Temperature acclimation modifies Na+ current in fish cardiac myocytes
University of Joensuu, Department of Biology, PO Box 111, 80101 Joensuu, Finland
* Author for correspondence (e-mail: matti.vornanen{at}joensuu.fi)
Accepted 20 May 2004
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Summary |
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Key words: fish heart, thermal acclimation, sodium current, Carassius carassius, Oncorhynchus mykiss, Lota lota
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Introduction |
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In previous studies we characterised cardiac function in three teleost fish
that have different temperature preferences and seem to use partially
different overwintering strategies in cold-temperate climates
(Matikainen and Vornanen,
1992; Aho and Vornanen,
1999
,
2001
; Tiitu and Vornanen,
2001
,
2002a
,b
).
Crucian carp Carassius carassius L. inhabit small and anoxic water
bodies where other fish are unable to survive. The crucian carp is a very
anoxia resistant and eurythermic species tolerating temperatures between
0°C and +36°C (Blazka,
1958
; Horoszewich,
1973
). This anoxia resistance is based on huge glycogen stores
(Hyvärinen et al., 1985
;
Vornanen, 1994
), alternative
metabolic pathways (Johnston and Bernard,
1983
; Holopainen and
Hyvärinen, 1985
) and especially on small energy consumption
that is reflected as inverse compensation in the heart function (Matikainen
and Vornanen, 1992
, 1994;
Tiitu and Vornanen, 2001
). In
contrast, rainbow trout Oncorhynchus mykiss is active in cold waters
and expresses several compensatory changes that are reflected in heart rate,
heart size, myofibrillar function as well as in the activity of sarcolemmal
ion channels (Graham and Farrell,
1992
; Driedzic et al.,
1996
; Aho and Vornanen,
1999
,
2001
). Rainbow trout is not as
eurythermic as crucian carp, but is able to tolerate temperatures between
0°C and +25°C (Taylor et al.,
1996
). Burbot Lota lota are cold-stenothermal fish that
are most active in the middle of winter and try to avoid warm waters (above
+13°C) in summer (Bernard et al.,
1993
). Structurally and functionally the burbot heart is more
similar to the heart of rainbow trout than crucian carp in that it is
relatively large and remains highly active in the cold (Tiitu and Vornanen,
2002a
,b
).
Voltage-gated Na+ channels are crucial for the excitability of
the heart by allowing fast Na+ influx. Sodium current
(INa) determines the amplitude and slope of the action potential
upstroke, which affect the threshold of excitability and are especially
important in the control of impulse conduction velocity
(Fozzard and Hanck, 1996).
INa is also involved in excitationcontraction coupling of
cardiac myocytes (Leblanc and Hume,
1990
; Maier et al., 2001). Considering the wide differences in
cardiac activity between cold-active (trout, burbot) and cold-dormant (crucian
carp) fish species, it would be expected that these differences might be
reflected in the properties of cardiac INa. Therefore, we examined
INa in ventricular myocytes of these fish to test the effects of
thermal acclimation on INa. More specifically, we hypothesised that
the density of INa would be larger and kinetics faster in
cold-active than cold-dormant species, and that thermal acclimation might
induce opposite changes in the function of INa in eurythermal
crucian carp and rainbow trout.
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Materials and methods |
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Myocyte isolation
Single ventricular myocytes were enzymatically isolated using previously
published methods (Vornanen and
Tuomennoro, 1999). Briefly, fish were stunned with a quick blow to
the head, the spine was cut and the heart was excised. A metallic cannula was
slipped through the bulbus arteriosus into the ventricle and the heart was
retrogradely perfused with a nominally Ca2+-free,
low-Na+ solution for 10 min and then with proteolytic enzyme
solution for 1015 min from a height of 50 cm. Solutions were
continuously gassed with 100% O2 and the enzyme solution was
recycled using a peristaltic pump. After enzymatic digestion the ventricle was
excised and placed in fresh isolating solution in a Petri dish. The ventricle
was chopped in small pieces with scissors and single cells were released by
agitating tissue pieces through the opening of a Pasteur pipette. Isolated
myocytes were stored in isolating solution at +6°C and used within 8 h
from the isolation. All procedures were in accordance with the local committee
for animal experimentation.
