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JEZ 796
THE JOURNAL OF EXPERIMENTAL ZOOLOGY 278:273–282 (1997)
Protein Synthesis Under Conditions of Anoxia
and Changing Workload in Ventricle Strips From
Turtle Heart
J.R. BAILEY* AND W.R. DRIEDZIC
Department of Biology, Mount Allison University, Sackville, Canada,
E0A 3C0
ABSTRACT
An earlier study determined that protein synthesis in isolated perfused turtle
(Trachemys [=Pseudemys] scripta elegans) hearts was three-fold lower under conditions of anoxia
than under conditions of normoxia. However, the earlier study did not attempt to define the role of
work in the isolated perfused preparation. In this study, the effects of varying workload, as defined by changing frequency of contraction, and anoxia on protein synthesis were examined. The
ventricle strip preparation allows for comparison of multiple strips from a single heart, which aids
in eliminating the variability found between individuals chosen from wild populations. Ventricle
strips forced to contract at 24 contractions·min–1 under anoxic conditions failed more rapidly than
strips forced to contract at 24 contrations·min–1 under normoxic conditions. Protein synthesis decreased by 32% when compared to normoxic controls. When stimulation was terminated after 2 hr
of contraction, the rate of protein synthesis in strips under anoxic conditions was similar to that
in strips under normoxic conditions. Also, returning strips to normoxic conditions after 2 hr of
anoxia restored protein synthesis to the level of the normoxic controls. A significant correlation
between pacing rate and protein synthesis was found under normoxic conditions but not under
anoxic conditions when strips were paced at 12, 18, and 24 contractions·min–1. Protein synthesis
increased by 30% at the 18 contractions·min–1 frequency and 45% at the 24 contractions·min–1
frequency over the rate at 12 contractions·min–1 frequency. Force-frequency studies revealed that
under normoxic conditions force generation did not change until above 24 contractions·min–1, but
under anoxic conditions there was a significant negative inotropic effect (20% decrease in force) at
24 contractions·min–1 and fell to 50% of initial at 36 contractions·min–1. These studies indicate
that, in the turtle heart, anoxia per se is not the only determinant of protein synthesis but rather
that work plays an important role in protein synthesis, as in the mammalian heart. J. Exp. Zool.
278:273–282, 1997. © 1997 Wiley-Liss, Inc.
Protein synthesis has been well studied in the
mammalian heart and is influenced by a variety
of factors including oxygen availability and workload (Sugden and Fuller, ’91) such that under
normoxic conditions workload causes an increase
in protein synthesis. When mammalian hearts are
exposed to severe hypoxic or anoxic conditions,
decreases in both protein synthesis and degradation are observed (Jefferson et al., ’71; Fuller and
Sugden, ’88). However, a problem encountered in
experiments with mammals is that these hearts
rapidly fail, and therefore, it is difficult to separate the effects of anoxia per se on metabolism
from overall contractile failure effects. It is possible to alter cardiac metabolism chemically with
blockers such as CO to induce chemical anoxia or
to inhibit contractile events by the action of excess K+. These manipulations give data that, while
useful, may not actually reflect metabolic events
© 1997 WILEY-LISS, INC.
in intact heart tissue. Studies of protein turnover
in hearts of non-mammalian vertebrates are limited but are considered to be of value in elucidating fundamental principles of control. For
example, in trout hearts, protein synthesis increases with power output (Houlihan et al., ’88),
and in cod heart, RNA efficiency increases at
higher temperatures (Foster et al., ’92).
In previous work (Bailey and Driedzic, ’95, ’96),
we have shown that the turtle heart can be used
as a model system to study protein metabolism
under anoxic conditions. The turtle heart is very
resistant to anoxia, and isolated heart preparations continue to contract under total anoxia for
*Correspondence to: Dr. John Bailey, Department of Biology, Mount
Allison University, Sackville, NB, Canada EOA 3C0.
