Regulation of cardiac contractility in a cold stenothermal fish, the burbot Lota lota L.
University of Joensuu, Department of Biology, PO Box 111, 80101 Joensuu, Finland
* Author for correspondence (e-mail: matti.vornanen{at}joensuu.fi )
Accepted 19 March 2002
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Summary |
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Key words: ryanodine sensitivity, restitution, rest-potentiation, myofibrillar ATPase, heart rate, burbot, Lota lota
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Introduction |
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In contrast to eurythermal fish species, relatively little is known about
cardiac contractile activity and Ca2+ management in cold
stenothermal fish, such as the burbot. Indeed, we know of no studies that
examine cardiac function in burbot. This is surprising as burbot are a
commercially valuable fish species, having the widest distribution of all
freshwater fish, ranging from the British Isles across Europe and Asia to the
Bering Strait and from Alaska across the North American continent to the
Atlantic coast (McPhail,
1997). Burbot are benthic, omnivorous fish that prefer cold waters
and are seldom found at temperatures above 13 °C
(Edsall et al., 1993
;
Carl, 1995
;
Pääkkönen and
Marjomäki, 2000
). In summer, burbot occupy hypolimnion of the
lakes and feed on invertebrates and fish. Indeed, burbot are more active and
better able to catch prey in winter than in summer. Although burbot are
piscivorous they are relatively poor swimmers and their metabolic rate is low
(Pääkkönen and
Lyytikäinen, 2000
). Spawning and embryonic development of
burbot occur from January to March under ice at water temperatures below 4
°C. Together, these features suggest that burbot is a cold stenothermal
species and therefore a suitable model animal for our study.
In this paper we report our investigations into the temperature-dependence of cardiac function and Ca2+ regulation of contractility in burbot atrium and ventricle, to test whether temperature adaptation in a cold stenothermal teleost involves similar functional changes and the same subcellular mechanisms that are typical for cold-acclimated eurythermal fish species.
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Materials and methods |
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Contractile properties
Experiments were conducted on perfused spontaneously beating whole hearts,
and atrial and ventricular strips. Fish were stunned by a blow to the head,
the spine was cut and the heart was carefully removed. Atrial (16.4±1.7
mg wet mass) and ventricular strips (27.2±1.7 mg wet mass) were mounted
on the bottom of a tissue bath with small needle electrodes and connected with
small stainless steel hooks and braided silk threads to the force transducers
(Grass FT03). A stimulus electrode was positioned in the tissue bath near the
preparations. The duration of the stimulus pulse was 10 ms and voltage 60 V.
The tissue bath was filled with 10 ml of oxygenated (100% O2)
physiological solution containing (in mmol l-1): NaCl, 150; KCl,
5.4; CaCl2, 1.8; MgCl2, 10; Hepes, 10; glucose 10; pH
7.64 at 1 °C. The temperature of the saline was regulated to 1±0.5
°C with the aid of a recirculating water bath. Some experiments were also
done at 7±0.5 °C (pH 7.57).
At the beginning of the experiment, the muscles were stretched stepwise to the length at which the developed force was close to maximum, after which the preparations were allowed to stabilize for a minimum of 30 min at a stimulation frequency of 0.25 Hz (1 °C) or 0.65 Hz (7 °C). Force of contraction was recorded on paper using a chart recorder (Grass 7D) and analog signals were digitized by an AD-converter (Digidata 1200, Axon instruments) and stored on the computer hard disk for later analysis. The recordings were analysed off-line using the Clampfit analysis program (Axon Instruments). Maximum developed force (Fmax), maximum rate of relaxation (dF/dtmin), time to peak force (TPF), time of half relaxation (T0.5R) and duration of contraction (TDC=TPF+T0.5R) were measured.
