Force development, energy state and ATP production of cardiac muscle from turtles and trout during normoxia and severe hypoxia
Department of Zoophysiology, Institute of Biological Sciences, University of Aarhus, DK 8000 Aarhus, Denmark
* Author for correspondence (e-mail: hans.gesser{at}biology.au.dk)
Accepted 10 March 2004
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Summary |
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Key words: oxygen consumption, lactate production, cellular energy state, phosphorylation potential, cost of contraction, twitch force, metabolic depression, Trachemys scripta, Oncorhynchus mykiss
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Introduction |
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The ability of cardiac muscle from turtles to tolerate anoxia is associated
with a superior maintenance of cardiac cellular energy state relative to most
other species of ectothermic vertebrates
(Wasser et al., 1990;
Jackson et al., 1995
;
Hartmund and Gesser, 1996
).
Furthermore, it seems that the myocardium preserves most contractile activity
during severe hypoxia/anoxia at low workload
(Wasser et al., 1990
;
Jackson et al., 1995
;
Hartmund and Gesser, 1996
).
This ability has been related to a high glycolytic capacity
(Bing et al., 1972
) and is
reflected in a high anaerobic metabolic capacity relative to aerobic capacity
(Christensen et al., 1994
).
Other studies, however, indicate that neither the glycolytic nor the
mechanical capacities of turtle hearts are exceptional, and it has been
suggested that their anoxia tolerance relates primarily to downregulation of
metabolic requirements so that cellular energy state is protected
(Arthur et al., 1997
;
Farrell et al., 1994
).
Studies of perfused hearts from turtle and trout indicate that work output
is achieved at similar rates of ATP consumption during full oxygenation and
severe hypoxia (Reeves, 1963b;
Arthur et al., 1992
,
1997
). However, recent studies
on rats, guinea pigs and frogs raised the possibility that the energetic cost
of developing tension during hypoxia is lower in hypoxia-tolerant vertebrates
(Joseph et al., 2000
). In
addition to inherent differences in the efficiency of the contractile system,
a low cost of contraction (i.e. overall cellular ATP consumption/force
developed) would also occur if the metabolic demands of non-contractile
processes are suppressed. This seems to be the case during anoxia in rabbit
papillary muscle, where cellular ATP production seemed insufficient to cover
energy-demanding processes other than contractility
(Mast and Elzinga, 1990
). As
metabolism of whole animals and tissue culture of turtles is markedly reduced
during anoxia (Jackson, 1968
;
Hochachka et al., 1996
;
Lutz and Nilsson, 1997
), it is
an interesting possibility that the apparent cost of cardiac contraction is
also reduced, either as a result of changes in the economy of the contractile
system or through a reduced cost of non-contractile processes.
Here, we investigate the relationships between cellular energy production,
force development and cost of non-contractile processes in ventricular muscle
strips from the turtle Trachemys scripta. Our preparation allowed for
simultaneous measurements of metabolism and force production in resting and
paced ventricular strips. Mechanical performance was assessed as isometric
twitch force, and cellular ATP production was derived from recordings of
oxygen consumption, lactate production and concentrations of cellular
high-energy phosphates. The preparations were exposed to adrenaline during
hypoxic recordings because substantial increases in plasma catecholamines have
been observed in both turtle and trout subjected to hypoxia/anoxia
(Wasser and Jackson, 1991;
Keiver et al., 1992
;
Reid and Perry, 2003
).
The results from the hypoxia-tolerant turtle were evaluated relative to a
parallel series of experiments performed on cardiac muscle from more
hypoxia-sensitive rainbow trout (Oncorhynchus mykiss). Relative to
the turtle heart, the trout heart has an inferior ability to maintain force
development and cellular energy state during hypoxia
(Arthur et al., 1992;
Hartmund and Gesser,
1996
).
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Materials and methods |
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Myocardial preparations
Animals were decapitated, and the hearts were rapidly excised and
transferred to an ice-cold physiological solution containing (in mmol
l-1) 125 NaCl, 2.5 KCl, 0.94 MgSO4, 1
NaH2PO4, 1.2 CaCl2, 10 glucose and 30 and 15
NaHCO3 for turtle and trout, respectively.