Received 7 November 1996; Revision accepted 27 January 1997
274
J.R. BAILEY AND W.R. DRIEDZIC
periods of up to 4 hr (Wasser et al., ’90). We found
that protein synthesis is not affected by 1 hr of
anoxia in the isolated perfused heart model
(Bailey and Driedzic, ’95) but that 2 or 3 hr of
anoxia produce a significant decrease in synthetic
rates without a concomitant decrease in contractility (Bailey and Driedzic, ’96). Also, there is an
apparent selective decrease in protein synthesis,
with synthesis of proteins destined for the mitochondria being preferentially decreased (Bailey
and Driedzic, ’96). This perfused heart model was
probably working at near maximal workloads
since input (atrial) pressure was very high compared to the in vivo condition and atrial pressure
has been shown to be a determinant of cardiac
work (Farrell et al.,’94; Jackson et al., ’95). However, we did not attempt to define the influence
of work on protein synthesis in the turtle heart
model system.
In this study, we utilized a ventricle strip preparation which allows for multiple strips from the
same heart, and thus, comparison of tissue responses from the same animal. This in turn allows for a greater insight into cellular events
unaffected by individual differences, which are always encountered when using samples from wild
populations. We report that there is a positive correlation between work and protein synthesis under normoxic but not under anoxic conditions. A
short recovery period under normoxic conditions
restores protein synthesis, and removing the work
component under anoxic conditions results in a
synthesis rate unchanged from the normoxic state.
MATERIALS AND METHODS
Animals
Adult red-eared sliders (Trachemys [=Pseudemys] scripta elegans) of either sex and mean
body mass of 683 ± 42 g were obtained from Carolina Biological Supply (Burlington, NC). Animals
were kept in flow-through aquaria at ambient
temperature (approximately 18°C) until use. They
were fed a mixed diet of greens and pieces of fish
ad lib. Aquaria were provided with basking platforms, allowing the animals to dry off periodically.
Photoperiod was 12 hr light:12 hr dark. All experiments were carried out in spring and early
summer seasons.Animals used in this study were
obtained at three separate times and probably
came from different wild populations.
Media used
Perfusion media consisted of a basic salt solution: 86.3 mM NaCl, 3.1 mM KCl, 2.3 mM CaCl2,
3.5 mM MgSO4 , 1.5 mM NaH2 PO4 , 43.5 mM
NaHCO3, and 10 mM glucose, pH 7.8 (basic medium) at 20°C. Basic medium pH was adjusted to
7.8 after 30 min gassing with either 5% CO2: 95%
O2 or 5% CO2:95% N 2. The basic medium was enriched by the addition of amino acids (Sigma
#6407, physiologic acidic and neutrals; Sigma
#6282, physiologic basics; Sigma Chemical Co., St.
Louis, MO) to a final concentration of 0.25 mM,
with the exception of cystine, which was 0.125
mM; proline, valine, and leucine, which were 0.5
mM; alanine and glycine, which were 1.0 mM; and
phenylalanine, which was 1.25 mM (amino acid
medium). In all cases, the experimental temperature was 20°C.
Experimental protocol
Animals were arbitrarily chosen from the holding tank and pithed and the plastron quickly removed. The heart was excised and placed in a
beaker of ice-cold medium. The atria were dissected free, the arteries exiting the ventricle removed, and the ventricle bisected. In the first
series of experiments, three strips were cut from
each section of ventricle for a total of six strips,
and in the remaining series of experiments, two
strips were cut from each section of ventricle for
a total of four strips. In all cases, each strip was
visually inspected and any obvious connective tissue removed. Each strip was then mounted in a
tissue bath between two platinum wire electrodes
and attached to an isometric force transducer
(Harvard Apparatus, Dover, MA), which was interfaced with a Biotronix BL 882 strip chart recorder. Strips were stimulated to contract with a
Grass (Quincy, MA) model 5D square wave generator at stimulus strength of 50 V and 200 msec
duration. Strips were stretched until maximal
peak height was observed. Each strip was allowed
to equilibrate under normoxic conditions in basic
medium for 15 min before the onset of the experimental period.
