Download Sarcoplasmic reticulum: a key factor in cardiac contractility of sea

J Comp Physiol B (2013) 183:477–489
DOI 10.1007/s00360-012-0733-0
ORIGINAL PAPER
Sarcoplasmic reticulum: a key factor in cardiac contractility
of sea bass Dicentrarchus labrax and common sole Solea solea
during thermal acclimations
N. Imbert-Auvray • C. Mercier • V. Huet
P. Bois
•
Received: 27 April 2012 / Revised: 19 November 2012 / Accepted: 23 November 2012 / Published online: 21 December 2012
Ó Springer-Verlag Berlin Heidelberg 2012
Abstract This study investigated the effects of acclimation temperature upon (i) contractility of ventricular strips
(ii) calcium movements in ventricular cardiomyocytes
during excitation–contraction coupling (ECC), and (iii) the
role of the sarcoplasmic reticulum (SR) in myocardial
responses, in two marine teleosts, the sea bass (Dicentrarchus labrax) and the common sole (Solea solea).
Because of the different sensitivities of their metabolism to
temperature variation, both species were exposed to different thermal ranges. Sea bass were acclimated to 10, 15,
20, and 25 °C, and common sole to 6, 12, 18, and 24 °C,
for 1 month. Isometric tension developed by ventricular
strips was recorded over a range of physiological stimulation frequencies, whereas the depolarization-induced
calcium transients were recorded on isolated ventricular
cells through hyperpotassic solution application (at 100 mM).
The SR contribution was assessed by ryanodine (RYAN)
perfusion on ventricular strips and by caffeine application
(at 10 mM) on isolated ventricular cells. Rates of contraction and relaxation of ventricular strip, in both species,
Communicated by H.V. Carey.
N. Imbert-Auvray (&) C. Mercier V. Huet
Littoral Environnement et Sociétés (LIENSs), UMR 7266,
CNRS-Université de La Rochelle, 2 rue Olympe de Gouges,
17042 La Rochelle Cedex 01, France
e-mail: [email protected]
P. Bois
Institut de Physiologie et Biologie Cellulaires, FRE 3511
CNRS-Université de Poitiers, Pôle Biologie Santé Bât B36,
BP 633, 1 rue Georges Bonnet, 86022 Poitiers, France
increased with increasing acclimation temperature. At a
low range of stimulation frequency, ventricular strips of
common sole developed a positive force–frequency relationship at high acclimation temperature. In both the species, SR Ca2?-cycling was dependent on fish species,
acclimation temperature and pacing frequency. The SR
contribution was more important to force development at
low acclimation temperatures in sea bass but at high
acclimation temperatures in common sole. The results also
revealed that high acclimation temperature causes an
increase in the maximum calcium response amplitude on
ventricular cells in both the species. Although sea bass and
common sole occupy similar environments and tolerate
similar environmental temperatures, this study indicated
that sea bass and common sole can acclimatize to new
thermal conditions, adjusting their cellular process in a
different manner.
Keywords Dicentrarchus labrax Solea solea Sarcoplasmic reticulum Ventricular strips Calcium-temperature Force–frequency relationship
Abbreviations
BSA
Bovine serum albumin
CICR
Ca2?-induced Ca2?-release
DMSO Dimethyl sulfoxide
ECC
Excitation-contraction coupling
EGTA
Ethylene glycol tetraacetic acid
ICaL
L-type Ca2?-current
NCX
Na?/Ca2?exchanger
PMCA
Sarcolemmal Ca2?-ATPase pump
RYAN
Ryanodine
SERCA SR Ca2?-ATPase pumps
SL
Sarcolemma
SR
Sarcoplasmic reticulum
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Introduction
Temperature exerts a powerful influence upon the rates of
physiological processes in ectothermic teleosts, but the
effects appear to be complex. In the heart, for example, an
acute increase in temperature elevates heart rate and the
velocity of cardiac contraction, but the force of contraction
is often depressed (Vornanen 1989; Moller-Nielsen and
Gesser 1992; Hove-Madsen 1992; Keen et al. 1994; Farrell
1997). As a consequence of this negative relationship
between force and frequency, the pumping capacity of
teleost hearts is low at extreme temperatures and generally
maximized at the optimal body temperature (Farrell 1997).
The cardiovascular physiology of rainbow trout
(Oncorhynchus mykiss) has been the most thoroughly studied in comparison to other fish. Rainbow trout remain active
over a temperature range of 1–25 °C, and cold acclimation
increases heart rate, decreases the duration of contraction,
and reduces the refractoriness of the heart (Driedzic et al.
1996; Aho and Vornanen 1999). These changes are induced
by the effects of temperature upon the complex mechanisms
that regulate excitation–contraction coupling (ECC) in cardiac cells. It has been shown that calcium influx across the
sarcolemma plays a critical role in the regulation of contractility. Calcium can also enter the sarcoplasm through the
sarcolemma via the L-type calcium current during depolarization, and possibly the Na?/Ca2? exchanger (Vornanen
1999). In mammals, the resulting influx of Ca2?, in turn,
induces the release of Ca2? from the sarcoplasmic reticulum
(SR). This combined sequence of Ca2? movements elicits
cell contraction. Cardiac muscle contractility is largely
determined by the amplitude of this systolic intracellular
calcium transient (Harwood et al. 2000). The following
decrease in intracellular calcium by means of SR
Ca2?-ATPase pumps (SERCA), sarcolemmal Ca2?-ATPase
pumps (PMCA), and Na?/Ca2? exchangers is responsible
for cell relaxation. In fish, most studies examining isometric
tensions at physiological relevant pacing frequencies have
supported the view that SR Ca2? release is a relatively
unimportant source of Ca2? for contraction (Hove-Madsen
1992; Moller-Nielsen and Gesser 1992; Keen et al. 1994;
Shiels and Farrell 1997). However, further studies reported
in teleost fish suggest that the relative SR contribution to a
rise in intracellular calcium varies with the species and
acclimation temperature.
The involvement of the SR in ECC should be particularly
important in species with high heart rates and short twitch
durations (Shiels et al. 1999, 2002; Shiels and Farrell 2000).
Acclimation temperature is one primary determinant of the
ability of SR to exchange Ca2? (Keen et al. 1994; Shiels
et al. 1998; Shiels et al. 1999) and, in cold-active fish
species, acclimation to low temperatures enhances both
Ca2?-release and Ca2?-uptake by the SR.