From each ventricle, 1 mm-thick ring-shaped preparations were obtained by
transverse cuts. The preparations were weighed, mounted on two hooks and
immersed in physiological solution continuously gassed with a mixture of 88%
O2, 2% CO2 and 10% N2 for turtle and 89%
O2, 1% CO2 and 10% N2 for trout. pH of the
physiological solution was 7.7 in both the turtle experiments at 20°C
and in the trout experiments at 15°C. This pH value accords reasonably
with in vivo recordings from freshwater turtles and trout at the same
temperatures (Nicol et al.,
1983
; Julio et al.,
1998
).
Muscular contractions were elicited by a Grass SD 9 stimulator (Quincy, MA,
USA), which delivered electrical square pulses of 5 ms and a voltage 50% above
that eliciting maximal twitch force. The preparation was stretched with a
micrometer screw to produce 90% of maximal twitch force, as this would
not overstretch the preparations while leaving almost all myosin heads
active.
Experimental setup
The experimental protocol was designed to evaluate the effects of severe
hypoxia on the energetic state, ATP production and its relation to force
production in ventricular preparations from turtle and trout in two
experimental series. In series 1, intracellular concentrations of high-energy
phosphates and lactate were measured. In series 2, ATP production was
calculated from the oxygen consumption during oxygenation and from both oxygen
consumption and lactate production during the period of severe hypoxia.
Series 1
This series was designed to measure tissue content of ATP, ADP, AMP,
phosphocreatine (PCr), creatine (Cr) and lactate together with twitch force.
Six ring-shaped preparations were mounted in parallel setups as open
ellipsoids around two hooks and immersed in 50 ml physiological solution in
thermostatted baths. The upper hook consisted of a thin glass rod connected to
a force transducer (Fort 10; World Precision Instruments, UK), while one of
two platinum electrodes used for pacing was used as the lower hook.
Series 2
The experimental setup for simultaneous measurement of twitch force, oxygen
consumption and lactate production was similar to that described by Kalinin
and Gesser (2002). Briefly,
ventricular rings were mounted around two hooks of stainless steel in a
tube-formed glass chamber (diameter of 12 mm and a volume of 2.56 ml) that
could be sealed with a glass stopper. The lower end of the ring-shaped
preparation was mounted on a stainless steel hook and the upper end was
mounted on a stainless steel rod attached to a force transducer through a 1 mm
hole in the stopper. Electrical stimulations were obtained from two
chloridized silver electrodes situated on opposite sides of the preparation
and connected to the stimulator through the stopper. To avoid polarization,
the polarity of the electrodes was alternated between pulses. A peristaltic
pump circulated physiological solution at a rate of 3 ml min-1
between the chamber and a reservoir through two stainless steel tubes
penetrating the stopper. The reservoir of 15 ml was continuously gassed with
the desired gas mixture. Oxygen tension in the chamber was recorded with an
oxygen electrode (Radiometer, E5046) placed horizontally so the tip was just
inside the chamber. The chamber, the oxygen electrode and the reservoir were
thermostatted to the desired temperature and the chamber solution was
continuously stirred using a glass-covered magnetic stir bar.
Signals from the force transducers and oxygen electrode were recorded with a Biopac MP100 data acquisition system (Biopac Systems, Inc., Goleta, CA, USA) at 50 Hz. At the experimental stimulation rate (30 min-1) the twitch duration was approximately 2 s and 1 s for the turtle and trout preparation, respectively.
Experimental protocol
The experimental protocol and time course of series 1 and 2 were chosen to
obtain measurement of twitch force, energy production and energy state after
stabilisation under oxygenation and severe hypoxia, respectively.
Series 1
When the ventricular rings had been mounted, they were stretched to produce
90% of maximal twitch force and allowed 30 min for twitch force to stabilise.
