Temperature dependence of cardiac sarcoplasmic reticulum function in rainbow trout myocytes
1 Simon Fraser University, Biological Sciences, Burnaby, British Columbia,
V5A 1S6, Canada
2 University of Joensuu, Department of Biology, PO Box 111, 80101 Joensuu,
Finland
* Author for correspondence at present address: School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK (e-mail: hollys{at}sfu.ca)
Accepted 21 August 2002
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Summary |
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Key words: L-type Ca2+ current (ICa), ICa inactivation, sarcoplasmic reticulum, Ca2+ load, excitationcontraction coupling, heart rate fish, rainbow trout, Oncorhynchus mykiss
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Introduction |
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In rainbow trout heart, Ca2+ delivery to the myofilaments during
excitationcontraction (ec) coupling primarily involves the
influx of extracellular Ca2+ across the sarcolemmal membrane (SL)
and secondarily involves the mobilization of the intracellular Ca2+
stores of the SR (Keen et al.,
1994; Aho and Vornanen,
1999
; Hove-Madsen et al.,
1999
,
2001
;
Harwood et al., 2000
;
Shiels et al., 2002a
). The
relative importance of the SR Ca2+ flux pathway in trout hearts is
generally considered to be minor but can vary depending on a number of
physiological parameters. For example, SR Ca2+ cycling appears to
play a greater role in ec coupling in (1) atrial tissue compared with
ventricular tissue, (2) at low (<0.6 Hz) compared with high (>0.6 Hz)
contraction frequencies, (3) after acclimation to cold temperatures
(<12°C) and (4) after an acute change to warm temperatures
(>18°C) (Hove-Madsen,
1992
; Keen et al.,
1994
; Shiels and Farrell,
1997
; Aho and Vornanen,
1999
; Shiels et al.,
2002a
).
In most adult mammalian hearts, the SR is the major source of activator
Ca2+ during ec coupling, with SL Ca2+ influx
serving primarily as the trigger for SR Ca2+ release
(Bers, 2001). The SR
Ca2+ flux pathways are known to be temperature-sensitive in many
mammals, and the inhibition of SR function at cold temperatures is a major
cause of cold-induced contractile dysfunction (Wang et al.,
1997
,
2000
). Cold temperatures
increase the open probability of the mammalian SR Ca2+ release
channels (ryanodine receptors) causing the leak of available SR
Ca2+ into the cytoplasm
(Sitsapesan et al., 1991
).
Cold-induced slowing of ion transport mechanisms prevents this Ca2+
from being easily removed from the cytoplasm, contributing to a loss of
Ca2+ homeostasis (Bers,
1987
; Sitsapesan et al.,
1991
).
Despite the fact that the fish heart continues to function at cold
temperatures, early research on whole-muscle contractility in trout ventricle
suggested that the teleost SR had a temperature dependency similar to that of
the mammalian SR. This is because contractile force was found to be sensitive
to ryanodine (an inhibitor of the SR Ca2+ release channel;
Rousseau et al., 1987) at warm
temperatures (18-25°C) but insensitive to ryanodine at cold temperatures
(<15°C) (Hove-Madsen,
1992
; Driedzic and Gesser,
1994
; Keen et al.,
1994
). However, a study with isolated trout atrial tissue showed a
20% reduction in developed force after ryanodine treatment at 4°C
(Aho and Vornanen, 1999
),
indicating maintained SR function in the cold and suggesting that, similar to
mammals (Bers, 2001
), SR
involvement may be greater in fish atrium than in fish ventricle. These
findings were supported in a recent cellular study that demonstrated
maintained SR function at cold temperatures in rainbow trout atrial myocytes
(Hove-Madsen et al., 2001
).