For whole-heart (0.54±0.06 g wet mass, N=8) experiments in vitro, an input cannula was secured through the bulbus arteriosus into the ventricle and the heart retrogradely perfused with oxygenated physiological saline (containing, in mmol l-1, NaCl, 150; KCl, 5.4; CaCl2, 1.8; MgCl2, 10; Hepes, 10; glucose 10; pH 7.4 at room temperature) with a pressure head of 4 cm. The heart was connected to a force transducer from the apex of the ventricle by a hook and short braided silk thread and tensioned to an afterload of 0.5 g. The heart was immersed in a temperature-regulated tissue bath, and the incoming perfusion saline and saline in the bath were regulated to the same temperature. Heart function was allowed to stabilize at 1±0.5 °C before heart rate and force contraction were recorded. The temperature was then raised from 1 to 18 °C in 2 °C steps. Contractile parameters were recorded at each temperature after the stabilization.
Mechanical restitution, relaxation restitution and
rest-potentiation
Atrial and ventricular preparations were paced with a frequency of 0.25 Hz
at 1±0.5 °C and the force was recorded on a computer. To measure
mechanical and relaxation restitution, the regular pacing was intervened by
test protocols. Each protocol consisted of six contractions at the
steady-state frequency (0.25 Hz) to keep a constant cellular Ca2+
load. With these six contractions, the steady-state force was reached. The
steady-state contractions were followed by a single extra stimulus with a
variable delay from the last steady-state pulse and a post-extrasystolic
stimulus with a constant delay of 4s from the extra stimulus. The
extrasystolic intervals were generated in the computer (Clampfit, Axon
Instruments) from where the trigger signals were delivered through a D/A board
(DigiData 1200, Axon Instruments) and a custom-made signal conditioner to the
stimulator (SD-7, Grass Instruments). The extrasystolic intervals covered a
range from the absolute refractory period to the steady-state interval (4s).
The force of the extrasystolic contraction (FES) was
normalized to the force of the preceding control contraction
(FC) and plotted as a function of a extrasystolic interval
(t) to produce a mechanical restitution curve. For relaxation
restitution, the first derivative of the force recording was generated in the
computer and relaxation restitution curves were constructed by plotting the
inverse of dF/dtmin for extrasystolic beat
(normalized to that of the preceding control beat) against extrasystolic
interval. Contraction and relaxation restitution curves were fitted to single
exponential equations to give rate constants of restitution (see
Aho and Vornanen, 1999).
For the determination of rest-potentiation at 1 °C, steady-state stimulation was interrupted for 10, 30 or 60 s, after which the normal pacing was resumed. The force of the first post-rest contraction was normalized to the force of the previous steady-state beat to give rest-potentiation.
Ca2+/Mg2+-ATPase activity of myofibrils
Atrium from six fish and one ventricle were needed for the purification of
one sample of myofibrils (Aho and Vornanen,
1999). Tissues were minced with scissors and then homogenized 3
times for 10 s in 20 volumes of ice-cold buffer 1 (containing in mmol
l-1: KCl, 100; Tris-HCl, 10; dithiothreitol, 1; pH 7.4), and
centrifuged at 10,000 g for 10 min. The pellets were
resuspended in the homogenization buffer 2 (buffer 1 + 1 % Triton X-100) and
were centrifuged again at 10,000 g for 10 min. The recovered
myofibrils were washed three times in buffer 1 and centrifuged at 600
g for 15 min between washes. The pellets from the last
centrifugation were suspended in 20 volumes of low-ionicstrength buffer
containing (in mmol l-1): imidazole, 45; KCl, 50; dithiothreitol,
1; pH 7.0. ATPase activities of the purified myofibrils were determined at
four different temperatures (1, 5, 10 and 15 °C) by liberation of
inorganic phosphate (Atkinson et al.,
1973
). The pH was allowed to change freely according to
temperature and was 7.73, 7.69, 7.64 and 7.59 at 1, 5, 10 and 15 °C,
respectively. Total ATPase activity was measured in a solution containing (in
mmol l-1): imidazole, 45; KCl, 50; EGTA, 5; MgCl2, 5;
Na2ATP, 3; CaCl2, 5; pH 7.0. Activity of the background
Mg2+-ATPase was determined in the same solution, but without
CaCl2. Activity of the myofibrillar
Ca2+/Mg2+-ATPase was obtained as a difference between
the total and the background activity. Protein concentration was determined by
the method of Lowry et al.
(1951
).