Solutions
A nominally Ca2+-free saline used for cell isolation contained
(mmol1): 100 NaCl, 10 KCl, 1.2 KH2PO4,
4 MgSO4, 50 taurine, 20 glucose and 10 Hepes at pH 6.9 at 20°C.
For enzymatic digestion, 0.75 mg ml1 collagenase (Type IA;
Sigma, St Louis, MO, USA), 0.5 mg ml1 trypsin (Type IX;
Sigma) and 0.5 mg ml1 fatty-acid-free bovine serum albumin
(BSA) were added to this saline. The physiological K+-based
external saline solution contained (mmol1): 150 NaCl, 5.4
KCl, 1.8 CaCl2, 1.2 MgCl2, 10 glucose and 10 Hepes (pH
adjusted to 7.7 with NaOH). The actual extracellular Cs-based solution used
for recording INa contained (mmol1): 20 NaCl, 120
CsCl, 1 MgCl2, 0.5 CaCl2, 10 glucose and 10 Hepes (pH
adjusted to 7.7 with CsOH). In addition, 10 µmol l1
nifedipine (Sigma) was added to both solutions to block L-type
Ca2+-currents. The pipette solution contained
(mmol1): 5 NaCl, 130 CsCl, 1 MgCl2, 5 EGTA, 5
Mg2ATP and 5 Hepes (pH adjusted to 7.2 with CsOH).
Patch-clamp experiments
A small portion of myocyte suspension was transferred to a recording
chamber (RC-26, Warner Instrument Corporation, Brunswick USA; volume 150
µl) and cells were allowed to settle on the chamber bottom before
superfusing with the external saline solution at the rate of 1.52.0 ml
min1. First, the myocytes were perfused with normal
K+-based saline so that gigaseal and whole-cell patch clamp
recording of the myocytes were established. Internal perfusion of the myocytes
with pipette solution was continued in this solution for at least for 3 min in
order to allow buffering of intracellular Ca2+ with 5 mmol
l1 EGTA. Then, solution flow could be switched to a
low-Na+ external solution without inducing contracture in the
patched myocyte. The experiments with myocytes of c.a. animals were conducted
at +4°C and +11°C and those with w.a. animals at +11°C and
+18°C. Accordingly, results were obtained not only at the physiological
body temperatures of the animals, but also at the common experimental
temperature of +11°C, which enabled direct comparison of results between
the two acclimation groups. The temperature of the saline was adjusted to the
desired temperatures by using two circulating water baths and was continuously
monitored by thermocouple positioned closed to the myocyte.
The whole-cell voltage clamp measurements of INa were performed
using an Axopatch 1-D amplifier with a CV-4 1/100 headstage (Axon Instruments,
CA, USA). The digitised data were stored on the hard drive of the computer
using the Clampex 8.2 software (Axon). The recordings were analysed off-line
with Clampfit 8.2 and SigmaPlot 6.0 (SPSS) software. Patch pipettes were
pulled from borosilicate glass (Garner, Claremont, CA, USA) using a vertical
two-stage puller (List-Medical, L/M-3P-A). Off-set potentials were zeroed just
before the formation of gigaohm seal and the pipette capacitance
(8.22±0.10 pF, N=101) was compensated for after the seal
formation. The membrane was ruptured by a short voltage pulse (zap) and
capacitive transients were eliminated by adjusting series resistance and cell
capacitance compensation circuits. Mean resistance of the electrodes and total
access resistance before compensation were 3.02±0.07 and
10.20±0.33 M (N=101), respectively. To ensure adequate
voltage control a minimum of 80% series resistance compensation was applied.
INa was elicited from the holding potential of 120 mV with
different pulse protocols and recorded at sampling rate of 10 kHz. The
recordings were low-pass filtered at 5 kHz. The calculated
liquidjunction potential of the electrodes was about 1.5 mV and was not
corrected in the results.
Steady-state activation and inactivation of INa
Steady-state inactivation was determined using a two-step protocol where a
500 ms conditioning pulse to potentials between 110 mV and 20 mV
was followed by a 15 ms test pulse to 20 mV. For the voltage dependence
of steady-state inactivation the normalized test pulse currents
(I/Imax) were plotted as a function of membrane
potential and fitted to the Boltzmann function:
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Recovery and development of INa inactivation
Time-dependent recovery of INa from inactivation was examined
using a paired-pulse protocol where two successive 100 ms pulses from
120 to 20 mV were separated by a variable (40400 ms)
delay at holding potential. The peak INa during the latter pulse
was plotted as a function of time and fit to a single exponential function
(y=y0+abt) to obtain the time
constant (=1/b) of recovery from inactivation.