However, the experimental conditions in this cellular study favoured a large
SR Ca2+ content, which could result in an overestimation of SR
capabilities in the cold. High intracellular Na+ concentrations
(approximately 17 mmol l-1) in conjunction with long depolarizing
pulses (>30 s) to positive membrane potentials were used in this study to
Ca2+-load the SR via the reverse mode (Ca2+ in,
Na+ out) of the sarcolemmal Na+/Ca2+
exchanger (NCX; Hove-Madsen et al.,
1998
,
1999
,
2001
). Indeed, mammalian and
teleost studies have shown that experimentally elevating intracellular
Na+ concentrations to a range of 15-20 mmol l-1
(concentrations in vivo are approximately 7 mmol l-1)
induces supra-physiological Ca2+ influx via the NCX
(Hove-Madsen et al., 2000
;
Bers, 2001
). Thus, it is
possible that previous studies with fish have overestimated the physiological
SR Ca2+ loading and release capabilities at cold temperatures. In
fact, cellular studies with permeabilized trout ventricular myocytes indicated
that the trout SR Ca2+-ATPase, which pumps cytosolic
Ca2+ back into the SR during relaxation, is temperature-dependent,
with an average Q10 of approximately 1.6 over a temperature range
of 5-20°C (Aho and Vornanen,
1998
; Hove-Madsen et al.,
1998
). As trout can experience acute temperature fluctuations of
as much as ±10°C either while transversing thermoclines or as a
result of diurnal changes in shallow streams
(Matthews and Berg, 1997
;
Reid et al., 1997
), the
temperature dependency of the SR Ca2+-ATPase, combined with
temperature-sensitive SR Ca2+ release channels, could limit the
utility of the SR as a source of activator Ca2+ during ec
coupling in trout heart.
In the present study, we were interested in assessing the physiological efficacy of the trout atrial SR at different temperatures. We acknowledge that it is difficult to be truly physiological using isolated voltage-clamped myocytes. However, to better approach the physiological situation, we included 10 mmol l-1 Na+ in our pipette solutions and, in addition to stimulating cells with conventional square (SQ) voltage-clamp pulses of constant dimensions, we applied physiological action potentials (APs) whose shape and rate of firing varied dynamically with changes in temperature. We assessed SR Ca2+ accumulation using rapid caffeine application, and SR Ca2+ release, by examining the effect of SR Ca2+ content on the inactivation kinetics of ICa (L-type Ca2+ channel current). We find that the shape of the AP and the rate of AP firing may be critical for ensuring adequate atrial SR Ca2+ cycling during temperature change in rainbow trout in vivo.
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Materials and methods |
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Isolated myocyte preparation
A detailed description of the myocyte preparation has been previously
published (Shiels et al.,
2000; Vornanen,
1998
). Briefly, fish were stunned with a blow to the head, the
spine was cut just behind the brain and the heart was excised. The heart was
then perfused first with the isolating solution for 8-10 min, and then with
the proteolytic enzyme solution for 20 min. After enzymatic treatment, the
atrium was removed from the ventricle and placed in a small dish containing
isolating solution. The atrium was cut into small pieces with scissors and
then triturated through the opening of a Pasteur pipette. The isolated
myocytes were stored in fresh isolating solution at 14°C.
Solutions
The isolating solution contained: NaCl, 100 mmol l-1; KCl, 10
mmol l-1; KH2PO4, 1.2 mmol l-1;
MgSO4, 4 mmol l-1; taurine, 50 mmol l-1;
glucose, 20 mmol l-1; and Hepes, 10 mmol l-1; adjusted
to pH 6.9 with KOH. For enzymatic digestion, collagenase (Type IA), trypsin
(Type IX) and fatty-acid-free bovine serum albumin (BSA) were added to this
solution. The physiological saline used as the extracellular solution for
recording Ca2+ currents (ICa) and NCX currents
(INCX) contained: NaCl, 150 mmol l-1; CsCl, 5.4 mmol
l-1; MgSO4, 1.2 mmol l-1;
NaH2PO4, 0.4 mmol l-1; CaCl2, 1.8
mmol l-1; glucose, 10 mmol l-1; and Hepes, 10 mmol
l-1; adjusted to pH 7.7 with CsOH. Additionally, 1 µmol
l-1 tetrodotoxin (TTX) was added to the perfusate to block fast
Na+ channels. Caffeine (10 mmol l-1) was added to the
extracellular solution to release the SR Ca2+ stores. For AP
recordings, the extracellular solution was modified by replacing the CsCl with
equimolar KCl, pH balancing with KOH and by omitting the TTX. The pipette
solution for ICa and INCX recordings contained: CsCl,
130 mmol l-1; MgATP, 5 mmol l-1; tetraethylammonium
chloride (TEA), 15 mmol l-1; MgCl2, 1 mmol
l-1; oxaloacetate, 5 mmol l-1;
Na2-phosphocreatine, 5 mmol l-1; Hepes, 10 mmol
l-1; EGTA, 0.025 mmol l-1; and Na2GTP, 0.03
mmol l-1; pH was adjusted to 7.2 with CsOH. For action potential
recordings, the pipette solution contained: K-aspartate, 125 mmol
l-1; KCl, 15 mmol l-1; MgCl2, 1 mmol
l-1; MgATP, 5 mmol l-1; EGTA, 0.05 mmol l-1;
Na2-phosphocreatine, 5 mmol l-1; and Hepes, 10 mmol
l-1; adjusted to pH 7.2 with KOH. All drugs, with the exception of
TTX (Tocris, Bristol, UK), were purchased from Sigma (St Louis, MO, USA).
