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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 123 478 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. 123 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). 123 480 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 123 J Comp Physiol B (2013) 183:477–489 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 123 482 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. References Aho E, Vornanen M (1998) Ca2? -ATPase activity and Ca2? uptake by the sarcoplasmic reticulum in fish heart: effects of thermal acclimation. J Exp Biol 201:525–532 Aho E, Vornanen M (1999) Contractile properties of atrial and ventricular tissue of the rainbow trout (Oncorhynchus mykiss) heart: effects of thermal acclimation. J Exp Biol 202:2663–2677 Bassani JW, Weilong Y, Bers DM (1995) Fractional SR Ca release is regulated by trigger Ca and SR Ca content in cardiac myocytes. Am J Physiol 268:C1313–C1329 Birkedal R, Christopher J, Thistlethwaite A, Shiels H (2009) Temperature acclimation has no effect on ryanodine receptor expression or subcellular localization in rainbow trout heart. J Comp Physiol B 179:961–969 Bowler K, Tirri R (1990) Temperature dependence of the heart isolated from the cold or warm acclimated perch (Perca fluviatilis). Comp Biochem Physiol A 96:177–180 Castilho PC, Landeira-Fernandez AM, Block BA (2007) Elevated Ca2? ATPase (SERCA2) activity in tuna hearts: comparative aspects of temperature dependence. Comp Biochem Physiol A 148:124–132 Chatelier A, Imbert N, Zambonino Infante JL, McKenzie DJ, Bois P (2006) Effects of oleic acid on the high threshold barium current in sea bass (Dicentrarchus labrax) ventricular myocytes. J Exp Biol 209:4033–4039 Claireaux G, Lagardere JP (1999) Influence of temperature, oxygen and salinity on the metabolism of European sea bass. J Sea Res 42:157–168 Cohen O, Kanana H, Zoizner R, Gross C, Meiri U, Stern M, Gerstenblith G, Horowitz M (2006) Altered Ca2_ handling and myofilament desensitization underlie cardiomyocyte performance in normothermic and hyperthermic heat-acclimated rat hearts. J Appl Physiol 103:266–275 Driedzic WR, Bailey JR, Septon DH (1996) Cardiac adaptations to low temperature in non-polar teleost fish. J Exp Zool 275: 186–195 Erickson JR, Sidell BD, Moerland TM (2005) Temperature sensitivity of calcium binding for parvalbumins from Antarctic and 123 488 temperate zone teleost fishes. Comp Biochem Physiol A 140:179–185 Farrell AP (1997) Effects of temperature on cardiovascular performance. In: Wood CM, MacDonald DG (eds) Global warming implications for freshwater and marine fish, society for experimental biology, seminar series 61. Cambridge University Press, Cambridge, pp 135–153 Galli GL, Lipnick MS, Block BA (2009) Effect of thermal acclimation on action potentials and sarcolemmal K? channels from Pacific bluefin tuna cardiomyocytes. Am J Physiol 297(2):R502–509 Galli GL, Lipnick MS, Shiels HA, Block BA (2011) Temperature effects on Ca2 ? cycling in scombrid cardiomyocytes: a phylogenetic comparison. J Exp Biol 214:1068–1076 Gamperl AK, Wilkinson M, Boutilier RG (1994) b-Adrenoreceptors in the trout (Oncorhynchus mykiss) heart: characterization, quantification and effects of repeated catecholamine exposure. Gen Comp Endocr 95:259–272 Gomez JP, Potreau D, Raymond G (1994) Intracellular calcium transients from newborn rat cardiomyocytes in primary culture. Cell Calcium 15:265–275 Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2? indicators with greatly improved florescence properties. J Biol Chem 260:3440–3450 Györke S, Terentyev D (2008) Modulation of ryanodine receptor by luminal calcium and accessory proteins in health and cardiac disease. Cardiovasc Res 77:245–255 Harwood CL, Howarth FC, Altringham JD, White ED (2000) Ratedependant changes in cell shortening, intracellular Ca2? levels and membrane potential in single, isolated rainbow trout (Onchorhynchus mykiss) ventricular myocytes. J Exp Biol 203:493–504 Haverinen J, Vornanen M (2009) Comparison of sarcoplasmic reticulum calcium content in atrial and ventricular myocytes of three fish species. Am J Physiol 297:R1180–R1187 Hove-Madsen L (1992) The influence of temperature on ryanodine sensitivity and the force–frequency relationship in the myocardium of rainbow trout. J Exp Biol 167:47–60 Hove-Madsen L, Llach A, Tort L (1998) Quantification of Ca2? uptake in the sarcoplasmic reticulum of trout ventricular myocytes. Am J Physiol 275:R2070–R2080 Keen JE, Farrell AP, Tibbits GF, Brill RW (1992) Cardiac physiology in tunas. II. Effect of ryanodine, calcium, and adrenaline on force–frequency relationships in atrial strips from skipjack tuna, Katsuwonus Pelamis. Can J Zool 70:1211–1217 Keen JE, Viazon DM, Farrell AP, Tibbits GF (1994) Effect of temperature acclimation on the ryanodine sensitivity of the trout myocardium. J Comp Physiol B 164:438–443 Landeira-Fernandez AM, Morrissette JM, Blank JM, Block BA (2004) Temperature dependence of the Ca2?