The development of rested-state inactivation was examined using a protocol consisting of a conditioning prepulse from 120 mV to 60 mV with variable (30360 ms) duration, followed by a short return (3 ms) to the holding potential and a test pulse to 20 mV for 30 ms. The peak INa elicited by test pulses was plotted as a function of the prepulse duration and fitted to a single exponential function to obtain the time constant for the development of rested-state inactivation.
Kinetics of INa inactivation
The time constants of INa inactivation kinetics were derived by
fitting the decay phase of the INa at different membrane potentials
(40 mV to +10 mV) with a single exponential equation using the
Chebyshev transformation procedure of the Clampfit software package.
Statistical analyses
Data are expressed as means ± S.E.M. Analysis of variance
(one-way ANOVA) with Tukey's HSD post hoc test was used for multiple
comparisons between species and acclimations groups. Effects of acute
temperature changes on INa were assessed by paired or unpaired
t-test. P<0.05 was considered statistically
significant.
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Results |
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Size of INa
The currentvoltage relationships and maximal conductance
(Gmax) of INa are shown in
Fig. 1 and
Table 2, respectively. In all
three species, INa activated between 70 and 60
mV,reached the peak value at 20 mV and reversed close to the
theoretical reversal potential (30.532 mV, depending on temperature) of
INa. In rainbow trout, cold-acclimation slightly shifted the
activation threshold of INa to more negative values. There were
marked species-specific differences in INa density and
Gmax of c.a. fish at the physiological body temperature
(+4°C). INa was clearly smallest in crucian carp and largest in
burbot, whereas the INa of trout heart was intermediate between
those of carp and burbot. Interestingly, thermal acclimation modified
INa in opposite manner in carp and trout; in the carp
cold-acclimation reduced the density of INa, while in the trout it
increased INa (Fig.
1B).
|
Acute temperature effects on the density of INa were bigger in c.a. than w.a. fish both for crucian carp (Q10=1.97 and 1.74, respectively) and rainbow trout (Q10=2.33 and 1.61, respectively). The Q10 for burbot INa was 2.20 between +4° and +11°C.
Steady-state activation and inactivation of INa
Steady-state activation and inactivation curves are shown in
Fig. 2 and statistical
evaluation of the data is presented in
Table 2. There were no
species-specific differences in half-voltage (V0.5) of
steady-state activation for c.a. fish at +4°C. In contrast, the
V0.5 of steady-state inactivation of c.a. crucian carp and
c.a. burbot were 16 and 10 mV more negative, respectively, than
that of the c.a. trout.
|
In rainbow trout myocytes, acclimation to cold caused a 6 mV shift of steady-state activation curve to left (Fig. 2B). This effect was specific for steady-state activation, since there was no difference in V0.5 of the steady-state inactivation between the acclimation groups.
Acute temperature change from the acclimation temperature to the common temperature of 11°C did not have any significant effect on V0.5 of steady-state activation or steady-state inactivation in any of the fish species. However, due to the tendency of rising temperature to shift the inactivation curve to right, the V0.5 of the steady-state inactivation curve of the c.a. carp was almost 10 mV more negative than in w.a. carp, when measured at the physiological body temperatures of the fish (Fig. 3B). Furthermore, acute increases in experimental temperature slightly increased the slope factor (S) of steady-state activation in w.a. and c.a trout and the slope factor of steady-state inactivation in w.a. trout.
|
Development of rested-state inactivation of INa
The amplitude of INa decreased as a function of prepulse
duration, possibly due to direct transfer of Na+ channels from
resting closed state to inactivated closed state without intervening opening
(Fig. 3,
Table 3). The large residual
currents after long prepulses indicate that a significant proportion of the
Na+ channels failed to enter the inactivated state within 360 ms.
The proportion of non-inactivated channels was especially large in w.a. and
c.a. trout and in w.a. carp. Neither thermal acclimation nor acute temperature
changes affected the time constant of rested-state inactivation. There were no
species-specific differences in the time constant of rested-state
inactivation.
|
Recovery of INa from inactivation
The recovery of INa from inactivation at +4°C was
significantly faster in c.a. trout in comparison to c.a. carp and c.a. burbot
(Table 3, Fig. 4). Acclimation to cold
made the recovery of Na+ channels from inactivation slower in carp
ventricular myocytes, but did not affect the INa of the trout
myocytes. Acute increases in temperature slightly accelerated the recovery of
the channels from inactivation in w.a. carp and c.a. burbot.
|
Inactivation kinetics of INa
There were some prominent species-specific differences in the rate of
inactivation (Fig. 5, see also
Table 3). In c.a. fish at
+4°C, inactivation kinetics was slower in burbot and carp in comparison to
trout. Furthermore, cold-acclimation increased the inactivation rate of
Na+ channels in rainbow trout ventricular myocytes, but did not
have any effect on Na+ channels of the carp cardiac myocytes.
|
Acute temperature increases accelerated the inactivation kinetics of Na+ channels in all species. The Q10-values at 20 mV were 0.48 and 0.40 for c.a. and w.a. carp, and 0.53 and 0.50 for c.a. and w.a. trout, respectively. The Q10 for the burbot INa was 0.32 between +4°C and +11°C.