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J Comp Physiol B (2013) 183:477–489
Despite some common features, there is much interspecific diversity in the contribution of the SR to ECC. For
example, at 18 °C the uptake rate of SR Ca2? pumps is
three- to four-fold higher in rainbow trout than in crucian
carp (Cyprinus carpio) (Aho and Vornanen 1998). The
calcium released from the SR is not, however, the major
source of the calcium required for contractile activation in
cardiac myocytes in either species under normal physiological conditions. In some active teleosts, such as tuna and
mackerel, on the other hand, most of the contractile Ca2? is
liberated from the SR (Shiels et al. 1999; Shiels and Farrell
2000; Shiels et al. 2002; Galli et al. 2011). This is also true
of some cold-stenothermal teleosts such as burbot (Lota
lota) (Tiitu and Vornanen 2001).
The aim of this study was to examine plasticity of ECC
in ventricular contractility as a function of acclimation
temperature in two poorly studied marine teleosts, the
European sea bass (Dicentrarchus labrax) and the common
sole (Solea solea). The sea bass and common sole are
abundant throughout the Mediterranean and Atlantic Europe. Although these species share the same environment,
they can be distinguished on the basis of the metabolic
susceptibility on a thermal range. For instance, the temperature range over which common sole can sustain a
metabolic rate corresponding to 80 % of their maximal
metabolic scope is 10 °C (Lefrançois and Claireaux 2003),
whereas it is only 5 °C in sea bass (Claireaux and Lagardere 1999). The thermal optimum for aerobic metabolic
rate is 22 °C for sea bass (Claireaux and Lagardere 1999)
and 18 °C for common sole (Lefrançois and Claireaux
2003). Maximal heart beat frequency is similar in both the
species and, between 15 and 25 °C, it increases by
approximately 80 %, from approximately 50 to over 90
beat min-1 (Lefrançois and Claireaux 2003). From an
ecological point of view, sea bass and common sole are
quite different. Whereas the former is an active pelagic
predator, the latter is a benthic ‘‘sit-and-wait’’ flatfish, so
they might be expected to exhibit differences in their cardiovascular physiology. Both the species are active all year
round. Each species was acclimated to four different temperatures within their thermal range and their cardiac
performance then evaluated at different levels of organizational complexity. Isometric force and rates of contraction/relaxation were measured on ventricular strips from
each species acclimated at four temperatures. Experiments
were conducted over a large range of pacing frequencies
(0.2–2 Hz). The role of the SR was investigated by ryanodine application (Rousseau et al. 1987). Therefore, to
assess the impact of acclimation temperature on calcium
movements, the calcium responses of isolated ventricular
cells were measured using laser cytofluorometry and the
calcium probe Indo-1. In addition, in order to determine
any effects of acute temperature changes upon Ca2?
J Comp Physiol B (2013) 183:477–489
movements, myocytes from fish acclimated at the four
acclimation temperatures were tested at 15 and 20 °C. The
maximum intracellular Ca2?-free mobilization was estimated through inducing cell membrane depolarization by
changing the K? gradient (Gomez et al. 1994). Total SR
Ca2? stores were evaluated by stimulation with caffeine
(Harwood et al. 2000).
Materials and methods
Fish
Sea bass (Dicentrarchus labrax) (n = 80) were obtained
from a local fish farm (St Clément, Isle of Ré) and common
sole (Solea solea) (n = 80) were caught by local fisherman
between La Rochelle and the Isle of Ré. Four groups of 20
sea bass were acclimated for a month to 10, 15, 20, or
25 °C, while four groups of 20 common sole were acclimated for a month to 6, 12, 18, or 24 °C. Fish were held in
400-l tanks supplied with aerated and biofiltered sea water
(salinity 28–30 %). They were exposed to a natural photoperiod and fed twice per week on commercial dry pellets
(sea bass) or fresh oyster and mussels (common sole). At
each acclimation temperature, half of the fish was used for
the ventricular strip work, the remainder being used for the
study of cardiomyocytes.
Ventricular tissue contractility
The preparation of cardiac tissues for measuring isometric
tension was performed using the method described in detail
by Shiels and Farrell (1997) and Mercier et al. (2002).
Briefly, at each acclimation temperature, 10 sea bass and
10 common sole were killed by cervical dislocation. The
heart was quickly excised and placed in a dish of icechilled, oxygenated physiological saline containing (in
mM): NaCl, 124; KCl, 3.1; MgSO4, 0.93; CaCl2, 2.52;
glucose, 5.6; Tes salt, 6.4; Tes acid, 3.6. The ventricle was
isolated and cut length-wise to expose the lumen. Two
1-mm-wide pieces of muscle (two ventricular strips) were
dissected from each heart. The duplicate strips were hung
in individual water-jacketed organ baths between an isometric force transducer (Somedic Medical Ltd, Sweden)
and a fixed bar. The tissue was immersed in oxygenated
saline and temperature was controlled. The muscle was
lengthened to remove slack and left for 30 min before
electrical stimulation, at a pacing frequency of 0.2 Hz, with
length adjusted to maximize tension production (i.e., such
that the preparation was operating on the plateau of the
length-tension curve, defined as Lmax). Strips were left to
stabilize at Lmax for a minimum of 30 min. Stimulation was
479
achieved using a Grass S9 stimulator (Grass Medical
Instruments, Quincy, Mass, USA) delivering current pulses
(40 V, 10 ms duration) via platinum plate electrodes
positioned on either side of the muscle. Signals from the
transducers were amplified (Somedic AB, Sweden) and
displayed on a computer with data collected and stored to
disk within LabView (version 5.0, National Instruments,
USA) at a sample rate of 10 Hz. The experimental design
tested effects of pacing frequency and ryanodine (RYAN)
on isometric tension in ventricular strips at each acclimation temperature. Because adrenaline (AD) is present at
nanomolar concentrations in the circulation of resting fish
(Randall and Perry 1992; Gamperl et al. 1994), a tonic
level of AD (1 nM) was utilized for the control situation.