Then, stimulation was switched off for three preparations while stimulation
was continued at 30 min-1 for three other preparations. Resting
heart rates of turtle and trout have been reported to be 25
min-1 for turtles and rainbow trout at 22°C and 15°C,
respectively (Hicks and Farrell,
2000
; Altimiras and Larsen,
2000
). In the present study, a rate of 30 min-1 was
chosen to obtain clearly increased energy consumption in the force-developing
preparations relative to that of the resting preparations. After 30 min at
high oxygenation, one contracting and one resting preparation were clamped
with aluminium pliers, pre-cooled in liquid nitrogen and the samples were
stored at 80°C. Immediately thereafter, stimulation frequency was
reduced to 12 min-1 and nitrogen was exchanged for oxygen, so that
the physiological solution in all baths was made severely hypoxic. After 30
min of severe hypoxia, 10 µmol l-1 adrenaline was added to the
baths. While this concentration of adrenaline is likely to be well above the
in vivo range for both species, it gives a well-defined and close to
maximal adrenergic stimulation of twitch force
(Nielsen and Gesser, 2001
). 10
min after addition of adrenaline, one contracting and one resting preparation
were sampled and stimulation frequency of the one remaining contracting
preparation was elevated to 30 min-1. After an additional 30 min
period of continued severe hypoxia, the two last preparations were sampled
(Fig. 1).
|
Series 2
In the experiments for determination of oxygen consumption, lactate
production and twitch force, the preparations were run as in series 1 with
respect to stimulation frequency, stretch, adrenaline exposure and gas
mixtures (Fig. 1). Here, every
other preparation was stimulated to contraction or left at rest, respectively.
Following the initial stabilization of twitch force, recirculation of chamber
solution was stopped for 30 min during which oxygen consumption was measured
from the gradual decline in oxygen tension. Then circulation was resumed with
the hypoxic solution, which resulted in a chamber oxygen partial pressure
(PO2) of 1.6 kPa within 1015 min.
After 30 min, 10 µmol l-1 adrenaline was added to the reservoir
and, 10 min later, circulation was stopped again for 30 min to allow for
recordings of oxygen consumption and lactate accumulation during hypoxia
(Fig. 1). The reported values
of respiration have been corrected for background changes in oxygen tension,
which were recorded in the absence of tissue in the chamber, with stimulation
either set at 30 min-1 or turned off. During full oxygenation, the
decrease in oxygen tension in the presence of preparation had to be corrected
by maximally 10% and during severe hypoxia by maximally 27%.
Lactate production was determined from the increase in lactate in the tissue and in the chamber solution over the 30 min of hypoxia. Tissue lactate content was assessed on the basis of the values recorded from the samples in series 1, while the increase in chamber solution lactate provided the lactate released from the preparation (Fig. 1). Tissue and solution samples were stored at 80°C until determination of lactate.
Mechanical activity
In the present experiments, mechanical activity was recorded in terms of
isometric twitch force, which was used as an indicator of heart function.
Thus, a previous study on turtle cardiac muscle showed that the relative
effect of anoxia on isometric twitch force and power output was almost
identical in preparations that were allowed to shorten after attaining 85% of
time to peak force (Shi and Jackson,
1997). The ring preparation was considered suitable for recordings
of energy state, energy production and twitch force. Its form, however, makes
it less suited for refined recordings of contractile performance. We aimed at
having the thickness and cross-section area of the preparations as constant as
possible so that twitch force given in mN could be assumed to be proportional
to twitch force related to cross-section area.
As an alternative, a time-tension integral has previously been used
(Mast and Elzinga, 1990). We
found, however, a significant correlation between the effects of severe
hypoxia on twitch force and the time-tension integral for both turtle and
trout preparations (data not shown).
Biochemical measurements
Myocardial high-energy phosphates were measured using HPLC
(Bøtker et al., 1994).
