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/ . Embryol exp. Morph. Vol. 28, 3, pp. 591-599, 1972
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59 \
Energy metabolism in isolated rat embryo hearts:
effect of metabolic inhibitors
By STEVEN J. COX1 AND DAVID L. GUNBERG 2
From the Department of Anatomy, University of Oregon
Medical School, Portland
SUMMARY
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.
INTRODUCTION
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
1
2
Author's address: Sacred Heart Medical Center, Spokane, Washington 99204, U.S.A.
Author's address: Anatomy Department, Faculty of Medicine, University of Malaya,
Kuala Lumpur, Malaysia.
592
S. J. COX AND D. L. GUNBERG
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 KrebsRinger 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 % N 2 , 5 % CO2 and for
anaerobic conditions included 95 % N 2 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 X 1 0 ~ 5 M
iodoacetate (IAA); (2) an inhibitor of the TCA cycle, 1-5 x 10~ 2 M malonate;
(3) an uncoupler of oxidative phosphorylation, 1 x 10~4 M 2,4-dinitrophenol
(2,4-DNP); and (4) a suspected inhibitor with an uncertain site of action,
1 x 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.
RESULTS
Inhibition of glycolysis and the TCA cycle (Figs. 1, 2)
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 x 10~ 2 M) 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).
Embryonic rat heart metabolism
11-day hearts
240
12-day hearts
•
• lodoacetate
O---O Malonatc
•
' • Control, no substrate
200
593
•
O
240
• lodoacetate
O Malonate
Control, no substrate
Inhibitors added
to 2 groups
200
Inhibitors added
to 2 groups
l l 60
?160
Q.
<L>
~ 120
2 120
3
X
X 80
80
40
40
0
0
Time (h)
Time (h)
Fig. 2
Fig. 1
Mean ± standard deviation
Substrate
No. in
group
No. of
litters
1h
2h
3h
4h
Control
IAA
Malonate
11
11
11
3
3
3
162 ±12
152 ±15
155± 16
149± 15
107± 12
149 ±13
137± 15
0
137+16
126+18
143 ±17
122 ±10
Fig. 1
Mean ± standard deviation
No. of
Substrate
No. in
group
litters
1h
2h
3h
4h
Control
IAA
Malonate
OS O O
3
3
3
199 ±2
197 ±6
197 ±5
187 ±16
119 ±18
157 ±19
161 ±16
0
120±18
150±21
119 ± 27
114 + 15
Fig. 2
Figs. 1, 2. The effects of malonate (1-5 x 10~2M) or iodoacetate (3 x 10~5M) on the
in vitro average contraction rates of 11- and 12-day embryonic hearts compared with
controls. No substrate was added to the media in these experiments. Note the
effects of pyruvate (1 x 10~2M) when added to the iodoacetate inhibited groups at
3h.
38
E M B 28
594
S. J. COX AND D. L. GUNBERG
12- day hearts
12-
11-day hearts
•
• Glucose and 2,4-DNP, O2
O
O Glucose and 2,4-DNP, N2
240
240
200 _
200
_
/
1 160
2,4-DNP added
to both groups
•
O
• Glucose and 2,4-DNP, O,
O Glucose and 2.4-DNP, N2
2,4-DNP added
to both groups
160
x^
X>
120
2 120
X 80
o
X
-
F-l,6-P2 to
N2 group
40 i
A
U
1
40
i
2
80
i
i
3
4
0
Time (h)
Fig. 3
Mean ± standard deviation
Substrate
No. in
group
glu, O2
glu, N2
No. of
litters
2h
lh
15O±18
153±18
12
11
3h
106± 19
107 ± 13
4h
93± 13
87±9
91 + 10
90 ±14
Fig. 3
Mean ± standard deviation
Substrate
glu, O2
glu, N2
No. in
group
No. of
litters
1h
Hh
2h
3h
4h
11
11
2
2
170+12
180±21
0
0
99+17
0
115 ± 17
0
104+14
0
Fig. 4
Figs. 3, 4. The effects of 2,4-dinitrophenol (1 x 10~4M) on the in vitro average
contraction rates of 11- and 12-day embryonic hearts incubated in a media containing
glucose (5 x 10~3M). Hearts cultured in an aerobic gas phase (65 % O2, 35 % N,
5 % CO2) are compared with organs incubated in an aenaerobic gas phase (95 % N,
5 % CO2). Note that the addition of fructose-l,6-diphosphate (5 x 10~3M) elicited no
effect on the contraction rates.
Embryonic rat heart metabolism
595
Uncoupling of oxidative phosphorylation (Figs. 3, 4)
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 KrebsRinger bicarbonate solution which contained 5 x 10~ 3 M 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 fructose1,6-diphosphate (5x 10~ 3 M).
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 x 10~ 3 M 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 x 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 fructose1,6-diphosphate (5x 10~ 3 M) 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 x 10~ 2 M 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.
38-2
596
S. J. COX AND D. L. GUNBERG
11-day hearts
•^—•
O
'P
Glucose
O Glucose+ trypan blue
160
c.
120
80
F-1.6-P2
(5x 10 3 \i) added
N,
40
Time (h)
Fig. 5. The effects of trypan blue (1 x 10~5M) on the average contraction rates of
11-day embryonic hearts incubated in a media containing glucose (5X10~ 3 M)
compared with controls. Note the effects of fructose- 1,6-diphosphate (5 x 10 3M)
added to both groups at 3 h. (Vertical bars represent S.D. Hatching represents
S.D. also.)
DISCUSSION
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
Embryonic rat heart metabolism
597
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.
598
S. J. COX AND D. L. GUNBERG
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 dyetreated 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 AdamsSmith (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 x 10~ 5 M) 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
Embryonic
rat heart metabolism
599
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.
REFERENCES
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KAPLAN, S. & JOHNSON, E. M. (1968). Oxygen consumption in normal and trypan bluetreated chick embryos. Teratology 1, 369-374.
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{Manuscript received 13 March 1972, revised 15 May 1972)