Series 1: force-frequency study
Four strips were set up and allowed to equilibrate in basic medium for 15 min at 12 contractions·min–1 under normoxic conditions. Strips
were then allowed to equilibrate for a further 18
min at 12 contractions·min–1 under either normoxic or anoxic conditions. Anoxic conditions were
achieved by changing the gassing mixture from
5% CO2:95% O2 to 5% CO2:95% N2. The PO2 of
the bathing medium was driven to less than 3
mm Hg after 5–10 min of vigorous gassing under
PROTEIN SYNTHESIS IN ANOXIC VENTRICLE STRIPS
these conditions. High-speed recordings were taken
for the last 30 sec of this period for contractiontime measurements. Frequency was increased in
steps of 6 contractions·min–1 at 3 min intervals up
to a maximum of 36 contractions·min–1. During
these increases in frequency, recordings were taken
as before for contraction-time measurements. At
the end of this period, strips were discarded.
Series 2: performance and protein synthesis
at set pacing rates
Anoxia. The effects of a set pacing rate and anoxia on strips from the same heart were determined. Following the end of the equilibration
period in basic medium, two strips were subjected
to anoxic conditions and two strips to normoxic
conditions. At T = 0 the medium was changed to
the amino acid medium and the strips were forced
to contract under either normoxic or anoxic conditions at a frequency of 24 contractions·min–1,
with high-speed recordings being taken every 15
min. The initial reference point was taken to be
T + 15 min to allow for readjustment to the mechanical disturbance following medium change.
Previous work has shown that protein synthesis
is significantly decreased under anoxic conditions
only after 2 hr of anoxia in the isolated perfused
heart preparation (Bailey and Driedzic, ’96); therefore, it was deemed necessary to have an equivalent equilibration time in the ventricle strip
preparations. At T + 120 min, 20 µl of 1.0 mCi·
min –1 L-[ring 2,6-3H]-phenylalanine (Amersham
Canada, Oakville, ON) were added to each bath. A
100 µl sample of the medium was immediately
taken and added to 10 ml of scintillation fluid
(CytoScint; ICN Pharmaceuticals Canada, Montreal, PQ) for the determination of specific activity. Strips were then forced to contract for a further
60 min at 24 contractions·min–1. At the end of the
experimental period, strips were taken down, rinsed
in cold basic medium, blotted dry, and immediately
processed for snalysis of phenylalanine uptake and
protein and RNA content.
Rest. A second set of experiments was carried
out to separate the effects of work and anoxia on
protein synthesis. Two strips were subjected to
normoxic and two strips to anoxic conditions in
amino acid medium for 2 hr at a frequency of 24
contractions·min–1. At T + 120 min the electrical
stimulus was discontinued; contractions ceased
and the ventricle strips entered a resting state.
At T + 175 min electrical stimulation was restarted, frequency as before, and contractions recorded for 5 min to determine the viability of the
275
strips. The experiment was then terminated and
the strips taken down. Recordings were taken, isotope was added, and the tissue treated as described in the anoxia experiment.
Recovery. A third set of experiments was used
to determine if the effects of anoxia on protein
synthesis were due to acute regulation during this
hour or due to a general deterioration of the protein synthetic mechanism. As before, two strips
were forced to contract under anoxic conditions
and two strips under normoxic conditions in amino
acid medium for 2 hr at a frequency of 24 contractions·min–1. The gassing mixture for the anoxic
medium was then changed to the normoxic gassing mixture (5% CO2:95% O2) at T + 120 min for
the remainder of the experimental period. Strips
were then forced to contract for a further 60 min
at 24 contractions·min–1. At the end of this period, strips were taken down. As before, recordings were taken, isotope added, and tissue treated
as described in the anoxia experiment.