Briefly, a 3060 mg piece of ventricle was homogenised in 1.6 ml of 0.42
mol l-1 perchloric acid in a glass-to-glass homogeniser. The
homogenate was then centrifuged for 10 min at 3400 g, and the
supernatant was separated into two 200 µl samples. The sample used for
measurement of creatine compounds (phosphocreatine and creatine) was
neutralised with 100 µl KOH (1 mol l-1), and the sample used for
subsequent measurement of adenylates (ATP, ADP and AMP) was neutralised with
100 µl of KHCO3 (2 mol l-1) and Tris (0.1 mol
l-1). After neutralization, both samples were kept on ice for 10
min to ensure precipitation of perchlorate. The neutralized portions were then
centrifuged for 5 min (3400 g) and the supernatant stored at
80°C until further analysis. Creatine compounds and adenylate
compounds were separated using HPLC (Waters; Minneapolis, MN, USA) with a 10
cm Crompack C18 microsphere-column of 3 µm particle size (Varian, Palo
Alto, CA, USA). Creatine compounds were measured at a wavelength of 210 nm
using a mobile phase of aqueous buffer containing 0.02 mol l-1
KH2PO4 and 2.3 mmol l-1 tetrabutyl ammonium
hydrogen sulphate (TBAHS) run at 1.5 ml min-1. Adenosine
nucleotides were measured at a wavelength of 254 nm using a mobile phase of
25% methanol and 75% aqueous buffer containing 0.06 mol l-1
KH2PO4 and 0.011 mol l-1 TBAHS run at 1 ml
min-1.
Muscle lactate concentration was measured from the same homogenates that
were used for measurements of adenosine products. Lactate concentration of
homogenates, reservoir solution (initial) and chamber (final) were determined
by transforming it quantitatively along the lactatedehydrogenase and
pyruvateamino acid transaminase catalysed reactions and recording the
amount of NADH formed spectrophotometrically
(Lowry and Passonneau,
1972).
Energy state
Gibb's free energy associated with hydrolysis of ATP
(GATP) varies with the phosphorylation potential
[ATP/(ADPxPi)], and Meyer
(1988
) argued that
phosphorylation potential could be estimated by the ratio of
PCr/Cr2, where PCr and Cr represent tissue concentrations of
phosphocreatine and creatine, respectively. As hydrogen ions participate in
the creatine kinase reaction, changes in
GATP may be
confounded by changes in intracellular pH, but this problem seems to be of
little importance in the type of preparations used in the present study
(Hartmund and Gesser,
1996
).
ATP production
Based on previous studies (Arthur et
al., 1997; Hansen et al.,
2002
), ATP production was assumed to be exclusively aerobic during
full oxygenation, whereas both lactate production and oxygen consumption were
considered during severe hypoxia. In our calculation of ATP production, we
assumed that 1 mole of O2 results in the formation of 6 moles of
ATP (Reeves, 1963b
;
Ferguson, 1987
;
Mast and Elzinga, 1990
) and
that 1 mole of lactate equals 1.5 moles of ATP. The lactate/ATP conversion
factor is based on the assumption that lactate is derived from glycogen, which
seems to be the case for hearts from turtle at low workloads
(Reeves, 1963a
). ATP
production was related to twitch force (reported in mN).
Statistics
Differences in high-energy phosphates or muscle lactate due to treatment
were examined using a one-way analysis of variance (ANOVA), where a post
hoc Fisher LSD test was employed to identify significant differences. If
data did not fulfil the requirement of normal distribution, a one-way ANOVA on
ranks was employed using an SNK post hoc test to identify
differences. ANOVAs were run separately for turtle and trout, and
species-specific differences were examined using Student's t-test.
Student's t-test was also applied to identify differences in ATP
production, twitch force and the relation between these two parameters. In all
cases, P-values below 0.05 were regarded as significant and all
results are presented as means ± S.E.M.
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Results |
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Turtle
As seen in Table 1, neither
stimulation nor hypoxia affected myocardial concentrations of ATP, ADP or
total adenylates (Adtot). During oxygenated and hypoxic conditions,
stimulation caused a decline in PCr and the phosphorylation potential
(PCr/Cr2). Hypoxia resulted in decreases in PCr, PCr/Cr2
and in the sum of high-energy phosphates (HEPtot), and in
stimulated hypoxic preparations this resulted in increased levels of Cr.
During hypoxia, PCr and PCr/Cr2 decreased to a new stable level in
both resting and stimulated preparations. As a result, the energy state of the
preparations did not change significantly between 40 and 70 min of hypoxia.
Muscle lactate levels increased under hypoxia and reached significantly higher
levels in the stimulated than in the resting preparations. Irrespective of
stimulation, the increases in muscle lactate levelled off with no significant
change between 40 and 70 min of hypoxia
(Table 1).