Blood analysis
Fish were slightly anaesthetised with 0.1 % MS 222 and arterial blood was
collected from the caudal vessel into heparinized syringes. Hematocrit (Hct)
was determined in 75 µl blood samples after centrifugation at 7000
g for 5 min (Hermle Z 231 M). Haemoglobin (Hb) was determined
with the cyan-methemoglobin method using a 20 µl sample of blood. Mean
cellular Hb concentration (MCHC) was calculated by dividing the Hb
concentration by the Hct value. Blood plasma was diluted 1:10 with 0.5 mol
l-1 NaOH and the protein concentration was determined
(Lowry et al., 1951) using
bovine serum albumin as a standard.
Statistics
All results are given as mean ± S.E.M. Differences between atrium
and ventricle were compared by one-way analysis of variance (ANOVA).
Statistical differences between treatments were evaluated with a paired
t-test. All percentage values were compared after
arcsin-transformation with Student's t-test and ANOVA. The
differences were considered to be significant at P0.05.
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Results |
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Contractile properties of paced atrial and ventricular muscle
Stable force production, for up to 4 h, was characteristic of both atrial
and ventricular preparations from burbot heart. Accordingly, no correction for
time-dependent deterioration of contractile function was needed when the
effects of the SR Ca2+ release channel inhibitor, ryanodine, were
examined. At the imposed preload of 0.5 g, there was no difference in the
absolute force between atrial (5.37±1.5 mN) and ventricular
(5.94±1.2 mN) muscle at 1 °C (P<0.05; note, however,
that the ventricular preparations were, on average, 65 % larger).
The duration of ventricular contraction was approximately double the duration of atrial contraction. TPF values in atrial and ventricular tissue at 1 °C were 610±18 and 1403±61 ms (P<0.05), respectively. The corresponding values for T0.5R were 383±17 and 563±30 ms (P<0.05). The rate of contraction increased with Q10 values between 1.9 and 3.8 when the temperature rose from 1 ° to 7 °C. Ryanodine (10 µmol l-1, 60 min), strongly suppressed Fmax in both atrial and ventricular preparations. At 1 °C, Fmax decreased by 16±3 (N=5) and 32±8% (N=6) (P<0.05) of the control in ventricular and atrial preparations, respectively (Fig. 2). At 7 °C, the inhibitory effect of ryanodine was even stronger as Fmax decreased 52±3 % (N=6) in atrial and 44±5 % (N=6) in ventricular preparations. Despite its strong inhibitory effect on Fmax, ryanodine had only marginal effects on the time course of contraction.
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Restitution
The fast component of mechanical restitution describes force production at
diastolic intervals shorter than the regular pacing interval. As expected on
the basis of twitch duration, mechanical refractory period (MRP; the shortest
extrasystolic interval where force was generated) was much shorter in atrial
(1108±130 ms) than ventricular muscle (1975±81 ms, N=6;
P<0.05). Ryanodine had no effect on MRP in either atrial or
ventricular preparations (Fig.
3A). Following MRP, the rate of force recovery () was very
similar in atrial (781±141 ms, N=6) and ventricular
(628±65 ms, N=6; P>0.05) preparations and was not
influenced by ryanodine (724±196 ms and 755±186 ms, for atrium
and ventricle, respectively; P>0.05). The force of the
post-extrasystolic contraction, following premature extrasystolic
contractions, was not potentiated in the burbot heart (not shown).
|
Analogous to the recovery of contractile force, cardiac relaxation also
follows a pattern of restitution, which can be determined from the time
derivative of the force recording, dF/dtmin
(Fig. 3B). Under control
conditions, the time constants () of relaxation restitution were
460±75 ms and 608±25 ms (N=6; P>0.05) for
atrium and ventricle, respectively. Ryanodine had no effect on the time
constants, which were 530±81 ms and 540±91 ms (N=6)
(P>0.05) for atrium and ventricle, respectively.