The involvement of the sarcoplasmic reticulum (SR) Ca2?
in tension development was assessed using RYAN, a specific and irreversible ligand for the Ca2?-release channel of
SR. When RYAN is applied to cardiac muscle in the
micromolar range (10 lM), it locks the SR-Ca2? release
channel in the open state, rendering it unable to contribute
Ca2? to tension production (Rousseau et al. 1987). As such,
tissue sensitivity to RYAN was considered to reflect the
dependence of contractility upon Ca2? release from SR.
The isometric force–frequency relationship was studied in
sea bass and common sole, using a range of frequencies
(0.2–2 Hz) including the physiological range of cardiac
frequencies of acclimation temperatures tested (Lefrançois
and Claireaux 2003). For each force–frequency trial, the
pacing frequency was increased from 0.2 to 0.5, 0.8, 1, 1.4,
1.6, 1.8, and 2 Hz (or until the preparation became
arrhythmic, depending on the acclimation temperature of
the fish). Thus, the maximum sustainable pacing frequencies were established. After each change in pacing frequency, the muscle was allowed to stabilize for 5 min
before taking tension measurements. The pacing frequency
was returned to 0.2 Hz prior to repeating the force–frequency trial under new test conditions 30 min later. Then,
RYAN was added to one ventricular strip, the other acting
as a control, with both exposed to the same force–frequency protocols. The response differences between the
‘‘control’’ and RYAN strips revealed tissue RYAN sensitivity. Moreover, the control strip allowed any changes in
tension development due to muscle fatigue to be accounted
for in subtracting the tension changes (as a percentage of
initial tension) from experimental results. At each pacing
frequency, peak tension (mN), time-to-peak tension, timeto-50 % relaxation, and the overall rates (Peak tensionbasal force (F)/time) of contraction and 50 % relaxation
were measured.
Only physiological range of pacing frequencies has been
kept according to acclimation temperature and fish concerned (Claireaux and Lagardere 1999; Lefrançois and
Claireaux 2003).
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Measurements of intracellular calcium responses
Ventricular cardiomyocyte isolation
Fish were anaesthetized (2-phenoxy-ethanol; dilution
0.2 ml l-1) and hearts rapidly excised. Single ventricular
cells were obtained by enzymatic dissociation using a
protocol similar to that described by Vornanen (1997) and
Chatelier et al. (2006). Briefly, a cannula was inserted
through the bulbus arteriosus into the ventricle and hearts
were rinsed for 5 min with a Ca2?-free solution, to disrupt
Ca2?-dependent cellular bonds. The Ca2?-free solution
contained (in mM): NaCl, 100; KCl, 10; KH2PO4, 1.2;
MgSO4, 6.7; taurine, 50; aD-glucose, 20; Hepes, 10
(adjusted to pH 7.1 using KOH). Hearts were then perfused
for 20 min with the Ca2?-free solution complemented with
collagenase (Type IA, 0.36 mg ml-1), trypsin (Type III,
0.24 mg ml-1), and BSA (1 mg/ml). Following enzymatic
treatment, the ventricle was cut into 8–10 small pieces and
dissociated with a Pasteur pipette in a control medium
containing (in mM): NaCl, 130; CsCl, 5.4; NaH2PO4, 0.4;
MgSO4, 2.5; CaCl2, 1.8; aD-glucose, 10; Hepes, 10
(adjusted to pH 7.4 using CsOH). All chemicals were
purchased from Sigma-Aldrich (St Quentin-Fallavier,
France).
Intracellular Ca2?-free concentrations
Intracellular Ca2?-free concentration was measured using
the permeant form of the fluorescent dye Indo-1 (Indo-1/
AM, Molecular Probes, Sigma) dissolved in DMSO. Cells
were incubated for 60 min in the dark at room temperature.
The probe was diluted in the control medium. After loading, cells were washed with the control solution. Fluorescence was recorded using an interactive laser cytometer
ACAS 570 (Meridium Instruments, Okemos, MI, USA).
Excitation of Indo-1 was set in the UV range (351 and
364 nm) by means of a laser beam (5 W pulsed Argon
laser) applied at a fixed point in the cell. Each data point
corresponded to an average of 256 consecutive measurements. The fluorescence emission of both the Ca2?-free
(485 nm) and bound (405 nm) forms of the dye was collected by means of a dichroic filter and two photomultiplier
tubes located behind the band-pass filters. The intracellular
calcium concentration ([Ca2?]i) related to the fluorescence
signal ratio (R = F405/F485) was calculated from the following equation (Grynkiewicz et al. 1985):
½Ca2þ i ¼ Kd b ½ðR Rmin Þ=ðRmax RÞ:
Rmin and Rmax were determined with the control solution
containing 10 lM ionomycin 0 Ca2? ? 10 mM EGTA for
Rmin and 10 lM ionomycin ?5 mM Ca2? for Rmax. b was
calculated for each calibration procedure as well as the
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ratio of the steady F485 signal obtained in Rmin solution and
its minimum value reached in Rmax solution.
In order to determine any effects of acute temperature
changes upon Ca2? movements, myocytes were also tested
at 15 and 20 °C. The temperature of cells’ medium was
controlled placing Petri dishes in a water jacket connected
to a thermostated bath (Ministat Huber, Germany). The Kd
of the Indo-1 probe was determined at 15 and 20 °C using
the following equation (Larsson et al. 1999):
0
pKd ¼ pKd logð0:00313 TÞ
0
½ðDH =ð2:303 RÞÞ ðð1= T Þ ð1= TÞÞ
where DH = 12.5 kJ mol-1, pKd is the negative logarithm
of experimental Kd value for Indo-1 at 37 °C (Kd = 250),
R is the universal gas constant (8.314 J mol-1), T0 is the
original temperature (K), and T is the experimental temperature (310 K).
Calibration procedures were performed for each group
of fish and for each test temperature.
Experimental solutions
During experiments, cells provided by each species acclimated at each of the four appropriate temperatures were
superfused with test solutions controlled to 15 or 20 °C by
means of a custom-designed microsuperfusion device. This
device allowed rapid and effective exchange of the cell
medium.
Cells were stimulated with 100 mM K? solution made
from a control medium in which 130 mM NaCl were
replaced by 100 mM K? and 30 mM NaCl (Gomez et al.