-ATPase (SERCA2) in the ventricles of tuna and mackerel. Am J Physiol 286:R398–R404 Larsson D, Larsson B, Lundgren T, Sundell K (1999) The effect of pH and temperature on the dissociation constant for Fura-2 and their effects on [Ca2?]i in enterocytes from a poikilothermic animal, Atlantic cod (Gadus morhua). Anat Biochem 273: 60–65 Lefrançois C, Claireaux G (2003) Influence of ambient oxygenation and temperature on metabolic scope and heart rate of common sole (Solea solea). Mar Ecol Prog Ser 259:273–284 Matikainen N, Vornanen M (1992) Effect of season and temperature acclimation on the function of crucian carp (Carassius carassius) heart. J Exp Biol 167:203–220 Mercier C, Axelsson M, Imbert N, Claireaux G, Lefrançois C, Altimiras J, Farrell AP (2002) In vitro cardiac performance in triploid brown trout at two acclimation temperatures. J Fish Biol 50:117–133 123 J Comp Physiol B (2013) 183:477–489 Moller-Nielsen T, Gesser H (1992) Sarcoplasmic reticulum and excitation-contraction coupling at 10 and 20 °C in rainbow trout myocardium. J Comp Physiol B 162:526–534 Orchard CH, Lakatta EG (1985) Intracellular calcium transients and developed tension in rat heart muscle. J Gen Physiol 86: 637–651 Pelouch V, Vornanen M (1996) Effects of thermal acclimation on ventricle size, protein composition, and contractile properties of crucian carp heart. J Therm Biol 2(I):1–9 Randall DJ, Perry SF (1992) Catecholamines. In: Hoar WS, Randall DJ, Farrell AP (eds) Fish physiology: the cardiovascular system. Academic Press, New York, pp 255–300 Rivaroli L, Rantin FT, Kalinin AL (2006) Cardiac function of two ecologically distinct Neotropical freshwater fish: Curimbata, Prochilodus lineatus (Teleostei, Prochilodontidae), and trahira, Hoplias malabaricus (Teleostei, Erythrinidae). Comp Biochem Physiol A 145(3):322–327 Rousseau E, Smith JS, Meissner G (1987) Ryanodine modifies conductance and gating behaviour of single Ca2? release channels. Am J Physiol 253:C364–C368 Shattock MJ, Bers DM (1987) Inotropic response to hypothermia and the temperature-dependence of ryanodine action in isolated rabbit and rat ventricular muscle: implications for E-C coupling. Cir Res 61:761–771 Shiels HA, Farrell AP (1997) The effects of temperature and adrenaline on the relative importance of the sarcoplasmic reticulum in the contributing Ca2? to force development in isolated ventricular trabeculae from rainbow trout. J Exp Biol 200:1607–1621 Shiels HA, Farrell AP (2000) The effect of ryanodine on isometric tension development in isolated ventricular trabeculae from Pacific mackerel (Scomber japonicus). Comp Biochem Physiol A 125:331–341 Shiels HA, Stevens ED, Farrell AP (1998) Effect of temperature, adrenaline and ryanodine on power production in trout (Oncorhynchus mykiss) ventricular trabeculae. J Exp Biol 201: 2701–2710 Shiels HA, Freund EV, Farrell AP, Block BA (1999) The sarcoplasmic reticulum plays a major role in isometric contraction in atrial muscle of yellowfin tuna. J Exp Biol 202:881–890 Shiels HA, Vornanen M, Farrell AP (2002) The force–frequency relationship in fish heart. A review. Comp Biochem Physiol A 132:811–826 Tiitu V, Vornanen M (2001) Cold adaptation suppresses the contractility of both atrial and ventricular muscle of the crucian carp heart. J Fish Biol 59:141–156 Tiitu V, Vornanen M (2003) Ryanodine and dihydropyridine receptor binding in ventricular cardiac muscle of fish with different temperature preferences. J Comp Physiol B 173(4): 285–291 Vornanen M (1989) Regulation of contractility of the fish (Carassius carassius L.) heart ventricle. Comp Biochem Physiol C 94: 477–483 Vornanen M (1997) Sarcolemmal Ca influx through L-type Ca channels in the ventricular myocytes of a teleost fish. Am J Physiol 272:1432–1440 Vornanen M (1998) L-type Ca2? current in fish cardiac myocytes: effects of thermal acclimation and b-adrenergic stimulation. J Exp Biol 201:533–547 Vornanen M (1999) Na?/Ca2? exchange current in ventricular myocytes of fish heart: contribution to sarcolemmal Ca2? influx. J Exp Biol 202:1763–1775 Vornanen M (2006) Temperature and Ca2?- dependence of [3H]ryanodine binding in the burbot (Lota lota L.) heart. Am J Physiol 290:R345–R351 J Comp Physiol B (2013) 183:477–489 Vornanen M, Shiels HA, Farrell AP (2002a) Plasticity of excitationcontraction coupling in fish cardiac myocytes. Comp Biochem Physiol A 132(4):827–846 Vornanen M, Ryökkynen A, Nurmi A (2002b) Temperature-dependent expression of sarcolemmal K? currents in rainbow trout atrial and ventricular myocytes. Am J Physiol 282:R1191–R1199 489 Yue DT (1992) Relationships between intracellular free calcium and force with changes of interval. In: Noble MI, Seed WA (eds) The interval-force relationship of the heart: bowditch revisited. Cambridge University Press., Cambridge, pp 95–109 123