Experimental procedures
Myocytes (average cell capacitance 36.2±0.92 pF, N=92) were
superfused at a rate of 2 ml min-1 with extracellular solution at
7°C, 14°C or 21°C. Each cell was tested at one experimental
temperature only. The extracellular solution and the solution flowing from a
rapid solution changing device (RS200, Biologic, Claix, France) was heated or
chilled by water bath circuits before emptying into the recording chamber
(RC-26, Warner Instrument Corp, Brunswick, USA; volume 150 µl). A
thermocouple was placed inside the recording chamber and positioned no less
than 5 mm from the cell to ensure it was experiencing the desired temperature.
Rapid application (approximately 50 ms) of caffeine was achieved by switching
between two barrels of the rapid solution changer; one containing control
extracellular solution and the other containing the same solution plus
caffeine, both flowing at approximately 0.5 ml min-1.
Whole-cell voltage-clamp experiments were performed using an Axopatch 1D
amplifier with a CV-4 1/100 headstage (Axon Instruments, Foster City, CA,
USA). Pipettes had a resistance of 2.4±0.11 M when filled with
pipette solution. Junction potentials were zeroed prior to seal formation.
Pipette capacitance (9.7±0.5 pF) was compensated after formation of a
G
seal. Mean series resistance was 6.1±0.2 M
. Membrane
capacitance was measured using the calibrated capacity compensation circuit of
the Axopatch amplifier. Signals were low-pass filtered using the 4-pole
lowpass Bessel filter on the Axopatch-1D amplifier at a frequency of 2 kHz for
ICa and 10 kHz for action potentials, and were then analyzed
off-line using pClamp 6.0 software (Axon Instruments).
Current-clamp and AP recording
Rainbow trout atrial myocytes were stimulated to elicit APs at 7°C,
14°C or 21°C at a frequency that corresponded to the resting heart
rate of rainbow trout in vivo at each of these temperatures. The
temperature/frequency pairs were as follows: 0.6 Hz at 7°C, 1.0 Hz at
14°C and 1.4 Hz at 21°C (Aho and
Vornanen, 2001; Tuurala et
al., 1982
; Farrell et al.,
1996
). APs were elicited by the minimum voltage pulse able to
trigger a self-sustained AP (1 ms, 0.8 nA). The resultant AP waveforms
(Fig. 1) were subsequently used
to provide a more physiologically relevant indication of how temperature and
frequency affected SR Ca2+ accumulation and release in rainbow
trout atrial myocytes. In all experiments, the capacitive transients resulting
from the AP waveform were compensated for by the amplifier.
|
Assessing SR Ca2+ content
We used either SQ pulses (-80 mV to +10 mV, 200 ms, interpulse holding
potential of -80 mV) or AP pulses (see Fig.
1) and varied both temperature and pulse frequency to examine
their effect on SR Ca2+ accumulation. SR Ca2+
accumulation was assessed by the application of caffeine, which induces the
release of Ca2+ from the SR. This Ca2+ is then extruded
from the cell via the NCX generating an inward current
(Fig. 2) that is directly
proportional to the Ca2+ released from the SR
(Varro et al., 1993). The time
integral of the caffeine-induced INCX current was used to calculate
the SR Ca2+ content (in pC) at the time of caffeine application.