1994). The high potassium solution perfusion changes the
potassium equilibrium potential (EK) resulting in a
decrease of inward rectifier potassium current (IK1) (Galli
et al. 2009) known to stabilize the resting membrane
potential. Thus, cardiomyocytes exposed to hyperpotassic
solution were depolarized inducing global calcium transient possibly by activation of L-type Ca2? channels and
Na?/Ca2? exchangers (in reverse mode). Caffeine solution
at 10 mM (Sigma-Aldrich; St Quentin-Fallavier, France)
was used to stimulate the release of calcium from the SR.
Caffeine is thought to release Ca2? from the sarcoplasmic
reticulum, and the amplitude of the induced [Ca2?]i transient can be used as an indicator of sarcoplasmic reticulum
Ca2? content (Bassani et al. 1995; Harwood et al. 2000;
Cohen et al. 2006). Briefly, cells were locally superfused
with incubation medium via a small delivery tube positioned near the cell (less than 1 mm). This solution was
rapidly exchanged for 100 mM K? or caffeine-containing
solution (one drop, 20 ll). Then, the stimulation of the
cardiomyocyte was stopped by the fast change of the test
solution (100 mM K? or caffeine) by the control medium.
J Comp Physiol B (2013) 183:477–489
481
Both applications were rapid to prevent excessive stimulation and possibly contracture followed by the cell death.
A
1000
Calculations and statistical analysis
800
[Ca2+]i (nM)
Analysis of isometric tension was performed as described by
Mercier et al. (2002). The baseline tension was subtracted
from the maximum tension and its associated time to calculate peak active tension and time-to-peak tension. The time
taken for relaxation to half of the recorded value from peak
tension was then recorded for time-to-50 % relaxation. The
overall rates (peak tension-basal force (F)/time) of contraction and 50 % relaxation (-F/t) were estimated by
dividing tension by time-to-peak tension and time-to-50 %
relaxation, respectively. Effects of temperature, pacing frequency and RYAN on peak tension, rate of contraction and
rate of 50 % relaxation were analyzed by multiway
ANOVA. Interactions between main factors were also tested.
A posteriori T tests for comparison of means (with P \ 0.05)
were applied following ANOVA. In order to obtain normality and homogeneity of variances (Kolmogorov–Smirnov
and Fmax tests, respectively), tension and rates of contraction
and 50 % relaxation were transformed as [SQRT(x)]-1. Data
were expressed as mean ± SEM (n fish).
The effects of acclimation temperature and test
temperature on cellular calcium content were tested using
two-way ANOVA (a = 0.05). Prior to ANOVA testing,
normality and homogeneity of variance were checked
using Kolmogorov–Smirnov and Fmax tests. Data were logtransformed when necessary. Following stimulation with
hyperpotassic or caffeine solutions, different variables
were considered, as represented in Fig. 1. The maximal
amplitude of calcium mobilized, the kinetics of the calcium
increase (ii) and the kinetics of the calcium decrease (id)
were analyzed by two-way ANOVA (a = 0.05), with the
acclimation temperature and test temperature being used as
sources of variation. When test temperature was not found
to have a significant effect, data sets were pooled and the
effect of acclimation temperature was tested using one-way
ANOVA. Following ANOVA tests, a posteriori T0 -tests for
comparison of means with P \ 0.05 were applied.
B
100 mM K+
600
Amplitude (nM)
Caffeine (10 mM)
τ d (sec)
τ i (sec)
400
200
0
10 s
10 s
Fig. 1 Calcium transients in 20 °C-acclimated sea bass. a In this
example, the ventricular myocyte was stimulated with hyperpotassic
solution (bold line indicates time superfusion, 100 mM K?) and
calcium movements were monitored using Indo-1 and laser cytofluorometry. The vertical arrow indicates the maximal amplitude of
calcium response (nM). si and sd (blue arrows) quantify, respectively,
the kinetics of increase and decrease of intracellular calcium
concentration. b Example of calcium transient obtained with caffeine
stimulation (bold line indicates the time superfusion, 10 mM)
progressive flattening of a negative relationship. At 24 °C,
the slope became positive, with force increasing with frequency for pacing rates \1 Hz. At stimulation frequencies
higher than 1 Hz, however, the tension generated
decreased with stimulation frequency. In sea bass, the same
general trend prevailed, but changes were less profound
and no clear inversion of the slope of the force–frequency
relationship was observed as temperature increased.
Contraction and relaxation rates
Ventricular tissue contractility
In sea bass, rates of contraction (RC) and rates of 50 %
relaxation (RR) increased progressively with increasing
acclimation temperature (P \ 0.05; Fig. 3). In common
sole strips stimulated between 0.2 and 0.8 Hz, RC and RR
were the fastest at 12 °C and the lowest at 6 °C (P \ 0.05;
Fig. 4). RC and RR in common sole acclimated to 18 and
24 °C were not significantly affected by stimulation frequency. Comparison of Figs. 3 and 4 reveals an interesting
difference between the two species. In sea bass, a 15 °C
rise in water temperature resulted in a three-fold increase in
RC and RR. On the other hand, in common sole, a temperature increase of the same order of magnitude had a
much more limited impact, casting doubt upon the overall
physiological relevance of the temperature effects in this
species, despite their statistical significance.
Force–frequency relationships of isometric contractions
Involvement of SR in tissue contractility
In both the species, the shape of force–frequency relationship was profoundly influenced by acclimation temperature (P \ 0.05; Fig. 2). In common sole, increasing the
acclimation temperature from 6 to 18 °C resulted in a
In both the species, development of isometric tension by
ventricular strips was significantly depressed by RYAN
(P \ 0.05; Fig. 5). In sea bass, RYAN reduced tension
development, particularly at low temperatures (-49 % at
Results
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J Comp Physiol B (2013) 183:477–489
Sea bass
180
300
25°C
20°C
160
250
15°C
140
Rate (mN.sec-1)
% of tension at low AD nd 0.2 Hz
A Sea bass
10°C
120
100
80
200
150
25°C
20°C
100
15°C
50
60
10°C
0
40
0.2
0.5
0.8
1
1.2
1.4
1.6
1.8
0.2
2
0.5
0
B Common sole
1
1.2
1.4
1.6
1.8
2
2
0.2
0.5
0.8
1
1.2
1.4
1.6
1.8
2
0
24°C
180
18°C
160
50
12°C
140
Rate (mN.sec-1)
% of tension at low AD and 0.2 Hz
0.8
Stimulation frequency(Hz)
Stimulation frequency (Hz)
6°C
120
100
80
100
150
60
200
40
0.2
0.5
0.8
1
1.2
1.4
1.6
1.8
2
Stimulation frequency (Hz)
Fig. 2 Effect of acclimation temperature on force–frequency relationships in sea bass (a) and common sole (b) ventricular strips.