Trout
In oxygenated trout preparations, stimulation reduced phosphorylation
potential without significantly affecting other parameters
(Table 1). During hypoxia, ATP
and Adtot levels were stable in resting preparations even though
ADP increased. PCr fell to 35% and HEPtot to 50% of the levels
during oxygenated conditions, and the decrease in PCr was attended by an
equimolar increase in Cr. The reduction in energy state during hypoxia was
exacerbated by stimulation, as ATP and Adtot fell while ADP
increased. Furthermore, PCr fell to 12% of the oxygenated value and, although
Cr increased, there was a decrease in Crtot. These changes were
associated with a marked reduction in phosphorylation potential and
HEPtot, which fell to 25% of initial values. Energy state in both
resting and stimulated preparations attained a new stable level after 40 min
with no changes occurring between 40 and 70 min of severe hypoxia.
Myocardial lactate concentration of resting preparations only increased slightly during hypoxia. Stimulation did not affect muscle lactate concentration under oxygenation but led to an approximate doubling of muscle lactate in hypoxia. Muscle lactate stabilized with no significant change between 40 and 70 min of hypoxia.
Turtle vs trout
Crtot, PCr and HEPtot were higher for trout than for
turtle during oxygenated conditions. However, PCr and HEPtot fell
to the same level as in turtle during hypoxia in resting preparations and
became even lower than in turtle in stimulated hypoxic preparations. The
larger changes in these parameters for trout were accompanied by significantly
lower phosphorylation potentials of the hypoxic trout preparation at both rest
and stimulation (Table 1).
The changes in muscle lactate due to stimulation and hypoxia were qualitatively similar for both species, although trout had significantly higher muscle lactate concentrations during oxygenation.
Oxygen consumption, lactate production and force
Stimulation at 30 min-1 led to a 23-fold increase in
oxygen consumption in both species. During severe hypoxia, anaerobic ATP
production was dominant, and oxygen consumption was reduced to less than 10%
of the aerobic level in both resting and contracting preparations from both
species (Fig. 2A).Stimulation
increased lactate production during severe hypoxia in both species
(Fig. 2B). In both turtle and
trout, hypoxia reduced ATP turnover to 3035% of the values during
oxygenation for resting preparations and to
25% for stimulated
preparations (Fig. 2C). There
were no significant differences between turtle and trout in either oxygen
consumption or lactate production in any of the experimental situations so
overall ATP production was similar in both species
(Fig. 2C). It should be noted
that lactate production was calculated exclusively on the basis of the change
in chamber solution, as tissue lactate was unchanged during the recording
period (Table 1).
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When compared with ATP production, twitch force is represented by the
average obtained over the 30 min when oxygen consumption and lactate
production were recorded (Fig.
1). All recordings during hypoxia were performed in the presence
of 10 µmol l-1 adrenaline that had been added 10 min before the
start of the 30 min period of recording. Within these 10 min, hypoxic twitch
force increased by 31±9% and 104±26% for turtle and trout,
respectively. Subsequently, twitch levelled off and then progressively
decreased during the following 30 min of hypoxia
(Fig. 1). As preparation
thickness and cross-section area were kept as constant as possible, the force
values recorded were assumed to be proportional to force related to
cross-section area. This assumption accords with the finding that twitch force
in the present study (Fig. 2)
and twitch force related to the estimated cross-section area (mN
mm-2) in a previous study on identical ring preparations under the
same control conditions (Kalinin and
Gesser, 2002) showed a similar insignificant tendency to be higher
for trout than for turtle.
In turtle, hypoxia reduced average twitch force by one-third, although this was not significant. In trout, average twitch force decreased significantly by two-thirds under hypoxia, and the change in twitch force due to hypoxia was significantly larger for trout than for turtle (Fig. 2D). The changes in twitch force during hypoxia in series 2 did not differ significantly from those recorded in series 1. During oxygenation, ATP production, twitch force and twitch force/ATP production ratio were similar in both species. However, during hypoxia, twitch force decreased less relative to ATP production in turtle and, consequently, the economy of force development appeared to increase as the twitch force/ATP production ratio increased more than twofold (Fig. 2E). In trout, the changes in ATP production and twitch force were of similar magnitude, leaving the twitch force/ATP production ratio, and thus the apparent cost of contraction, unchanged.