This value was expressed per unit capacitance (pC pF-1). The SR
Ca2+ content was also expressed in µmol l-1 and was
calculated from the integral of INCX and the cell volume. Cell
volume was calculated from cell surface area [obtained by measurements of cell
capacitance (pF) and assuming a specific membrane capacitance of 1.59 µF
cm-2] and the surface:volume ratio of 1.15 [determined
experimentally in previous studies; see Vornanen
(1997
)]. Finally, SR
Ca2+ content was expressed as a function of myofibrillar volume
(40%), as determined previously (Vornanen,
1998
).
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Interaction between the SL L-type Ca2+ channel and the SR
Ca2+ release channel
Possible interaction between extracellular Ca2+ influx
via the L-type Ca2+ channel (ICa) and
intracellular Ca2+ release through the SR Ca2+ release
channel was examined by investigating the effect of SR Ca2+ content
on the inactivation of ICa. Previous work has shown that if there
is interaction between these Ca2+ fluxes, ICa
inactivation is faster due to SR Ca2+-dependent inactivation
(Lipp et al., 1992;
Sham, 1997
). To test whether
this mechanism occurs in trout atrium, we fitted either a double
(
f and
s; SQ) or single (
; AP)
exponential function to the inactivating portion of ICa immediately
after the SR Ca2+ stores were depleted with caffeine (loading pulse
1) and then again after SR Ca2+ content had reached steady-state
(after 25 pulses). In some cells, the effect of progressive SR Ca2+
accumulation on ICa with each pulse was examined between the first
and 100th pulse. Single and double exponential fits were calculated using the
standard exponential fitting procedure with the Chebyshev transformation with
Clampfit 6.0 software (Axon Instruments).
Statistical Analysis
One-way repeated measures analysis of variance (RM ANOVA) was used to
compare the effect of pulse number on SR Ca2+ content. One-way
ANOVA was used to test the effects of temperature, frequency and shape of the
stimulation pulse on SR Ca2+ accumulation. Significant differences
(P<0.05) were assessed with StudentNewmanKeuls (SNK)
post-hoc analysis. The effects of steady-state SR Ca2+
content on the inactivation kinetics of ICa were tested using
paired Student's t-tests.
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Results |
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At 21°C, SR Ca2+ accumulation was more variable, as only four out of eight cells demonstrated SR Ca2+ accumulation under these experimental conditions. Those cells that accumulated Ca2+ into the SR also reached a steady-state of approximately 1 pC pF-1 after 25 pulses (equivalent to 460±76 µmol l-1 Ca2+) and did not change significantly with an additional number of pulses up to 100. Only the cells that were able to accumulate Ca2+ into the SR are shown in Fig. 3A (broken line).
Effect of frequency and temperature on steady-state SR
Ca2+ accumulation
Because the frequency of cell depolarization should affect the
Ca2+ available for uptake into the SR, our next experiment examined
SR Ca2+ content at each temperature when the 200 ms SQ pulses were
applied at a frequency that corresponds to the heart rate of the rainbow trout
in vivo at each temperature. These results are given in
Fig. 3B. In contrast to the
results with 1.0 Hz stimulation, when frequency was increased to 1.4 Hz at
21°C, all cells displayed significant SR Ca2+ accumulation and
reached a steady-state of 0.78±0.09 pC pF-1 (equivalent to
367±42 µmol l-1 Ca2+) after 25 pulses
(Fig. 3B). At 14°C, the
physiologically relevant frequency was 1.0 Hz and the results were no
different from those presented in Fig.
3A. At 7°C, a physiologically relevant stimulation frequency
was 0.6 Hz, and the SR Ca2+ content after 25 pulses and 50 pulses
was similar to that at 1.0 Hz but increased significantly (P<0.05,
N=8) after 100 pulses (Fig.
