Acute temperature change can be cardioplegic to mammals, yet certain ectotherms maintain their cardiac scope over a wide temperature range. To better understand the acute effects of temperature on the ectothermic heart,we investigated the stimulus-induced change in intracellular Ca2+concentration ([Ca2+]i; cytosolic Ca2+transient) in isolated rainbow trout myocytes at 7°C, 14°C and 21°C. Myocytes were voltage-clamped and loaded with Fura-2 to measure the L-type Ca2+ channel current (ICa) and[Ca2+]i during physiological action potential (AP)pulses at frequencies that correspond to trout heart rates in vivo at 7°C, 14°C and 21°C. Additionally, [Ca2+]iand ICa were examined with square (SQ) pulses at slow (0.2 Hz) and physiologically relevant contraction frequencies. The amplitude of[Ca2+]i decreased with increasing temperature for both SQ and AP pulses, which may contribute to the well-known negative inotropic effect of warm temperature on contractile strength in trout hearts. With SQ pulses, [Ca2+]i decreased from 474±53 nmol l-1 at 7°C to 198±21 nmol l-1 at 21°C,while the decrease in [Ca2+]i with AP pulses was from 234±49 nmol l-1 to 79±12 nmol l-1,respectively. Sarcolemmal Ca2+ influx was increased slightly at cold temperatures with AP pulses (charge transfer was 0.27±0.04 pC pF-1, 0.19±0.03 pC pF-1 and 0.13±0.03 pC pF-1 at 7°C, 14°C and 21°C, respectively). At all temperatures, cells were better able to maintain diastolic Ca2+levels at physiological frequencies with AP pulses compared with 500 ms SQ pulses. We suggest that temperature-dependent modulation of the AP is important for cellular Ca2+ regulation during temperature and frequency change in rainbow trout heart.

Cardiac contractility is profoundly influenced by temperature in all vertebrates. This is not surprising as the transient rise in intracellular Ca2+ concentration ([Ca2+]i), which underlies excitation—contraction coupling, represents the culmination of many temperature-sensitive cellular processes; i.e. action potential configuration(Møller-Nielsen and Gesser,1992; Puglisi et al.,1999; Coyne et al.,2000), myofilament Ca2+ sensitivity(Harrison and Bers, 1990; Churcott et al., 1994; Gillis et al., 2000) and Q10 effects on protein pumps and ion channels(Sitsapesan et al., 1991; Herve et al., 1992; Xue et al., 1999; Kim et al., 2000; Shiels et al., 2000). In most vertebrates, the transient change in [Ca2+]i in response to stimulation is the product of both extracellular Ca2+ influx across the sarcolemmal membrane (SL) and intracellular Ca2+ release from the sarcoplasmic reticulum (SR), although the relative Ca2+contribution from each source may vary(Fabiato and Fabiato, 1978; Driedzic and Gesser, 1985;Bers, 1985, 1989; Tanaka and Shigenobu, 1989; Hove-Madsen, 1992; Bassani et al., 1994).

In mammals, the temperature sensitivity of extracellular and intracellular Ca2+ fluxes are well characterized. Transsarcolemmal Ca2+ influx via the L-type Ca2+ channel current(ICa) is strongly influenced by temperature, with a Q10ranging from 2 to 3.5 (Cavalie et al.,1985; Herve et al.,1992). Cold temperatures reduce SR function in many mammalian hearts, impairing both release (Bers,1987; Sitsapesan et al.,1991) and uptake (Liu et al., 1991, 1997; Wang et al., 2000) of SR Ca2+ stores. Under most conditions, excitation—contraction coupling in rainbow trout heart is thought to be driven primarily by extracellular Ca2+ influx via ICa, with SR Ca2+ release playing a secondary role(Tibbits et al., 1992; Hove-Madsen, 1992; Keen et al., 1994; Shiels and Farrell, 1997). Recent studies of ICa in trout myocytes indicate that the temperature sensitivity of peak current amplitude is not very different from that of mammals (Q10 of approximately 1.7-2.1; Kim et al., 2000; Shiels et al., 2000) but that the total transsarcolemmal Ca2+ influx is fairly well maintained during temperature change (Shiels et al.,2000). Furthermore, the fish cardiac SR has been shown to uptake and release Ca2+ at temperatures well below those known to inhibit SR function in most mammals (Aho and Vornanen, 1999; Hove-Madsen et al., 2001; Shiels et al.,2002a; Tiitu and Vornanen,2002).