Charge transfer by INa
Na+ channels are the most significant Na+ entry
pathway in the myocyte, and this may have an affect on
excitationcontraction coupling and Na+ load imposed on the
Na+/K+-pump. Integration of INa at 20
mV indicated that the charge transfer per excitation through Na+
channels is about four times larger in burbot ventricular myocytes than in
trout or carp myocytes (Fig.
6).
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Discussion |
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Density of INa
In agreement with our hypothesis, cold acclimation reduced the density of
INa in crucian carp ventricular myocytes and increased it in
rainbow trout myocytes. The cold-induced increase in the density of trout
INa is in line with the well-known compensatory changes in size and
function of the trout heart that counteract the direct decelerating effects of
low temperature on circulation (Farrell and
Jones, 1992; Driedzic et al.,
1996
; Aho and Vornanen,
1999
,
2001
). However, thermal
compensation was only partial as the density of INa at +4°C was
59% of the density at +18°C. Similarly, the decrease in the density of
INa in carp myocytes is in agreement with the previously reported
cold-induced depression in heart rate and velocity of cardiac contraction
(Matikainen and Vornanen,
1992
; Vornanen,
1994
; Tiitu and Vornanen,
2001
). In carp, the density of INa at +4°C was only
21% of the value at +18°C, suggesting that only one fifth of the current
amplitude of the w.a. summer fish is needed to maintain cardiac excitability
in c.a. winter fish. It should be noted that due to the very negative position
of the steady-state inactivation curve in the voltage axis, all Na+
channels might not be available for opening at 120 mV and therefore
INa of the c.a. carp might have been slightly underestimated. This
does not, however, affect the conclusions since a similar reduction in
INa would be expected to occur in the physiological context.
Indeed, a temperature-dependent shift in the availability may be the means of
achieving downregulation of INa. In brief, acclimation-induced
changes in the density of INa would mean that in winter the rate of
upstroke and conduction velocity of action potentials are only moderately
depressed in rainbow trout but severely depressed in the carp.
The differences in response to acclimation between trout and carp
INa are not explained by the acute effects of temperature on
INa, and therefore must involve acclimation-induced changes in the
number of functional channels. Whether this is due to temperature-dependent
changes in transcription, translation, rate of protein degradation or
trafficking of channels to the sarcolemma remains to be clarified
(Herfst et al., 2004).
Furthermore, the significance of temperature related changes in membrane
lipids on Na+ channel function should be also examined.
Steady-state activation
In ventricular myocytes of rainbow trout, acclimation to cold caused a 6 mV
shift of the steady-state activation curve to more negative voltages. The
negative shift of steady-state activation will probably decrease stimulus
threshold for eliciting an action potential. Thus, the temperature-induced
change in voltage-dependence of steady-state activation will further improve
on that obtained by the partial compensation of current density, and thereby
maintain excitability in c.a. trout. Such a change was not observed in crucian
carp myocytes, whose Na+ channels are inherently activated at
slightly more negative voltages, as are those of the burbot myocytes.
Furthermore, the shallow slope of the activation curve in c.a. carp actually
also decreases the action potential threshold at negative voltages.
Inactivation of INa
Na+ channels were completely inactivated by a 100 msdepolarising
pulse to 20 mV and all channels recovered from inactivation within
100200 ms. Accordingly, only relatively short diastolic intervals are
needed for full recovery of cardiac excitability, and therefore it might be
anticipated that little compensatory adjustments are required upon cold
acclimation. Indeed, in rainbow trout there were no temperature-dependent
changes in time constant of recovery from inactivation. In crucian carp, the
recovery of INa from inactivation was about 44% slower in c.a. than
w.a. fish, which is in line with the depressed cardiac function in
cold-dormant crucian carp (Matikainen and
Vornanen, 1992), although its physiological importance is not
immediately clear. The slow recovery of channels from inactivation might,
however, explain the negative shift of the steady-state inactivation of the
c.a. carp at +4°C, and thus the depression of INa density.