Series 3: protein synthesis with increasing
workload (normoxia vs. anoxia)
Three strips were set up and allowed to equilibrate for 15 min in basic medium. Each strip was
paced at a different frequency, i.e., one strip at
12, one strip at 18, and one strip at 24 contractions·min–1, and forced to contract for 2 hr under
either normoxic or anoxic conditions. The basic
medium was replaced with amino acid medium
at T = 0. In all cases, the initial reference point
was taken to be T + 15 following the change in
the medium to allow the strips to readjust to the
mechanical disturbance. At T + 120 min radiolabeled phenylalanine was added to each bath. A
100 µl sample of the medium was immediately
taken and added to 10 ml of scintillation fluid for
the determination of specific activity. Strips were
forced to contract for a further 1 hr. At the end of
the experimental period, strips were taken down,
rinsed in cold basic medium, blotted dry, and immediately processed for analysis of phenylalanine
uptake and protein and RNA content.
Whole tissue analysis
All extraction procedures are based on the
method of Fuller and Sugden (’88). Briefly, each
tissue was homogenized in nine volumes of 0.56
M perchloric acid. Crude homogenates were centrifuged at 10,000g for 15 min. The supernatant
was discarded, while the pellet was washed three
times with 2 ml of 0.56 M perchloric acid. The
pellet was then digested in 2.25 ml of 0.3 M NaOH
276
J.R. BAILEY AND W.R. DRIEDZIC
at 37°C for 1 hr. Digestion was regarded as complete when there was no visual evidence of any
solid material.
Phenylalanine incorporation
One hundred microliters of 100% (w/v, 1 g/ml)
trichloroacetic acid (TCA) were added to a 1 ml
aliquot of the digestate to precipitate proteins.
This mixture was centrifuged at 10,000g for 15
min and the supernatant discarded. The protein
pellet was washed with 1 ml diethyl ether and
centrifuged and the supernatant discarded. The
pellet was then dried in a 37°C water bath and
digested in 200 µl of 1 M NaOH in a boiling water bath for approximately 30 min, then added to
10 ml of scintillation fluid and counted in a
RackBeta scintillation counter.
RNA determination
Two hundred and fifty microliters of 2.5 M
perchloric acid were added to a 1 ml aliquot of
the digestate and the mixture centrifuged. The
supernatant was reserved for RNA analysis using the orcinol method (Munro and Fleck, ’66).
RNA from Torula yeast (Sigma #R6625) was
used as a standard.
Protein content
The remaining 250 µl aliquot of the digestate
was analyzed for protein content by a comercial
method (Biorad D C Protein Assay #500-0116,
Biorad, Richmond, CA).
Data analysis
Specific activity of the isotope-labeled perfusate
was calculated by determining disintegrations per
minute and dividing by concentration of phenylalanine in the medium. The amount of phenylalanine incorporated into protein pools was calculated
from the extracellular specific activity. This is the
standard method of data analysis and is based on
the findings that extracellular phenylalanine rapidly equilibrates with the intracellular pool, which
in turn equilibrates with phenylalanine tRNA
(McKee et al., ’78; Sugden and Fuller, ’91). The
rate of phenylalanine incorporation into total protein is expressed as a percentage of normoxic controls. Contractility was calculated as a percentage
of the initial (T + 15 min) post-equilibration period peak height from the strip chart records.
Time to peak tension and time to 50% relaxation were calculated from the strip chart records.
Relative isometric work was calculated as a function of contraction frequency and relative force at
each frequency from the force-frequency curve.
Means between normoxic and anoxic conditions
were compared using the Mann-Whitney U test.
In all cases, P < 0.05 was considered significant.