Isometric tension is presented as a percentage of the tension measured
at 0.2 Hz in saline with 1 nM adrenaline. All results are means ± SEM (n = 10). Statistical differences are described in the text
10–15 °C vs -23 % at 20–25 °C; P \ 0.05). When stimulation frequency increased, the impact of RYAN significantly
decreased at 15, 20, and 25 °C (P \ 0.05), but not at 10 °C.
In common sole, the depressive effect of RYAN upon
isometric tension was strong at high temperatures (63 and
49 %, respectively, at 18 and 24 °C) but was weaker at low
acclimation temperatures (24 and 19 %, respectively, at 6
and 12 °C). In both the species, cold-acclimation (10 °C in
sea bass; 6 and 12 °C in common sole) was associated with
weakly positive relationships between RYAN-related inhibition of tension and stimulation frequency. In warm-acclimated fish (15, 20, and 25 °C in sea bass; 18 and 24 °C in
common sole), a strong negative slope was observed.
Fig. 3 a Effect of acclimation temperature on the relationship
between the rate of contraction and stimulation frequency in sea
bass ventricular strips. b Effect of acclimation temperature on the
relationship between the rate of 50 % relaxation and stimulation
frequency. All results are means ± SEM (n = 10). Statistical differences are described in the text
20 °C) had no significant effect upon the maximal amplitude
of the calcium responses in cells stimulated with the hyperpotassic solution (100 mM K?). That is why, data from
the two test temperatures were pooled (Fig. 6). On the other
hand, acclimation temperature did influence the maximal
amplitude of calcium responses (P \ 0.05 for sea bass and
P \ 0.0001 for common sole, Fig. 6). On average, the
maximal amplitude of calcium responses observed in common sole was nearly twice as large as in sea bass. Unlike sea
bass, acclimation temperature modified the kinetics of Ca2?
movements in common sole (P \ 0.05; Table 1). The
kinetics of the increase (si) and decrease (sd) in intracellular
calcium following hyperpotassic stimulation were 45 and
53 % higher, respectively, at 24 °C compared with 6 °C.
Responses of ventricular cells to caffeine
Intracellular Ca2?-free concentrations
Maximal Ca2?-responses by ventricular cells
Hyperpotassic solution (100 mM K?) was used to induce
depolarization of cardiomyocytes and thus Ca2? mobilization. In both the species, acute temperature change (15 or
123
In order to evaluate the calcium content of the SR, in both
the species, according to the temperature (acute and
acclimation), caffeine (10 mM) was used as it was known
to induce the release of calcium content from the SR. In sea
bass, both acute temperature change and the underlying
acclimation temperature had a significant effect upon the
J Comp Physiol B (2013) 183:477–489
Common sole
70
A Sea bass
6°C
90
60
12°C
50
24°C
% reduction in tension due to
ryanodine
Rate (mN.sec-1)
483
18°C
40
30
20
25°C
20°C
15°C
60
10°C
30
0
10
0
0.2
0.5
0
0.2
0.5
0.8
1
1.2
1.4
1.6
1.8
0
2
1
1.2
1.4
1.6
1.8
Rate (mN.sec-1)
20
30
40
50
60
Fig. 4 a Effect of acclimation temperature on the relationship
between the rate of contraction and stimulation frequency in common
sole ventricular strips. b Effect of acclimation temperature on the
relationship between the rate of 50 % relaxation and stimulation
frequency. All results are means ± SEM (n = 10). Statistical differences are described in the text
amplitude of the response to caffeine (P \ 0.05; Fig. 7a).
A significant interaction between the two temperature
factors was also found. When cells were tested at 15 °C,
the maximal amplitude of caffeine-responses increased
significantly between acclimation temperatures of 15 and
20 °C. Nevertheless, when cells were tested at 20 °C, this
effect was not observed. In common sole, acute temperature change had no effect. On the other hand, the maximal
amplitude of caffeine responses increased significantly
with acclimation temperature (from approximately 370 nM
at 6 °C to 900 nM at 12–24 °C; P \ 0.05, Fig. 7b).
In both the species, the kinetics of caffeine-responses
were not affected by either acute temperature change or by
acclimation temperature (Table 1). Nevertheless, si and sd
were higher in common sole (approximately 23 s over the
range of acclimation temperatures tested here) by comparison with sea bass (16 and 13 s, respectively, for si and sd).
Discussion
The aim of this study was to examine the specific plasticity
of the ventricular contractility function to the temperature
0.8
1
1.2
1.4
1.6
1.8
2
B Common sole
2
90
% reduction in tension due to
ryanodine
0.8
0.5
Stimulation frequency (Hz)
Stimulation frequency (Hz)
10
0.2
24°C
18°C
12°C
60
6°C
30
0
0
0.2
0.5
0.8
1
1.2
1.4
1.6
1.8
2
Stimulation frequency (Hz)
Fig. 5 Inhibition of ventricular strip isometric tension following
treatment by ryanodine in sea bass (a) and common sole (b).