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Discussion |
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Energy metabolites and twitch force
During severe hypoxia, cardiac ATP levels were generally well defended and
only decreased significantly in stimulated hypoxic trout preparations. There
was, however, a reduction in the energy state (estimated from
PCr/Cr2), as observed previously in cardiac strips and in
situ perfused hearts (Wasser et al.,
1990; Arthur et al.,
1992
; Jackson et al.,
1995
; Hartmund and Gesser,
1996
). The reduction in energetic state during hypoxia was
considerably smaller in turtle than in trout but, for both species, energy
state stabilised at a higher level during rest than during stimulation. The
larger reduction in cellular energy state of trout not only indicates a
greater drop in
GATP (free energy liberation of ATP
hydrolysis) but also a greater increase in the cellular concentration of
inorganic phosphates, [Pi]. Furthermore, trout cardiac muscle has a
higher concentration of creatine compounds than turtle
(Christensen et al., 1994
;
Hartmund and Gesser, 1996
)
that allows for larger increases in [Pi], which may explain the
larger reduction of twitch force during hypoxia in trout compared with turtle.
Increased [Pi] has, together with intracellular acidosis, been
suggested as the main cause of contractile failure when cellular energy
production is compromised (e.g. Allen et
al., 1985
; Godt and Nosek,
1989
; Arthur et al.,
1992
; Hartmund and Gesser,
1996
), but this effect may vary among species and may depend on
energy state (Jensen and Gesser,
1999
).
Cellular energy production and twitch force
After 40 min of severe hypoxia, ventricular ATP production had decreased to
similar rates in the two species, but the stabilisation occurred at a higher
cellular energy state in the turtle. Hence, a balance seemed to be attained at
which a similar rate of energy production supported a higher energy state in
turtle than in trout cardiac muscle. While it is unclear how this difference
between turtle and trout cardiac muscle is achieved, overall ATP consumption
during the initial period of anoxia decreases faster in turtle than in trout
(Hartmund and Gesser, 1996).
The ability of the turtle heart to balance ATP production and consumption
during hypoxia with small reductions in energetic state may be due to their
superior ability to downregulate metabolic requirements (e.g.
Lutz and Nilsson, 1997
;
Jackson, 2000
,
2002
). Indeed, metabolic rate
of anoxic whole turtles, turtle brain slices and turtle hepatocytes is reduced
to 1040% of normoxic levels
(Jackson, 1968
;
Hochachka et al., 1996
;
Lutz and Nilsson, 1997
). A
metabolic depression resembling that found in other tissue also seems to occur
in resting turtle cardiac muscle, where the ATP turnover fell to 35% of
oxygenated values. However, hypoxic metabolic depression is, at least
regarding cardiac muscle, not unique for turtle as we recorded similar
reductions in resting metabolic rate of hypoxic trout preparations.
While ATP production in hypoxic stimulated preparations fell by the same
proportions in turtle and trout, twitch force decreased more in trout.
Consequently, cardiac force development relative to ATP production was
markedly increased during hypoxia for turtles while essentially unchanged in
trout. The cost of contraction is estimated on the basis of the total rate of
ATP production and the force developed. Suppression of energy-demanding
processes not related to contraction would, however, enlarge the fraction of
ATP production available for force development. Thus, given the decreased ATP
turnover at rest, the cost of contraction should appear to decrease. The
results from turtle myocardium accord qualitatively with this, although the
increase in the force/ATP production ratio seems too large to be explained
exclusively by suppression of non-contractile processes. By contrast, the
force to ATP ratio was unchanged in trout despite a considerable decrease in
ATP consumption of resting preparations. It seems, therefore, that costs of
contractile processes increase in trout. In the estimate of an apparent cost
of contraction, overall ATP production of contracting preparations was used
directly. It may seem more appropriate to use the ATP consumption remaining
after subtraction of that in resting preparations. However, considering the
interdependence of different cellular processes, the assumption that ATP
production due to contractility and other processes are additive is, in all
likelihood, a crude over-simplification. For instance, a substantial part of
the oxygen consumption in resting cardiac cells probably supports a proton
leak (Mortensen and Gesser,
1999), which is transferred to ATP production in contracting cells
(Rolfe and Brand, 1997
).