Rest-potentiation represents the slow phase of mechanical restitution at diastolic intervals longer than the regular pacing interval. The first contraction after the prolonged diastolic interval (10-60 s) is bigger than the preceding control contraction and the potentiation dissipates gradually during the consecutive 15 beats (Fig. 4A). The maximum rest-potentiation was achieved at the diastolic interval of 60 s and was slightly stronger in atrial (154±8 %) than ventricular (122±2 %) preparations (P<0.05) (Fig. 4C). The rest-potentiation was completely abolished by ryanodine and, in fact, turned it to a rest-decay; the first contraction after the rest was smaller than the control contraction and was followed by gradual increase of force to the steady-state level during the following 15 beats (Fig. 4C). At the rest-period of 60 s, the force of the first post-rest contraction in ryanodine-treated preparations was only 72±2% in atrium and 63±9% in ventricle from the force of the control (P<0.05) (Fig. 4A). In atrial muscle, the duration of the first post-rest contraction was the same as that of the preceding control contraction and ryanodine increased both. In ventricular preparations, the first post-rest contraction was longer in duration than the control contraction and it became shorter during the following 15 beats, irrespective whether the force declined (control) or increased (ryanodine). Thus, the duration of contraction was positively correlated with Fmax in control preparations but negatively correlated in ryanodine-treated preparations (Fig. 4B).
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Myofibrillar Ca2+/Mg2+-ATPase activity
The activity of myofibrillar ATPase was determined at four different
temperatures. The Ca2+/Mg2+-ATPase activity was
significantly higher in atrial than in ventricular preparations
(P<0.05) and the temperature optimum of the ATPase was 10°C in
both tissues (Fig. 5).
Furthermore, the temperature dependence of the ATPase activity was remarkably
strong between 1 and 10°C (Q10 as great as 15) and much less
between 10 and 15°C (Q10 approx. 0.5).
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Blood composition
Blood composition was determined from nine burbot. Blood analysis showed a
Hb value of 67.2±4.0 gl-1 and Hct was 31.6±1.63%,
yielding MCHC 212.7±7.4 gl-1. The concentration of plasma
protein was 9.4±0.7 gl-1.
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Discussion |
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Temperature tolerance of cardiac function is similar in burbot and
cold-acclimated (4°C) (Aho and
Vornanen, 1999) rainbow trout, since arrhythmic contractions
appear at approximately 18°C in both species. More experiments are needed,
however, to clarify whether acclimation to higher temperatures can increase
thermal tolerance of the burbot heart, as happens in eurythermal rainbow trout
and crucian carp (Matikainen and Vornanen,
1992
; Aho and Vornanen,
2001
). Such experiments would shed light on whether the
contractile characteristics of the burbot heart also include a non-genetic
component.
Pumping capacity, the product of heart rate and peak force, can be used as
an index of power output for isolated muscle preparations since it integrates
the effects of changes in tension and heart rate, and usually achieves the
maximum value at the preferred temperature of the fish
(Matikainen and Vornanen,
1992; Shiels and Farrell,
1997
). In agreement with those studies, pumping capacity of the
burbot heart was maximum at the acclimation temperature (1 °C) and
declined at higher temperatures. This strongly suggest that the burbot heart
is best able to propel blood through the vasculature at near freezing
temperatures.
The viscosity of blood is the major factor for the resistance to blood flow
(Guyton and Richardson, 1961)
and is largely determined by the number of red blood cells and concentration
of proteins in the plasma. In most teleosts the Hct values are greater than 20
% and Hb concentration is between 4 and 15 g % (see
Gallaugher and Farrell, 1998
).
The Hct (32 %) and Hb (6.7 g %) of the burbot blood seem to be slightly less
than in many active teleosts, but not compared to the values of other sluggish
fish (see Gallaugher and Farrell,
1998
). Some caution is needed, however, when making interspecies
comparisons, because handling stress and method of blood sampling can effect
Hct values (Franklin et al.,
1993
). The total protein concentration in the burbot plasma (10.7
g l-1) is remarkably low when compared to the concentration (20 to
80 g l-1) of most other teleosts (see
McDonald and Milligan, 1992
).
On the basis of Hct and protein values, it seems the burbot blood is slightly
diluted, which could reduce its viscosity and the resistance to blood flow at
the expense of oxygen-carrying capacity. In this respect the burbot heart has
similarities to the `volume pumps' of the haemoglobin-free Antarctic ice-fish,
which circulate large volumes of dilute blood at sub-zero temperatures
(Tota et al., 1998
).