Ryanodine (10 lM) was added to the organ bath under tonic
adrenergic stimulation (1 nM). Results are means ± SEM. Statistical
differences are described in the text
Fig. 6 Effects of acclimation temperature on the maximal amplitude
of intracellular calcium responses ([Ca2?]i) in sea bass and in
common sole. Ventricular myocytes were stimulated by hyperpotassic
solution (100 mM K?). Results are means ± SEM with the number
of cells tested indicated above each mean. ANOVA analysis revealed
no significant effect of test temperature. Consequently, data obtained
on cells tested at 15 and 20 °C were grouped for each acclimation
temperature. Bars sharing the same letter are not significantly
different (P [ 0.05)
123
484
J Comp Physiol B (2013) 183:477–489
Table 1 Effects of acclimation temperature on the kinetics of increase (ii) and decrease (id) of intracellular calcium concentration following
stimulation of sea bass and common sole ventricular myocytes with caffeine (10 mM) or hyperpotassic solution (100 mM)
Stimulation calcium kinetics
K-Evoked calcium increase (100 K)
ii (s)
Caffeine
id (s)
ii(s)
id (s)
Sea bass, 10 °C
23.8 ± 5.0 (15)
25.4 ± 6.4 (8)
17.2 ± 3.3 (10)
12.4 ± 2.7 (13)
Sea bass, 15 °C
23.4 ± 5.7 (10)
22.8 ± 6.5 (9)
6.7 ± 1.3 (7)
12.3 ± 1.5 (8)
Sea bass – 20 °C
Sea bass, 25 °C
26.2 ± 2.8 (16)
16.9 ± 2.9 (12)
19.0 ± 3.8 (15)
26.4 ± 5.1 (12)
22.6 ± 4.3 (8)
15.6 ± 3.3 (7)
15.6 ± 5.2 (8)
9.8 ± 3.4 (4)
Common sole, 6 °C
17.3 ± 2.9 (26)
17.2 ± 4.3 (15)
21.6 ± 3.1 (14)
14.7 ± 4.6 (8)
Common sole, 12 °C
25.5 ± 2.8 (19)
15.5 ± 4.7 (14)
21.6 ± 4.7 (15)
14.6 ± 5.0 (13)
Common sole, 24 °C
36.4 ± 3.5* (10)
26.0 ± 10.1* (8)
19.0 ± 5.0 (12)
12.4 ± 4.9 (8)
All results are means ± SEM. Number of cells is indicated in parentheses
Maximal amplitude (nM)
* Values are statistically different from those obtained at 6 °C (P \ 0.05)
1200
Sea bass tested at 15°C
1000
Sea bass tested at 20°C
b15
800
b9
600
400
a12
a9
10
15
a12
a9
a9
a8
200
0
20
10
25
15
20
25
Maximal amplitude (nM)
Acclimation temperature (°C)
b20
1200
Common sole
b15
1000
800
600
a18
400
200
0
6
12
24
Acclimation temperature (°C)
Fig. 7 Effects of acclimation and test temperatures on the maximal
amplitude of intracellular calcium responses ([Ca2?]i) of ventricular
cardiomyocytes of sea bass (a) and common sole (b) stimulated with
caffeine (10 mM). Results are means ± SEM with the number of
cells tested indicated above each mean. ANOVA analysis revealed no
significant effect of test temperature in common sole. Consequently,
data obtained on cells tested at 15 and 20 °C in common soles were
grouped for each acclimation temperature. Bars sharing the same
letter are not significantly different (P [ 0.05)
in the European sea bass (Dicentrarchus labrax) and the
common sole (Solea solea). To assess the specific thermal
constraints on their cardiac performance, studies were
conducted at different levels of organization. This study
reports in two poorly studied marine teleosts, sea bass and
common sole, a significant effect of acclimation temperature upon calcium-handling capacity and contractility at
both cellular and tissue levels. The main fact is that sea
123
bass and common sole can acclimatize to new thermal
conditions, adjusting their cellular process in a different
manner. In this study, we showed that SR can be an
important source of activation for the contraction, according to (i) acclimation temperature, (ii) pacing frequencies,
and (iii) specie.
Previous studies have extensively examined and discussed force–frequency relationships (Shiels and Farrell
1997; Aho and Vornanen 1999; Shiels et al. 1999; Vornanen 1999). The negative force–frequency relationship
was usually a typical phenomenon in cardiac muscle of
many teleosts. In both the species already studied, the
shape of force–frequency relationship was profoundly
influenced by acclimation temperature. Ventricular strips
of sea bass and common sole show a positive force–frequency relationship for low frequencies when acclimation
temperature increased.
In common sole, increasing the acclimation temperature
resulted in a progressive flattening of a negative relationship with a positive slope at 24 °C for pacing rates less than
1 Hz. In sea bass, the same general trend prevailed but
even if changes were less profound. Moreover, ventricular
strips from both the species exhibited more rapid rates of
contraction and relaxation with increasing acclimation
temperature. At the cellular level, the maximal amplitude
of calcium responses in depolarized ventricular myocytes
increased significantly with increasing acclimation temperature. In both the species, we found significantly greater
quantities of calcium in the SR at high acclimation temperatures. However, the SR contribution was more
important to force development at low acclimation temperatures in sea bass but at high acclimation temperatures
in common sole.
Changes in force development are directly related to the
intracellular calcium transients involved in ECC (Orchard
and Lakatta 1985; Shattock and Bers 1987; Yue 1992)
during action potential. The sarcolemmal depolarization
J Comp Physiol B (2013) 183:477–489
induces the activation of both L-type Ca2? channels and
Na?/Ca2? exchangers (in reverse mode). The resulting
influx of calcium into the cell, in turn, induces the release
of Ca2? (CICR) from the sarcoplasmic reticulum (SR).
This sequence of Ca2? movements contributes to generate
contraction. Conversely, the decrease in intracellular calcium by means of SR Ca2?-ATPase pumps (SERCA),
sarcolemmal Ca2?-ATPase pumps (PMCA) and Na?/Ca2?
exchangers (forward mode) is responsible for relaxation.
Temperature influences myocardial twitch force development by several mechanisms. In sedentary teleosts, the
sarcolemmal calcium current is the most temperaturesensitive cellular mechanism involved in ECC (Vornanen
1998, 1999; Vornanen et al. 2002a). At low acclimation
temperatures, thermal acclimation had no effect on the
density of cardiac ICaL in trout (Vornanen 1998), but the
rate of current inactivation was accelerated. Consequently,
the contribution of sarcolemmal Ca2? flux to the total
intracellular calcium concentration was less in cold-acclimated trout than in warm-acclimated trout (Vornanen
1998; Vornanen et al. 2002a). In this study, we did not
directly measure calcium currents. However, decreased
maximum amplitudes of calcium responses in cold-acclimated sea bass and common sole are consistent with a
similar thermal effect upon the activity of L-type calcium
channels. In addition to L-type, Na?/Ca2? exchangers
could make a significant contribution to contractile Ca2?
entry (33–50 % of the total sarcolemmal Ca2? influx at
22 °C) in fish cardiac myocytes (Vornanen et al. 2002a).
But, previous studies on salmonids and carp revealed a
relatively weak effect of acclimation temperature upon the
density of NCX in isolated ventricular cells (Hove-Madsen
et al. 1998; Vornanen et al. 2002a). Further studies are
needed to clarify the contribution of the NCX and/or ICaL
to sarcolemmal calcium influx and their temperature
dependency in sea bass and in common sole.
