(5xlO"3M to 5X10" 2 M)
+
-
++
++
+
-
+
-
++
++
+
-
-
-
-
++
++
++
++
++
++
++
++
—
—
—
+
—
—
—
—
+
++
++
—
—
++
-
-
-
++
-
-
_
_
-
-
_
_
-
_
_
_
_
.
.
.
.
.
.
.
.
—
—
.
+
+
-
+
_
_
_
_
Mean heart rates are given at hours 1 and 3 (representative of anaerobic and aerobic conditions, respectively) for the glucose and no substrate controls. All other substrates were compared with these. The
symbol (—) indicates no significant difference (P > 005) between that substrate and the no substrate
control. The symbol ( + ) indicates the mean heart rates were significantly greater than the no-substrate
controls, but significantly less than the glucose controls (P < 005). The symbol (+ +) indicates no significant difference (P > 005) between that substrate and the glucose control mean rate. The data for each
group was obtained from 7-12 hearts taken from two or more litters. A dot implies insufficient data to
make a comparison for that group.
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.
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 by16-2
238
S. J. COX AND D. L. GUNBERG
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.
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 without 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
/-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.
Embryonic rat heart metabolism
S.D., 0 2
• — • Glucose, O2
200 p 0---0 Glucose, N2
S.D., N 2
239
11-day hearts
160
120
200
(109---.,
- 160
-f-f.H
« 120
200
13-day hearts
160
120
100
Time (h)
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.
RESULTS
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 12and 13-day hearts, with the 13-day hearts exhibiting the greatest retardation in
rate (Fig. 2).
240
S. J. C O X A N D D . L. G U N B E R G
12-dav hearts
200
•
•Glucose (10 :.u)
o
O Fructose (10~ 2.\t)
•
•• K-R salts
160
= 12 °
/
•
•
/ /
8r
80
Time (h)
Mean ± standard deviation
Substrate
Glucose
Fructose
L-Salts
No. in No. of
group litters
24
23
20
6
6
6
1h
2h
165 ±12
85 ± 9
89 ±12
163 ±16
155±35
141 ±29
3h
162± 18
142 ±24
121 ±18
4h
152± 14
142±25
110±15
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.
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
Embryonic rat heart metabolism
241
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.
Similar experiments were undertaken to test each of the following compounds:
galactose, mannose, glucose-1-phosphate, glucose-6-phosphate, fructose-6phosphate, 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 alphaglycerophosphate 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.
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 experi-
242
S. J. COX AND D. L. GUNBERG
12-day hearts
OH-i.-prolinc (5 x 10~ 3 M)
200
K-R salts
r
160
S 120
80
O,
N,
Time (h)
Mean ± standard deviation
Substrate
OH-proline
L-Salts
No. in No. of
group litters
10
10
lh
91 ±10
90 ±17
2h
178± 17
150± 15
3h
164± 15
127±16
4h
144 ±7
108 ±14
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.
ments 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.
Embryonic rat heart metabolism
243
DISCUSSION
Differences in the utilization of hexoses by developing chick brain were noted
by Spratt (1950 a) 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-1,6diphosphate 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
244
S. J. COX AND D. L. GUNBERG
& 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 12and 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.
Embryonic rat heart metabolism
245
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