3B). As a result, SR Ca2+ content was 2.1±0.3 pC
pF-1 or 972±142 µmol l-1 Ca2+ after
100 pulses at 0.6 Hz, which was almost double the steady-state amount observed
after stimulation with 100 SQ pulses at 1.0 Hz (550±90 µmol
l-1 Ca2+) (Fig.
3A).
Effect of AP shape on steady-state SR Ca2+
accumulation
The experiments with SQ-voltage-clamp pulses indicated that SR
Ca2+ content can vary as a function of temperature and frequency.
However, in vivo, extracellular Ca2+ entry into the
myocyte does not occur during a SQ-voltage pulse but rather during an AP. As
both the shape and rate of APs vary considerably with temperature
(Coyne et al., 2000;
Harwood et al., 2000
;
Shiels et al., 2000
), studies
with AP clamp may be essential for understanding the physiological capacity of
the SR as a function of temperature in fish hearts. Indeed, we observed a
pronounced effect of acute temperature change on AP duration, which is clearly
illustrated in Fig. 1. AP
duration at 50% repolarization varied by more than 50% among temperatures
(increasing from approximately 90 ms at 21°C to 160 ms at 14°C and to
290 ms at 7°C; Fig. 1).
Thus, our next experiments set out to examine the Ca2+-accumulating
abilities of the trout SR during AP stimulation.
When myocytes were stimulated at a physiologically relevant frequency, with the AP waveform specific for the test temperature, SR Ca2+ content reached a steady-state after 25 pulses at all three temperatures (2.8±0.3 pC pF-1, 2.7±0.3 pC pF-1 and 2.3±0.2 pC pF-1 at 7°C, 14°C and 21°C, respectively; Fig. 3C) and the steady-state temperature and frequency-dependent changes in SR Ca2+ content observed with SQ pulses were no longer evident. The Ca2+ content of the SR was greater (1043±189 µmol l-1 Ca2+, 1138±173 µmol l-1 Ca2+ and 1095±142 µmol l-1 Ca2+ at 7°C, 14°C and 21°C, respectively) when cells were stimulated with AP pulses compared with 200 ms SQ pulses (664±180 µmol l-1 Ca2+, 474±75 µmol l-1 Ca2+ and 367±42 µmol l-1 Ca2+ at 7°C, 14°C and 21°C, respectively) applied at the same frequency (Fig. 3B).
Interaction between ICa and SR Ca2+
release
ICa records initiated immediately after depletion of SR
Ca2+ by caffeine allowed us to monitor the effects of SR
Ca2+ re-accumulation on ICa. Inactivation kinetics of
ICa were significantly faster when the SR was loaded with
Ca2+, and this observation is consistent with a greater SR
Ca2+ release resulting in greater SR-Ca2+-dependent
inactivation. Fig. 4 shows a
representative recording of the progressive quickening of inactivation as SR
Ca2+ content increases with each pulse between 1 and 25 at
21°C. After 25 pulses, inactivation kinetics did not change significantly
with additional pulses, supporting the finding that SR Ca2+ content
had reached a steady-state. Fig.
5 shows representative ICa currents with both SQ and AP
pulses with the SR empty and at a steady-state at 14°C. The mean
inactivation kinetics of ICa for each condition and temperature are
given in Tables 1,
2. At 21°C, fast
inactivation kinetics (f) and slow inactivation kinetics
(
s) were significantly faster than at 14°C and were
significantly faster under the SR-loaded condition
(Table 1). At 7°C, the
inactivation of ICa was a better fit with a single exponential
function. Inactivation of ICa at 7°C was faster as the SR
refilled (Table 1). This
suggests that, even with the reduced amplitude of ICa at 7°C
(see Shiels et al., 2000
),
ICa elicited by a SQ stimulation pulse is still of sufficient
magnitude to cause Ca2+-dependent inactivation of SL
Ca2+ influx.
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The Ca2+ current is more complex during AP clamp (Fig. 5B). The effect of SR Ca2+ content on inactivation kinetics was examined by fitting a single exponential to the first part of the inactivation of ICa [first 200 ms after peak current at 14°C (see Fig. 5B) and first 100 ms after peak at 21°C (not shown)]. The AP waveform could not be consistently modelled at 7°C and so inactivation kinetics at 7°C are excluded from this part of the analysis. At both 14°C and 21°C, the inactivation of ICa elicited by AP pulses was faster with a steady-state Ca2+ SR content (Table 2).