RESULTS
Force-frequency study
A force-frequency study was conducted in order
to have a better understanding of the relative
workloads at different frequencies. Although it is
possible to calculate an estimate of the force exerted by the strips, force exerted is expressed in
relative terms since the composition of the turtle
ventricle is not uniform and the amount of spongy
vs. compact tissue may vary from section to section (Brady and Dubkin, ’64). Strips subjected to
normoxic conditions maintained contractility at
approximately 90% of the initial level over the 18
and 24 contractions·min–1 frequencies, but force
fell off rapidly above 24 contractions·min–1 (Fig.
1A). However, anoxic strips tended to lose contractility after 18 contractions·min–1 and fell off
more rapidly than did the strips under normoxic
conditions (Fig. 1A). There was a significant negative correlation between time to peak tension and
increasing frequency under both normoxic and
anoxic conditions (Fig. 1B). Also, there was a significant difference in time to peak tension between
normoxic and anoxic conditions at the 12 and 18
contractions·min–1 frequencies but not the remaining frequencies (Fig. 1B). In this particular experiment, normoxic and anoxic strips were from
different hearts. This may account for the observed difference in time to peak tension as these
preparations may have developed different absolute levels of force. Time to 50% relaxation also
displayed a significant negative correlation with
increasing frequency in both conditions, but there
was no difference between anoxic and normoxic
conditions at any frequency (Fig. 1C).
Protein synthesis and RNA and
protein content
In series 2, three independent experiments were
conducted involving a variety of experimental perturbations with paired preparations. In no case
was there a significant difference in protein content between the control strips run under normoxic conditions, mean value 88.2 ± 4.3 mg
protein·g–1 tissue (N = 19), and the experimental
srips run under anoxic conditions, mean value 89.0
± 4.7 protein mg·g–1 tissue (N = 19). Similarly,
there was no difference in RNA content between
PROTEIN SYNTHESIS IN ANOXIC VENTRICLE STRIPS
277
control strips run under normoxic conditions,
mean value 23.6 ± 1.5 µg RNA·mg–1 protein and
experimental strips run under anoxic conditions,
mean value 22.7 ± 2.0 µg RNA·mg–1 protein. In
series 3 experiments (N = 11), there was no significant difference in protein content of strips subjected to normoxic (86.8 ± 2.0 mg·g–1 tissue) or
anoxic (86.0 ± 2.0 mg·g–1 tissue) conditions. Also,
RNA values did not differ significantly, mean value
24.1 ± 1.3 µg·mg–1 protein for normoxic conditions
and 24.6 ± 1.8 µg·mg–1 protein for anoxic conditions.
Protein synthesis rates were quite variable between experimental groups ranging from 0.37 ±
0.04 to 2.1 ± 0.2 nmol phenylalanine incorporation·mg–1 protein·hr for normoxic conditions
and 0.25 ± 0.02 to 1.38 ± 0.2 nmol phenylalanine
incorporation·mg–1 protein·hr for anoxic conditions.
However, values were extremely consistent within
experimental groups. Consequently, rates of protein synthesis are expressed on a relative basis
in terms of the normoxic control for each experiment in series 2. In experiments involving increasing frequencies (series 3), protein synthesis is
expressed relative to the lowest frequency, 12
contractions·min–1.
Performance and protein synthesis at
set pacing rates
Under anoxic conditions the force of contraction
exerted by the strips decreased by 50% over the 3
hr time period (Fig. 2A). Under anoxic conditions
protein synthesis during the last hour was depressed by 30% when compared to normoxic conditions (Fig. 3, left bar). In an effort to assess if
the decrease during the last hour was due to reduced work or the lack of oxygen per se, electrical stimulation was discontinued during the last
hour. If the strips were not stimulated to contract
for the third hour of either normoxia or anoxia,
contractility was restored to the anoxic strips (Fig.
2B). When the strips were unpaced and, consequently, did zero isometric work, protein synthesis was unchanged from the normoxic state (Fig.
3, middle bar). Also, once a normoxic environment
was restored, the strips regained contractility (Fig.