It was hypothesised that, similar to the current density, the effect of temperature acclimation on INa kinetics might be opposite in trout and carp; however, this was found to be only partially correct. In rainbow trout, cold-acclimation induced a partial compensation in inactivation kinetics of INa, while no change was observed in carp. As a consequence of thermal acclimation the inactivation kinetics of Na+ channels is decelerated 1.8-fold and 2.6-fold in c.a. trout and c.a. carp, respectively, in comparison to w.a. species mates.
Na+ channels can directly enter the inactivated closed state
from the resting closed state without opening. The rate of rested-state
inactivation was measured at the prepulse voltage of 60 mV where
Na+ channels have a low probability of opening and where the
decrease in INa is, therefore, likely to depend on a direct
transition of Na+ channels from the resting closed state to the
inactivated state. If rested-state inactivation occurred at voltages close to
the resting membrane potential (80 mV;
Paajanen and Vornanen, 2004),
a small depolarisation of membrane would significantly compromise the
excitability of the heart. Indeed, a significant proportion of the
Na+ channels in fish cardiac myocytes was inactivated by the
60 mV prepulse, and therefore transient subthreshold depolarisation
would decrease excitability of the fish hearts. At physiological body
temperatures, the rested-state inactivation was more extensive in c.a. carp
than in w.a. carp, in agreement with more negative steady-state inactivation
curve of the c.a. carp. In contrast, there were few differences in the rate of
development of the rested-state inactivation either between acclimation groups
or between different species.
Species-specific differences
All three species of fish were acclimated and examined at +4°C, which
allowed direct comparison between their INa properties.
INa of the burbot heart resembles that of the trout heart with
respect to high current density. On the other hand, with respect to voltage
dependence of steady-state activation and inactivation, it is closer to those
of the crucian carp myocytes. The high current density and negative position
of the steady-state activation curve make the Na+ channels of the
burbot heart especially suitable for maintaining membrane excitability in the
cold.
Recovery from inactivation and the kinetics of inactivation of the burbot
heart are similar to those of the crucian carp and slower than those of the
rainbow trout. The slow kinetics of inactivation, together with high current
density, results in an approximately fourfold larger charge transfer per
excitation in burbot cardiac myocytes in comparison to those of carp and
trout. This will impose a high Na+ load on
Na+/K+-pump of the burbot cardiac myocytes and will be
energetically expensive. It might be expected that the evolutionary
preservation of such an energetically expensive mechanism in cold-adapted
species would serve some important physiological function. Intracellular
Na+ is needed for the reverse mode of
Na+/Ca2+ exchange, and therefore it could be speculated
that large INa will be used to activate Ca2+ influx
through Na+/Ca2+ exchange. Sarcolemmal Ca2+
influx by Na+/Ca2+ change might directly activate
myofilaments or serve as a trigger for further Ca2+ release from
the SR. It should be noted that in burbot ventricle significant portion of the
Ca2+ comes from the SR (Tiitu
and Vornanen, 2002a; Vornanen
et al., 2002
), while the sarcolemmal Ca2+ current is
relatively small (M. Vornanen, unpublished results). Therefore, the reverse
mode Na+/Ca2+ exchange might serve as an additional
trigger for Ca2+ release from the SR
(Vornanen et al., 1994
; Han et
al., 2001). Increased Ca2+ handling capacity of the cardiac SR (for
a recent review, see Vornanen et al.,
2002
) and enhanced aerobic ATP production of cardiac myocytes
(Rodnick et al., 1997) in cold-acclimated fish has been documented, and could
support such an excitationcontraction coupling mechanism. However, this
possibility should be directly examined by appropriate methods.
Conclusions
In ventricular myocytes of rainbow trout heart, excitability of the heart
is supported in the cold by an acclimation-induced increase in the density of
INa and leftward shift of the steady-state activation curve. In the
heart of c.a. crucian carp, the leftward shift of the steady-state activation
was absent and INa density was depressed. This difference in
response to cold acclimation may be associated with the different life styles
and habitat conditions of the two eurythermal species in the cold. Depression
of INa density and associated Na+ pumping might be
necessary manoeuvres for crucian carp to deal with the hypoxic energy
shortage, but unnecessary for trout living in oxygen-rich waters.
Due to its high density and low voltage threshold, the INa of the cold stenothermal burbot is perhaps best able to maintain cardiac excitability and conductivity in the cold. Unlike INa of trout and carp, the burbot INa allows a large Na+ influx, which suggests an additional function of INa in the burbot heart.
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Acknowledgments |
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