Subcellular mechanism of cold-adaptation
In addition to the characteristics of the volume pump, in many respects
(heart rate, contraction velocity, ryanodine sensitivity) the burbot heart
resembles the `power pump' of the cold-acclimated rainbow trout heart and
other cold-active fish. Some of the mechanisms that underlie the improved
contractility in cold-acclimated fish heart include enhanced Ca2+
management by the SR (Bowler and Tirri,
1990; Keen et al.,
1994
; Aho and Vornanen,
1998
,
1999
). In cold-acclimated
rainbow trout, the rate of Ca2+ uptake into the SR is enhanced
(Aho and Vornanen, 1998
),
thereby increasing the relative importance of SR Ca2+ release in
excitationcontraction coupling
(Keen et al., 1994
;
Aho and Vornanen, 1999
). SR
Ca2+ uptake was not directly assessed in the burbot heart, but
ryanodine inhibition of contraction force indicates that Ca2+
release from the SR contributes to the activation of contraction in this
cold-active, stenothermal fish. Indeed, the ryanodine inhibition of force is
somewhat larger in burbot (1 °C) than trout (4 °C) heart, in both
atrial (32 % versus 20 %) and ventricular (17 % versus 6 %)
muscle (present results; Aho and Vornanen,
1999
). Thus, it is clear that in the cold-active fish, cardiac
excitationcontraction coupling is modified so that the myocyte relies
more on the SR as a source of activator Ca2+.
Since the effect of ryanodine is frequency-dependent and the burbot
preparations were paced at a slightly slower rate (15 beats min-1)
than the real heart rate (25 beats min-1), the extent of ryanodine
inhibition under physiological conditions remains elusive. It appears,
however, that the role of the SR in excitationcontraction coupling of
the burbot heart is even more important at warmer (7 °C) temperatures,
which agrees with findings from rainbow trout
(Hove-Madsen, 1992;
Keen et al., 1994
;
Shiels and Farrell, 1997
) and
mackerel (Scomber japonicus) heart
(Shiels and Farrell, 2000
).
Indeed, at 7 °C (at physiological heart rate) ryanodine abolished about 50
% of the force production, which is similar to the values discovered in the
hearts of highly active tunas (Keen et
al., 1992
; Shiels et al.,
1999
). Thus movement of the burbot across the thermocline from
cold to warm water may change the functional characteristics of the heart from
volume pump to those of power pump, which might be more suitable for
circulating the less viscous blood.
Quantitative estimations of the relative significance of SR and sarcolemma
(SL) Ca2+ management on the basis of ryanodine inhibition are not
completely accurate and may underestimate the real SR Ca2+ release.
This is because negative feedback of SR Ca2+ release on the SL
Ca2+ influx is absent in the presence of ryanodine. Furthermore,
other Ca2+ cycling pathways may compensate for the inhibition of SR
in the presence of ryanodine. Direct measurements of intracellular
Ca2+ on single cardiac cells are needed to resolve these issues.
Nevertheless, the present experiments show that the SR Ca2+ release
is physiologically important for excitationcontraction coupling in the
cold-stenothermal burbot and is in agreement with electron-microscopic
documentation of well-developed dyadic couplings in the burbot heart
(Tiitu and Vornanen, 2002).
Furthermore, the present findings indicate clearly that cardiac ryanodine
receptors of the ectothermic fish are functional at near freezing temperatures
(1 °C) and do not allow leakage of Ca2+ from the SR. This is a
remarkable difference between fish and mammals. In mammalian cardiac myocytes,
ryanodine receptors are locked in the open state at 1 °C and
Ca2+ leaks out of the SR
(Sitsapesan et al., 1991
). The
molecular basis of this interesting difference remains to be shown.