2C) and protein synthesis remained the same as
under 3 hr of normoxic (Fig. 3, right bar). There
were no significant differences in either time to
peak tension or time to 50% relaxation when
strips under experimental conditions were compared to paired controls (data not shown).
Fig. 1. Force-frequency study of turtle ventricle strips. All
values are means ± SEM, N = 12. Circles represent normoxic
conditions and squares represent anoxic conditions. *P < 0.05
between normoxic and anoxic conditions. A: Force genera-
tion with increasing frequency. Force is calculated as a percentage of the initial force. B: Time to peak tension with increasing frequency. C: Time to 50% relaxation with increasing
frequency.
278
J.R. BAILEY AND W.R. DRIEDZIC
Fig. 3. Protein synthesis in turtle ventricle strips under
various conditions. All values are means ± SEM. N = 6 for
anoxia at 24 bpm, N = 6 for anoxia at 0 bmp, and N = 7 for
normoxia at 24 bpm. The three conditions relate to Figure
1A, 1B, and 1C, respectively. Anoxia at 24 bpm refers to strips
that were forced to contract for 3 hr under anoxic conditions.
Anoxia at 0 bpm refers to strips that were forced to contract
for 2 hr under anoxic conditions and then were unpaced for a
further 1 hr of anoxia. Normoxia at 24 bpm refers to strips
that were forced to contract under anoxic conditions for 2 hr
followed by 1 hr of normoxia. Protein synthesis rate is normalized as a percentage of paired normoxic controls.
Protein synthesis at increased workload
(normoxia vs. anoxia)
Fig. 2. Force generation by turtle ventricle strips under
conditions of normoxia and anoxia. 3H-Phenylalanine was
added at times indicated by arrows. All values are means ±
SEM. A: Strips paced at 24 contractions·min–1 for 3 hr. Circles
represent normoxic conditions and squares represent anoxic
conditions. N = 6 for each data point. B: Strips paced for 2 hr
followed by 1 hr of rest under normoxic or anoxic conditions.
Circles represent normoxic conditions and squares represent
anoxic conditions. N = 6 for each data point. C: Strips paced
at 24 contractions·min–1 for 3 hr. Circles represent strips
paced under normoxic conditions and squares represent strips
paced under anoxic conditions for 2 hr followed by 1 hr of
normoxic conditions. N = 7 for each data point.
The series 2 experiments suggest that oxygen
availability alone is not the only determinant of protein synthesis. In order to further investigate the
relationship between isometric work, anoxia, and
protein synthesis, the effect of changing frequency
was examined. Under normoxic conditions increasing frequency of contraction resulted in a slight decrease, approximately 10%, in the ability of the
strips to maintain contractility when paced at 24
contractions·min–1 but contractility remained relatively unaffected at 12 and 18 contractions·min–1
(Fig. 4A). Under anoxic conditions the strips paced
at 12 contractions·min–1 were able to maintain contractibility, but the strips paced at 18 and 24
contractions·min–1 showed an approximate 50% decrease in force after 3 hr (Fig. 4B). There was no
significant change in either time to peak tension or
time to 50% relaxation over the time period under
either normoxic or anoxic conditions at any pace
rate (data not shown).
PROTEIN SYNTHESIS IN ANOXIC VENTRICLE STRIPS
279
tions·min–1 (Fig. 5). Under anoxic conditions, however, the relationship between frequency and protein synthesis was different in that there was no
change in protein synthesis as a function of contraction frequency (Fig. 5).