Over the range of acclimation temperatures considered,
the amplitude of the calcium responses was greater in
common sole than in sea bass. Moreover, the kinetics of the
increase (si) and decrease (sd) in intracellular calcium
transients were significantly higher in common sole acclimated to high temperature, but did not change in sea bass.
These results indicate that there are interspecific differences in the temperature-dependence of the mechanisms
involved in ECC. At high acclimation temperatures, the
membrane permeability of calcium could be higher in
common sole than in sea bass. Indeed, caffeine-induced
Ca2?-release experiments showed that the SR of sea bass
and common sole can store significant amounts of Ca2? at
high acclimation temperatures. Consequently, when the
sarcolemma was depolarized by 100 K solution, SL Ca2?
entry can induce the release of Ca2? from the SR via CICR.
At high acclimation temperature, the calcium amount
485
released from the SR could be higher in common sole than
in bass, as shown by the higher calcium transients induced
by caffeine.
Sea bass and common sole are active all year round.
Several studies suggest that, in species that remain active at
low ambient temperature (rainbow trout, burbot), adaptation to cold is associated with an increased Ca2?-handling
capacity of the SR (Aho and Vornanen 1999; Keen et al.
1994). For example, cold acclimation is accompanied in
trout by increases in rate of force development and faster
mechanical restitution, allowing the trout heart to attain a
higher maximum pacing frequency at a colder temperature
(Shiels and Farrell 1997; Aho and Vornanen 1999). In
cold-acclimated perch or trout, the proliferation of SR
increases SR-Ca2? uptake and, consequently, the amount
of calcium available for contractile myofilaments (Bowler
and Tirri 1990; Aho and Vornanen 1999). On the other
hand, a decrease in the cardiac Ca2?-ATPase (SERCA2)
activity was observed in cold-dormant species such as carp
(Aho and Vornanen 1998). Low SERCA2 activity at low
temperature is likely to slow down relaxation rates in the
ventricle. Tuna species that inhabit cold water would have
an increased-SR-Ca2? intake capacity compared with
tropical tunas (Landeira-Fernandez et al. 2004; Castilho
et al. 2007). Our study revealed a pattern of temperaturesensitivity that is roughly different. In this study, it is
notable that, except at 12 °C in common sole, kinetics of
isometric contractions are faster at warmer acclimation
temperatures. This cannot be attributed to an increase in the
rate of calcium transients or with a greater participation of
SR as, at 12 °C, contraction is less affected by ryanodine
than at 18 or 24 °C. The fast kinetics of isometric contractions could be associated with a positive thermal
compensation in the activity of myofibrillar ATPase (Aho
and Vornanen 1999).
The difference between acclimation groups may reflect
temperature-induced changes in calcium regulation. Our
results at the multicellular level are consistent with a higher
and faster calcium mobilization observed in ventricular
cells of warm-acclimated sea bass and common sole. The
more rapid mobilization of great quantities of calcium
in common sole at high acclimation temperatures (i.e.,
increase of kinetics si and sd) associated with a greater
SR-involvement might also explain why twitch force was
more efficient and increased at low pacing frequency.
In trout ventricular cells, Harwood et al. (2000) observed a
decrease of approximately 30 % in intracellular calcium
concentration after stimulation at increasing pacing frequency (from 0.2 to 1.4 Hz). In salmonids, this decrease in
intracellular calcium at high stimulation frequency has
been linked to the activity of ICaL, NCX, and SR (Shiels
et al. 2002). For example, when stimulation frequency was
increased from 0.2 to 1 Hz, the calcium current amplitude
123
486
(ICaL) decreased by 20 % in ventricular myocytes of
rainbow trout (Harwood et al. 2000). Moreover, increasing
stimulation frequency reduced the duration of action
potential by increasing the K? current (IKr) density and,
consequently, decreased the influx of calcium through the
NCX (Shiels et al. 2002; Vornanen et al. 2002b). In the
majority of teleosts, the rates of uptake and recapture of
Ca2? by SR have been found to be inconsistent with
maintaining appropriate calcium release at high stimulation
frequency. Taken together, the above limitations are put
forward to explain the negative force/frequency relationship described in salmonids (see for review Shiels et al.
2002). Unlike salmonids, the slope of the relationship
between isometric force and stimulation frequency was
sensitive to both acclimation temperature and frequency in
our study. In common sole, this interaction was quite
obvious, with the slope of the force–frequency relationship
increasing from -80 at 6 °C to ?70 at 24 °C for pacing
frequencies \1 Hz. In sea bass, the same trend was
observed, although it was less marked. These results suggest that the ability of both common sole and sea bass
myocytes to exchange calcium improves with rising temperature, possibly allowing increased cardio-vascular work
at physiologically representative stimulation rates. These
responses were associated with a marked increase in the
rates of contraction and relaxation with increasing acclimation temperatures in sea bass, a trend that was not as
clear in common sole. In common sole, acclimation to high
temperatures increased the kinetics of the rise and fall in
intracellular calcium during cell depolarization, unlike the
case for sea bass. Moreover, the amplitude of calcium
responses by ventricular cells was quantitatively more
important in warm-acclimated common sole than in warmacclimated sea bass. These results suggest that common
sole can increase force-generating ability at high acclimation temperatures and for low stimulation frequencies by
increasing (i) the amount of calcium mobilized and (ii) the
kinetics of the calcium transients. Positive force–frequency
relationships have previously been described in very active
teleosts such as tuna (Keen et al. 1992; Shiels et al. 1999)
and mackerel (Shiels and Farrell 2000). A negative force–
frequency relationship was observed in cold-acclimated
(5 °C) carp, whereas warm-acclimated (15 °C) carp displayed a positive force–frequency relationship (Matikainen
and Vornanen 1992). In both the species studied here, the
release of calcium from the SR represents a greater fraction
of total sarcolemmal calcium influx than in salmonids.