Because the voltage waveform (either SQ or AP) was the same for each pulse and the amplitude of ICa was not significantly different, the change in inactivation kinetics observed in these experiments is unlikely to result from voltage-dependent inactivation or Ca2+-dependent inactivation of Ca2+ entry through the channel. Thus, we conclude that there is SR Ca2+-dependent inactivation of the SL ICa in rainbow trout atrial myocytes and that it is independent of temperature between 7°C and 21°C.
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Discussion |
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The increase in AP duration at cold temperatures, and the increase in AP
firing rate at warm temperatures, may underlie the temperature independence
observed in the present study (see Fig.
1). Indeed, the duration of the AP plateau will profoundly affect
SL Ca2+ influx and thus the Ca2+ available for uptake
into the SR. Additionally, changes in stimulation frequency affect diastolic
Ca2+ load in trout atrial myocytes (see
Shiels et al., 2002b), which
could also influence the activity of the SR Ca2+ ATPase. Thus, we
suggest that temperature-dependent modulation of the AP shape and firing rate
may help to offset the known temperature sensitivity of the SR Ca2+
ATPase (Q10 of approximately 1.6;
Aho and Vornanen, 1998
;
Hove-Madsen et al., 1998
).
Our results show greater SR Ca2+ accumulation with AP
stimulation than with SQ pulses in trout atrial myocytes. This may be
explained by the fact that ICa generated by an AP has a sustained
phase during the AP plateau. The sustained phase is a result of an increased
driving force for Ca2+ as a result of repolarization (to
approximately 0 mV) after the peak and the reactivation of L-type
Ca2+ channels during the AP plateau (window current)
(Arreola et al., 1991). Indeed,
the relatively depolarized resting membrane potential in the AP experiments
(approximately -50 mV) may have increased Ca2+ influx via
the window current under AP conditions. A -50 mV resting membrane potential is
typical for isolated rainbow trout atrial myocytes
(Shiels et al., 2000
) and
results from the loss of cholinergic tonus and also reflects the lower density
of inward rectifier current (IK1) in fish atrial cells compared
with ventricular cells (Vornanen et al.,
2002a
). However, recent measurements in vivo suggest that
the resting membrane potential in rainbow trout atrial cells is approximately
-65 mV (Vornanen et al.,
2002a
). Therefore, future studies could consider adding tonic
levels of acetylcholine to solutions when recording APs from isolated
myocytes.
In previous studies (Shiels et al.,
2000), we have measured the ICa window current in trout
atrial cells at 7°C, 14°C and 21°C and found a peak
Ca2+ contribution at -10 mV at all temperatures. However, we also
found that the relative Ca2+ contribution from the window current
was greater (0.08 relative units) and had a larger voltage window (-40 mV to
+30 mV) at 7°C compared with at 14°C and 21°C (0.055 relative
units, -30 mV to +20 mV). A larger window current at 7°C may explain, in
part, the large SR Ca2+ content observed at 7°C with the SQ
pulses at 0.6 Hz (Fig. 3B).
Additionally, because kinetics are slower at 7°C (see
Shiels et al., 2000
),
prolongation of the diastolic period at 0.6 Hz could allow for more complete
recovery of ICa, thereby increasing Ca2+ influx and SR
loading.
We report a high steady-state SR Ca2+ content during AP
stimulation (approximately 1250 µmol l-1). However, when trout
atrial myocytes are depolarized to +50 mV for 32 s, the SR can accumulate even
larger quantities of Ca2+ (approximately 2340 µmol
l-1; Hove-Madsen et al.,
1998,
1999
). In the adult mammalian
heart, SR Ca2+ release predominates over SL Ca2+
delivery during e-c coupling and yet the steady-state SR Ca2+
content in mammalian myocytes (100-150 µmol l-1;
Bassani et al., 1995
;
Negretti et al., 1995
;
Díaz et al., 1997
) is
an order of magnitude less than in fish. An intriguing question for future
study emerges: why does the SR of rainbow trout have such a large maximal and
steady-state capacity to store Ca2+ when isometric muscle studies
indicate that Ca2+ release from the SR contributes a rather small
amount of the Ca2+ involved in contraction?