Activity of myofibrillar ATPase is a significant determinant of contraction
rate (Barany, 1967). In
accordance with previous findings from mammals (see
Minajeva et al., 1997
) and
ectotherms (Deng and Gesser,
1997
; Aho and Vornanen,
1999
), the myofibrillar ATPase activity was higher in atrial than
ventricular muscle and explains, to a large extent, the faster contraction of
the atrial muscle. The temperature optimum of the burbot ATPase (10 °C) is
much lower than those of cold-acclimated trout and cold-acclimated crucian
carp (>15 °C) (Aho and Vornanen,
1999
; Tiitu and Vornanen,
2001
) and underscores the narrow thermal tolerance of the burbot
heart. Furthermore, temperature dependence of the myofibrillar ATPase of the
burbot heart was particularly marked below 10 °C. Also in the trout heart,
temperature dependence of the myofibrillar ATPase is high at low temperatures
(Aho and Vornanen, 1999
).
Although it is a general biological rule that Q10 values are high
near zero temperatures (Bennett,
1984
), it is unlikely that temperature-sensitivity of the ATPase
activity would be as high in vivo as we observed in vitro
since there were no dramatic and abrupt changes in the duration of contraction
at temperatures below 10 °C. There may be some modulating factors absent
from the purified myofibrils that might regulate ATPase activity in
vivo. A similar discrepancy between the temperature dependence of
myofibrillar ATPase activity and unloaded velocity of shortening was found in
myotomal muscle of the bullrout (Myoxocephalus scorpius L.)
(Johnston and Sidell, 1984
).
Myosin structure and function of the fish heart are poorly understood and
require further research.
Restitution
During cardiac contraction, molecular mechanisms responsible for the
initiation of contraction are inactivated. Recovery from inactivation occurs
gradually with time and determines the forceinterval relationship of
cardiac muscle. Mechanical restitution represents the increase in force of
contraction associated with progressively longer extrasystolic intervals and
is linearly related to time-dependent increases in intracellular
Ca2+ activator (Wier and Yue,
1986; Cooper and Fry,
1990
). In mammalian heart, the activator Ca2+ comes
primarily from the SR and therefore the time course of restitution is assumed
to be due to time-dependent restoration of Ca2+ release from the
SR. Ca2+ release from the SR is influenced by the rate of
Ca2+-uptake, extent of Ca2+ loading and availability of
Ca2+ release channels (Fabiato,
1983
) and, in principle, any factor involved in these processes
could contribute to mechanical restitution. Thus, in mammals, when
Ca2+ release from the SR is impaired by ryanodine, the rate of
restitution increases. This is because SL Ca2+ influx through
L-type Ca2+ channels, with faster recovery kinetics than SR
processes, becomes the limiting step in restitution
(Cooper and Fry, 1990
;
Prabhu, 1998
).
Although ryanodine decreased the force of steady-state contraction in
ventricular (16 %) and atrial (32 %) muscle, the rate of restitution in burbot
heart was not influenced by ryanodine, which contrasts with previous findings
on trout cardiac muscle. In the cold-acclimated rainbow trout, where the
steady-state force of contraction was inhibited by 6 % and 17 % in ventricle
and atrium, respectively, ryanodine clearly prolonged MRP and reduced the rate
of restitution (Aho and Vornanen,
1999). The findings on trout cardiac preparations indicate that
mechanical restitution is a relatively sensitive indicator of the contribution
of SR Ca2+ to contraction and that in the trout heart the
ryanodine-sensitive component of restitution is faster than the
voltage-dependent component (i.e. the opposite to the situation in mammals).
The present findings with burbot heart suggest that either the contribution of
SR Ca2+ release to the rate of restitution is rather small and has
therefore previously been unnoticed, or the recovery rates of SR and SL
mechanisms from inactivation are similar and not easily separated from each
other. In this context it should be noted that rate constants of mechanical
restitution are strongly temperature-dependent
(Aho and Vornanen, 1999
), and
are much larger in the burbot (approximately 700 ms at 1 °C) than trout
(200-300 ms at 4°) heart. Furthermore, in the burbot heart, the rate of
restitution was very similar in atrium and ventricle, whereas in trout atrial
restitution was much faster than ventricular restitution
(Aho and Vornanen, 1999
).