When SR calcium cycling is blocked by RYAN, a 20 %
reduction in ventricular strip tension development is
observed in pacific mackerel (Shiels and Farrell 2000),
30 % in skipjack tuna (Keen et al. 1992), and 40 % in
yellow tuna (Shiels et al. 1999). However, it would be
wrong to conclude that the role of SR Ca2? cycling was
123
J Comp Physiol B (2013) 183:477–489
more important in common sole compared to other species
because, in this study, relative force was used and not
absolute force (Shiels et al. 2002). Over the range of
acclimation temperatures tested here, the percentage of
tension depression following RYAN application ranged
from 20 to 75, depending on the stimulation frequency. In
cold-acclimated common sole (6 and 12 °C), inhibition by
RYAN was small at low pacing frequency and tended to
increase with stimulation frequency. The same was
observed in 10 °C-acclimated sea bass. In warm-acclimated common sole and sea bass, on the other hand,
increasing pacing frequency reduced the RYAN-related
inhibition of force development.
How stimulation frequency and acclimation temperatures interact to affect the SR is unclear in teleosts. Shiels
et al. (1999) found an increased contribution of SR calcium
release to force production at high pacing frequencies in
yellowfin tuna. Similar to our finding on sea bass, numerous studies suggest a higher contribution of the SR to ECC
in cold-acclimated fish (rainbow trout, Keen et al. 1994;
Hove-Madsen et al. 1998; Aho and Vornanen 1999; carp,
Aho and Vornanen 1998; Vornanen et al. 2002a). At low
acclimation temperatures (4 °C), the velocity of calcium
release from SR and of calcium uptake is increased in carp.
In perch (Perca fluviatilis), the fractional volume of cardiac
cells occupied by sarcoplasmic reticulum was greater in
cold-acclimated fish in comparison with warm-acclimated
fish (Bowler and Tirri 1990) and the amount of calcium
released increased. However, although cardiac enlargement
was induced by cold in the crucian carp (Pelouch and
Vornanen 1996), caffeine increased the force of contraction
more in the hearts of warm-acclimated than cold-acclimated fish. The differences in SR responses revealed in
common sole by comparison with sea bass may reflect
differences in the expression of SR calcium-release channels, differences in the sensitivity to RYAN (Vornanen
2006) and/or differences in the expression/activity of calcium pump ATPase in the SR. Aho and Vornanen (1998)
have demonstrated a clear species-specific difference in SR
calcium uptake rate between crucian carp and rainbow
trout heart, with calcium pumps of the latter being three- to
four-fold more active.
In common sole, following treatment of isolated myocytes with caffeine, we found total SR calcium stores to
have faster release and uptake kinetics. This faster release
of calcium at high acclimation temperature (24 °C) may be
explained by a higher calcium sensitivity of the RYAN
receptor (Vornanen 2006) during CEC, whereas an
increased SERCA2 protein content (Ca2?-ATPase) could
be associated with the higher Ca2?-uptake by SR (Landeira-Fernandez et al. 2004). Although the physiological
responses of SR established by treating ventricular strips
with RYAN differed between the two species, the effect of
J Comp Physiol B (2013) 183:477–489
warm acclimation on total SR calcium stores was similar
when assessed at the myocyte level using caffeine. Acclimation to high temperatures (20–25 °C) elevated total SR
calcium stores in both the species. As previously observed
(Haverinen and Vornanen 2009), our study confirmed that
fish cardiac myocytes can contain important SR Ca2?
stores, whereas SR makes a relatively small contribution to
contraction as already supposed by Haverinen and Vornanen (2009). These results could be explained by the action
of temperature upon SR membrane permeability (Castilho
et al. 2007). The total number of RYAN receptors seems
not to be affected by temperature acclimation (Tiitu and
Vornanen 2003, crucian carp; Birkedal et al. 2009, rainbow
trout). Acclimation temperature could also affect the
affinity of the calcium-binding proteins (Erickson et al.
2005) and, more especially, the calcium capacity of calsequestrin, the SR Ca2? buffer known to modulate the SR
Ca2? load and the activity of the ryanodine receptor
(Györke and Terentyev 2008).
Moreover, cardiomyocytes from sea bass acclimated at
high acclimation temperatures (20 and 25 °C) were more
sensitive to acute change of temperature than cardiomyocytes from common sole, especially when measure of
calcium loading of the SR was tested with caffeine at 15 °C
rather than 20 °C. In common sole, calcium content
released by caffeine application did not show any significant difference regardless the test temperature (acute
change 15 or 20 °C), whereas calcium mobilization due to
caffeine was significantly higher when stimulation was
done at 15 °C rather 20 °C in cardiomyocytes from sea
bass. As mentioned previously, we have shown that
acclimation to cold increased RYAN sensitivity of contraction in the sea bass heart with a maximum effect when
fish were acclimated at 15 °C. This means that in sea bass,
the contribution of SR to contraction is the most important
when fish are acclimated at 15 °C compare to other
acclimation temperatures. These results suggest that in sea
bass (i) acclimation temperature affects calcium-loading
capacity of the SR and (ii) acute change of temperature
affects calcium mobilization of SR content. Confirmation
of this hypothesis would require testing the effects of acute
changes of temperature on ventricular strips from sea bass
acclimated at various temperatures.
In conclusion, during seasonal temperature variations,
sea bass and common sole can acclimatize to new thermal
conditions, adjusting their cellular process. This study
shows that, in both the species, SR Ca2?-cycling is
dependent on fish species, acclimation temperature and
pacing frequency. At high acclimation temperatures, isolated ventricular cells mobilized a larger quantity of calcium and the isometric force developed by ventricular
strips increased with the pacing frequencies. Nonetheless,
sea bass and common soles exhibited some differences in
487
thermal sensitivity of the mechanisms involved in ECC. In
common sole, the involvement of the SR was more
important at high temperatures, but not in sea bass. We can
hypothesize that involvement of the SR in ECC in warmacclimated common soles allows high heart rates. In sea
bass, like other active fish species at cold temperatures, SR
could compensate for the effects of low temperatures on
ventricular contractility. This hypothesis reinforces the
statement that a functional SR would be an intrinsic characteristic of species presenting high swimming performance (Keen et al. 1992; Aho and Vornanen 1999; Shiels
et al. 1999; Rivaroli et al. 2006). Quite clearly, more
studies are needed at the molecular level to improve (i) our
understanding of the diversity and plasticity of cardiac
physiology in teleost fish and (ii) the physiological significance of these acclimation responses.
Acknowledgments We are indebted to the Aquarium of La
Rochelle and N.Vallee for their technical assistance. The study was
funded by the Région Poitou–Charentes, the CNRS and IFREMER.
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