In accordance with our results on turtles, Mast and Elzinga
(1990) reported an increased
ratio between force and overall ATP consumption in anoxic rabbit papillary
muscle due to an apparently complete suppression of non-contractile processes.
The increased force to ATP consumption ratio in turtle is, however, not fully
explained by suppressed non-contractile processes. Alternatively, a decrease
in the apparent cost of muscle contraction may reside in the contractile
system itself. It was recently reported that the cost of force development
increased under hypoxia in isolated hypoxia-sensitive cardiac muscle from rat
while it remained unchanged in the more hypoxia-tolerant cardiac muscle from
guinea pig and frog (Joseph et al.,
2000
). Decreases in the efficiency of the contractile system
during hypoxia appear immediately easier to conceive than increases, and it
should be stressed that there does not seem to exist any report on improved
efficiency of cross bridge formation during hypoxia. Given the findings of the
present study, it would be of interest to examine force development and rate
of ATP hydrolysis in skinned myocardial tissue of turtle to evaluate cost of
contraction directly.
Apart from the contractile system, differences in energy economy may reside
in the excitationcontraction coupling. The excitationcontraction
coupling in contracting cardiac muscle may account for a considerable fraction
of energy consumption, which in mammals amounts to 30% of energy turnover
(Kammermeier, 1997
).
Furthermore, the relative importance of Ca2+ regulation by the
sarcoplasmic reticulum and sarcolemmal Ca2+ transport varies
substantially among ectothermic vertebrates
(Tibbits et al., 1991
;
Driedzic and Gesser, 1994
).
Thus, possible differences in the excitationcontraction coupling
between turtle and trout myocardium may influence the economy of the
contracting myocardium.
Our finding of a decreased apparent cost of contraction expressed as
increased ratio of force development to ATP production in the turtle cardiac
muscle during hypoxia deviates from previous studies on perfused hearts of
turtle and trout in which work output relative to ATP formation was similar
under severe hypoxia and full oxygenation
(Reeves, 1963b; Arthur et al.,
1992
,
1997
). An explanation of the
differences found between previous studies as well as between turtle and trout
pertains to the assumptions used in the assessment of ATP production. Thus,
comparing ATP regeneration during full oxygenation and severe hypoxia involves
a risk that oxidative ATP formation is overestimated in fully oxygenated
preparations, as a proton leak across the inner mitochondrial membrane has
been reported to account for a substantial fraction of oxygen consumption in
isolated trout atrial myocytes (Mortensen
and Gesser, 1999
). This fraction is likely to be lowered during
severe hypoxia, as indicated by mitochondrial experiments
(Gnaiger et al.,
2000
). The possibility therefore exists that an overestimation of
ATP production during oxygenation would exaggerate the apparent improvement of
economy of contraction observed for turtle during hypoxia. By a similar
argument, the economy of contraction would not stay unchanged but would be
reduced for trout during hypoxia. While a comparison of turtle and trout would
be obscured by a difference in mitochondrial proton leak, no evidence for such
a species-related difference appeared, as turtle and trout preparations had
similar force to ATP ratios during oxygenation. Thus, the economy of
contraction during hypoxia differs between turtle and trout, and it seems
likely that the ratio between force and ATP production does, indeed, increase
in turtle.
Due to differences in living and acclimation temperatures, the turtle and
trout experiments were carried out at 20 and 15°C, respectively. In a
previous study on isolated trout cardiac muscle, twitch force and energy state
were found to attain similar values after 60 min of anoxia at 10 and 20°C
(Hartmund and Gesser, 1992).
Thus, the qualitative differences between the two preparations should remain,
although the 5°C difference in experimental temperature may have small
quantitative effects.
In conclusion, this study raises the interesting possibility that turtle cardiac muscle relative to cardiac muscle of other species protects both the cellular energy state and force development during severe hypoxia in an energetically more efficient way. It is unclear how this relates to a downregulation of non-contractile energy demand, as the decrease in resting energy production was, somewhat surprisingly, about the same for trout as for turtle myocardium. However, a superior energy economy and energetic state may contribute to the ability of turtle cardiac muscle to maintain mechanical activity over long periods of severe hypoxia/anoxia.
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Acknowledgments |
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References |
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