DISCUSSION
Performance
Force-frequency study
Above 24 contractions·min–1 turtle heart ventricle strips display a significant negative inotropic isometric twitch response with increasing
frequency. This response is not unique to turtle
hearts as both fish and amphibian hearts show a
similar response (Driedzic and Gesser, ’85, ’88;
Morad and Cleeman, ’87). The reason for this decrease in twitch force remains unclear but is probably related to the poorly developed sacroplasmic
reticulum in these hearts (Santer, ’85; Driedzic
and Gesser, ’88). Consequently, for contraction, calcium must diffuse across the plasma membrane
and then be pumped out against a concentration
gradient before relaxation can occur. It is known
that increasing calcium concentrations can pro-
Fig. 4. Force generation of turtle ventricle strips under
normoxic and anoxic conditions paced at different frequencies. 3H-Phenylalanine was added at times indicated by arrows. All values are means ± SEM. Force generated is
expressed as a percentage of initial value. Squares represent
12 contractions·min–1, circles represent 18 contractions·min–1,
and triangles represent 24 contractions·min–1. A: Normoxic conditions. N = 5 for each data point. B: Anoxic conditions. N = 6
for each data point. Both the 18 and 24 contractions·min–1
frequencies show a significant negative correlation with time.
Under normoxic conditions protein synthesis increased with the increase in frequency. There was
a significant (P < 0.05) linear correlation such that
the ratio of protein synthesis at 24 contractions·min–1 was 45% higher than at 12 contrac-
Fig. 5. Protein synthesis in turtle ventricle strips paced
at different frequencies under normoxic and anoxic conditions.
All values are means ± SEM. Circles represent normoxic conditions (N = 5) and squares represent anoxic conditions (N =
6). Protein synthesis is normalized as a percentage of the
synthetic rate at 12 contractions·min–1. Synthesis under
normoxic conditions shows a significant positive correlation
with increasing frequency.
280
J.R. BAILEY AND W.R. DRIEDZIC
tect against this loss of contractility at elevated
frequencies (Driedzic and Gesser, ’85), which indicates that the limiting step is the inward diffusion of calcium. It is quite likely that a similar
situation exists in the turtle heart since the response to increasing frequency is similar. The important finding here is that the force-frequency
curve is steeper under anoxic conditions than under normoxic conditions. The reason for this is unclear, but it is possible that the inward Ca2+ flux
is compromised under anoxia as a result of a
depencence on anaerobic metabolism and a consequent fall in intracellular pH.
Long-term anoxia
In both normoxic and anoxic preparations, there
is a negative inotropic effect associated with increasing frequency of contraction and length of the experimental period, but this effect is more pronounced
under anoxic conditions. Strips were able to regain
contractility and force generation very quickly once
normoxic conditions had been restored, indicating
that the 3 hr of anoxia did not result in a permanent loss of functionality. In addition, ventricle strips
were able to regain contractility after 3 hr of anoxia
when the work component was removed.
Under anoxic conditions both the 18 and 24
contractions·min–1 frequencies resulted in an approximate 50% decrease in the ability of the strips
to develop force over the experimental period (Figs.
2, 4). The energy for contraction under anoxic conditions is supplied by anaerobic glucose metabolism
with a concomitant buildup of lactic acid (Reeves,
’63) and a lowering of intracellular pH (Wasser et
al., ’90). These factors in turn could influence the
ability of the strips to maintain contractility, although in isolated, perfused heart preparations
anoxia alone is not sufficient to produce a decrease
in cardiac output (Jackson et al., ’95; Bailey and
Driedzic, ’96). This preparation’s specific response
may relate to a better washout of H+ ions in perfused hearts than in ventricle strips.
Anoxia and set pacing rates
In the initial experiment, in which strips were
stimulated at a frequency of 24 contractions·min–1,
the rate of protein synthesis decreased by about 30%
in association with a decrease in force development. However, protein synthesis did not decrease
when the work component was removed, even
though the strips were still in an anoxic environment. These data show that anoxia per se is not
the sole determinant in the decreased rate of protein synthesis since the ventricle strips can main-
tain protein synthesis under anoxic conditions
when the work component is removed. In contrast,
rat hearts are unable to maintain protein synthesis under anoxic conditions when the work component is removed by arresting these hearts with
tetrodotoxin (Jefferson et al., ’71). When normoxic
conditions were restored to the turtle ventricle
strip preparation, both contractility and protein
synthetic rates were restored to control levels.