Relaxation restitution is the increase in the rate of atrial and
ventricular relaxation with progressively longer extrasystolic intervals. In
mammalian heart, where the major part of activator Ca2+ is recycled
through the SR, relaxation restitution is governed by SR
Ca2+-uptake. Accordingly, the rate of relaxation restitution is
decreased by ryanodine and increased in transgenic mice lacking phospholamban,
the inhibitory regulator of SR Ca2+-ATPase
(Prabhu, 1998;
Hoit et al., 2000
). The
absence of any ryanodine effect on relaxation restitution in burbot suggests
that SR Ca2+ uptake is not an important determinant for the
recovery of relaxation from inactivation in this species. Although over 30% of
the activating Ca2+ in the atrial muscle recycles through the SR at
the pacing rate of 0.25 Hz, no ryanodine-sensitive component of relaxation
restitution was found. It is, however, possible that the ability of the SR to
take up Ca2+ is impaired at short extrasystolic intervals and that
the inhibitory effect of ryanodine appears only at the steady-state frequency
of 0.25 Hz or lower. The SL Na+-Ca2+ exchange and SR
Ca2+-pump are the major Ca2+ removal pathways in cardiac
myocytes and therefore the ryanodine-resistant component of relaxation
restitution in burbot is probably related to the operation of the
Na+-Ca2+ exchange, with little contribution by the SR
Ca2+ pump. As the Na+-Ca2+ exchange is
voltage-dependent, the rate of relaxation restitution should describe the
recovery of the action potential from inactivation. The similarity of time
constants for mechanical and relaxation restitution suggests that both
processes might be controlled by the same mechanisms, possibly the membrane
potential of the SL. Clearly single-cell experiments are needed to clarify the
excitation-contraction coupling of the burbot heart.
The slow phase of mechanical restitution appears as rest-potentiation. In
mammals, increase in force at the post-rest contraction is associated with the
larger release of Ca2+ from the SR
(Lewartowski and Zdanowski,
1990; Bassani et al.,
1995
). The potentiation is not, however, associated with a higher
SR Ca2+ load, but is due to a larger fractional release, i.e. the
same Ca2+ trigger releases a larger proportion of the SR
Ca2+ content (Bassani and Bers,
1994
; Bouchard and Bose,
1989
). The force of post-rest contraction decreases with the
duration of rest period, which is known as rest-decay
(Allen et al., 1976
;
Bers, 1985
), and is related to
the leak of SR Ca2+ into the cytoplasm, where it is extruded by the
Na+-Ca2+ exchange
(Bers and Christensen, 1990
).
Furthermore, rest-potentiation and rest-decay are interconvertible by
modulating the intracellular Na+ concentration, suggesting that SR
Ca2+-pump and the Na+-Ca2+ exchange compete
for the same Ca2+ and, depending on the relative competitiveness of
the two systems, either rest-potentiation or rest-decay is expressed.
Accordingly, ryanodine abolishes rest-potentiation in the mammalian cardiac
muscle and reveals the underlying rest-decay
(Bers, 1985
). Like mammalian
myocardium, the burbot atrium and ventricle show rest-potentiation, which is
transformed into rest-decay in the presence of ryanodine. According to the
mammalian restitution model, under normal control conditions the SR
Ca2+-pump is more powerful than Na+-Ca2+
exchange in removing Ca2+ from the cytosol of the resting myocytes,
but after ablation of the SR by ryanodine, Na+-Ca2+
exchange remains the only relaxation mechanism and extrudes Ca2+
from the cell. In addition to the SR, other intracellular Ca2+
buffers are also partially depleted of Ca2+ during the rest, since
the first post-rest contraction is weaker than the steady-state contraction.
The depletion of intracellular Ca2+ buffers might explain the
inability of SL mechanisms to activate full-strength twitch immediately after
the rest and the negative correlation between force and duration of
contraction. In brief, the strong effect of ryanodine on the force of the
first post-rest contraction indicates the potential power of the SR in
regulating myoplasmic Ca2+, but at the same time reveals the
effectiveness of SL mechanisms in regulating the Ca2+ management of
the myocyte with only a small reduction in amplitude and without changes in
time-course of contraction.
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Conclusions |
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Acknowledgments |
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References |
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Aho, E. and Vornanen, M. (1998).
Ca2+-ATPase activity and Ca2+ uptake by sarcoplasmic
reticulum in fish heart: effects of thermal acclimation. J. Exp.
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