The Ca2+-storing capacity of the SR is determined by the volume
fraction of the SR in the myocyte, the concentration of low-affinity
Ca2+ buffers inside the SR and the concentration gradient of free
Ca2+ across the SR membrane
(Shannon and Bers, 1997). It
is unclear which of these factors contributes to the enhanced
Ca2+-storing capacity of the trout SR. It is possible that
functional differences in the rainbow trout SR Ca2+ release
channels, possibly at the luminal side of the channel, or differences in SR
Ca2+ buffers may account for the difference in maximal SR
Ca2+ content. However, to date there is no information available to
accept or reject this idea. In mammals, it is difficult to increase
steady-state SR Ca2+ above approximately 100 µmol l-1
without spontaneous release (Bassani et
al., 1995
), probably because of a direct effect of luminal
Ca2+ on the SR Ca2+ release channel
(Lipp et al., 1992
;
Sitsapesan and Williams, 1994
;
Bassani et al., 1995
). One
possibility is that the SR Ca2+ release channels in fish heart are
approximately 10-fold less sensitive to luminal Ca2+ than those in
mammals. Indeed, experiments with carp (Cyprinus carpio) suggest that
their SR Ca2+ release channels may be an order of magnitude less
sensitive to Ca2+ than mammals
(Chugun et al., 1999
).
Furthermore, indirect studies of SR function do suggest striking differences
between fish and mammalian SR Ca2+ release channels with respect to
cold sensitivity. The results of the present study confirm earlier work
(Bowler and Tirri, 1990
;
Hove-Madsen et al., 1998
;
Aho and Vornanen, 1999
;
Tiitu and Vornanen, 2002
)
indicating that the teleost SR Ca2+ release channels remain
functional at cold temperatures whereas the mammalian SR Ca2+
release channels open such that Ca2+ leaks out of the SR
(Bers, 1987
;
Sitsapesan et al., 1991
).
One speculation as to why rainbow trout store large amounts of
Ca2+ in their SR may be related to their burst mode of swimming.
Burst swimming has been shown to decrease blood pH by as much as 0.5 pH units,
and an extracellular pH change of such magnitude will significantly inhibit
cardiac performance (Milligan and Farrell,
1986; Churcott et al.,
1994
). Although there is no information at present, a large SR
Ca2+ store, if releasable, could serve as a safety factor during
acidosis to override decreased myofilament Ca2+ sensitivity.
Indeed, we know that adrenergic stimulation can protect force development in
the acidotic rainbow trout myocardium
(Farrell et al., 1985
;
Farrell and Milligan, 1986
)
although the mechanisms have not yet been defined. However, under normoxic
conditions, a 30-60% release of the steady-state SR Ca2+ content in
trout (as reported for mammals; Bassani et
al., 1995
) could cause severe contracture and could damage the
contractile machinery. It may be that, regardless of SR Ca2+
content, there is an upper limit to the amount of Ca2+ that is
releasable via Ca2+-induced Ca2+ release, as
the absolute amount of Ca2+ released in fish and mammalian
cardiomyocytes at 21°C is comparable: 35-60 µmol l-1
Ca2+ (Bassani et al.,
1995
; Janczewski et al.,
1995
; Hove-Madsen et al.,
1998
).
In summary, the present study has demonstrated that the rainbow trout
atrial SR Ca2+ content reaches a steady-state after approximately
25 stimulation pulses over a physiological range of temperatures and heart
rates during AP stimulation. Furthermore, SR Ca2+ accumulation and
release are not compromised by temperature change, indicating significant
differences between rainbow trout and mammalian SR. Finally, because the
temperature changes used in the present study represent a realistic
physiological challenge for the rainbow trout heart, our results suggest that
the plasticity of the rainbow trout contractile machinery (see
Vornanen et al., 2002b for a
recent review) may help to maintain SR Ca2+ content during
temperature change in vivo.
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Acknowledgments |
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References |
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