This indicates that the decrease in protein synthesis was due to an acute regulatory mechanism
and not to a deterioration of the protein synthetic
machinery during the period of anoxia. Unlike the
isolated perfused heart preparation (Bailey and
Driedzic, ’96), there was no drop in cellular RNA
levels, so the decrease in protein synthesis cannot be attributed to an impairment at that gross
a level. It is known that a fall in intracellular pH
may adversely affect protein synthesis in mammalian hearts (Fuller et al., ’89; Sugden and
Fuller, ’91) and that anoxia does result in a decrease in intracellular pH in working turtle hearts
(Wasser et al., ’90; Jackson et al., ’95). We suggest that intracellular pH may be the control factor in protein synthesis in these experiments. It
has long been known that the turtle heart can
cycle large quantities of glucose through glycolysis to maintain energy production (Reeves, ’63)
and that ATP levels remain relatively constant
over at least 4 hr of anoxia (Wasser et al., ’90).
Therefore, we do not view ATP itself as an important intracellular regulator under these conditions. If there is no mechanical work being done,
the degree of acid production is probably reduced
since energetic requirements would be significantly reduced when the strips are in a noncontractile state. The restoration of normoxic
conditions could result in a rapid resetting of intracellular pH and, thus, a restoration of protein
synthesis. These experiments indicate that there
is a complex interaction between the variables of
oxygen availability and mechanical work on protein synthesis in these preparations.
Anoxia and increasing workload
The rate of protein synthesis, as measured by
phenylalanine incorporation, displays a positive
correlation with frequency of contraction in normoxic strips. We are not able to quantitate work
since there is not a common set point for normalization for all strips, but we can provide a qualitative estimate of work by taking into account the
differences in frequency and the impact of contraction frequency alone on force development.
PROTEIN SYNTHESIS IN ANOXIC VENTRICLE STRIPS
These data are presented in Figure 6A and B. The
12 contractions·min–1 frequency is set to 100%, the
18 contractions·min–1 frequency is multiplied by
1.5, and the 24 contractions·min–1 frequency is
multiplied by 2. Both of the latter are then adjusted for the decrease in force observed in the
force-frequency study. This analysis reveals that
the strips paced at the higher frequencies do more
work than those paced at the lowest frequency
(Fig. 6A). Therefore, we are confident in stating
that under normoxic conditions protein synthesis
281
increases in concert with an increase in work. This
coupling of work and protein synthesis also has been
reported for rat heart (Jefferson et al., ’71) and for
fish heart (Houlihan et al., ’88) and may be a common principle applicable to all vertebrates.
Under anoxic conditions an increase in contraction frequency did not result in an increase in protein synthesis. The qualitative analysis of work
suggests that strips paced at 18 and 24 contractions·min–1 under anoxic conditions do less work
than the strips paced at 12 contractions·min–1 (Fig.
6B). If protein synthesis and work were coupled
under anoxic conditions, there should be a reduction in protein synthesis under anoxia, but there
was no change from the protein synthesis rate
observed at the 12 contractions·min–1 frequency.
These data indicate that work and protein synthesis become uncoupled under anoxia, and
thus, control mechanisms may differ under the
two conditions.
In summary, in ventricle strips prepared from
turtle hearts, work and protein synthesis are
coupled under normoxic conditions. Under anoxic
conditions protein synthesis is depressed and,
moreover, protein synthesis and work become uncoupled. It is known that intracellular pH is depressed under anoxic conditions in turtle heart,
and this may mediate the uncoupling of work and
protein synthesis. At present, work to elucidate
the effects of acidotic conditions on protein synthesis in turtle heart is being carried out in this
laboratory.
ACKNOWLEDGMENTS
This study was supported by the Natural Sciences and Engineering Research Council of Canada and the New Brunswick Heart and Stroke
Foundation.
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