Despite the fact that the individual Ca2+ flux pathways in trout hearts seem to demonstrate some tolerance to temperature change, in situ heart and in vitro cardiac muscle strip studies show both inotropic and chronotropic responses to temperature change(Keen et al., 1994; Shiels and Farrell, 1997; Aho and Vornanen, 1999). We wanted to understand to what extent these responses might be mediated by changes in cellular Ca2+. The transient change in[Ca2+]i during excitation—contraction coupling represents the sum of both extracellular and intracellular Ca2+flux, and its amplitude is directly related to the strength of cardiac contraction (Yue, 1987). Thus,the focus of the present study was to examine [Ca2+]i at different temperatures in rainbow trout atrial myocytes. Previous studies have shown that trout experience acute temperature fluctuations of as much as±10°C either while transversing thermoclines or as a result of diurnal changes in shallow streams (Reid et al., 1997; Matthews and Berg, 1997). Thus, we conducted our studies on 14°C-acclimated rainbow trout at 7°C, 14°C and 21°C. By simultaneously quantifying[Ca2+]i and ICa at each of these temperatures, we were able to assess the effects of temperature on[Ca2+]i and indirectly separate the effect of temperature on extracellular and intracellular Ca2+ fluxes.

In fish hearts, ambient temperature also directly modulates heart rate(Farrell and Jones, 1992). In turn, changes in contraction frequency affect many of the cellular processes that underlie the transient change in [Ca2+]i(Møller-Nielsen and Gesser,1992; Hove-Madsen and Tort,1998). For example, increasing the contraction frequency from 0.2 Hz to 1.4 Hz causes significant reductions in the amplitude of ICa,as well as reductions in both the amplitude and duration of the AP in trout ventricular myocytes (Harwood et al.,2000). Because the frequency of stimulation will modulate cellular Ca2+ cycling, it is critical to consider the physiologically relevant contraction frequency associated with a particular temperature when trying to understand the implications of temperature change on[Ca2+]iin vivo. Thus, we examined[Ca2+]i and ICa in trout atrial myocytes that were stimulated with physiological APs at physiologically relevant contraction frequencies for the applied temperature. We also examined the effect of temperature on [Ca2+]i and ICa in response to conventional square (SQ) voltage clamp pulses at slow (0.2 Hz) and physiologically relevant frequencies to gain mechanistic insight and to facilitate comparison of our results with earlier studies.

### Fish origin and care

Rainbow trout Oncorhynchus mykiss Walbaum (mean mass 416.7±17.2 g, N=12) were obtained from a local fish farm(Kontiolahti, Finland) and held in the laboratory at 14±1°C for a minimum of four weeks prior to experimentation. They were fed daily on commercial trout pellets (Biomar, Brande, Denmark) and exposed to a 15 h:9 h L:D cycle. All procedures were in accordance with local animal handling protocols.

### Isolated myocyte preparation

A detailed description of the rainbow trout myocyte preparation has been published previously (Vornanen,1998; Shiels et al.,2000). Briefly, myocytes were obtained by retrograde perfusion of the heart, first with an isolating solution for 8-10 min and then with a proteolytic enzyme solution for 20 min. After enzymatic treatment, the atrium was removed from the ventricle, cut into small pieces and then triturated through the opening of a Pasteur pipette. The isolated cells were stored for up to 8 h in fresh isolating solution at 14°C.

### Solutions

The isolating solution contained: NaCl, 100 mmol l-1; KCl, 10 mmol l-1; KH2PO4, 1.2 mmol l-1;MgSO4, 4 mmol l-1; taurine, 50 mmol l-1;glucose, 20 mmol l-1; and Hepes, 10 mmol l-1; adjusted to pH 6.9 with KOH. For enzymatic digestion, collagenase (Type IA), trypsin(Type III) and fatty-acid-free bovine serum albumin (BSA) were added to the isolating solution. Standard internal and external solutions were used to eliminate contaminating Na+ and K+ currents. Tetrodotoxin (TTX, 1 μmol l-1) was included in all external solutions to effectively eliminate fast Na+ currents(Vornanen, 1998). Cs+-based internal and external solutions with 15 mmol l-1 tetraethylammonium chloride (TEA) in the pipette were used to block K+ currents (Hove-Madsen and Tort, 1998). The extracellular solution for recording ICa contained: NaCl, 150 mmol l-1; CsCl, 5.4 mmol l-1; MgSO4, 1.2 mmol l-1;NaH2PO4, 0.4 mmol l-1; CaCl2, 1.8 mmol l-1; glucose, 10 mmol l-1; and Hepes, 10 mmol l-1; adjusted to pH 7.7 with CsOH. Additionally, 5 nmol l-1 adrenaline was added to emulate the tonic level of adrenaline circulating in the blood of resting rainbow trout in vivo(Milligan et al., 1989). The pipette solution contained: CsCl, 130 mmol l-1; MgATP, 5 mmol l-1; TEA, 15 mmol l-1; MgCl2, 1 mmol l-1; oxaloacetate, 5 mmol l-1; Hepes, 10 mmol l-1; EGTA, 0.025 mmol l-1; Na2GTP, 0.03 mmol l-1; and K5-Fura-2, 0.1 mmol l-1; pH adjusted to 7.2 with CsOH. All drugs, with the exception of TTX (Tocris, Bristol, UK),were purchased from Sigma (St Louis, MO, USA).

### Patch-clamp recordings

Whole-cell voltage-clamp experiments were performed using an Axopatch 1D amplifier with a CV-4 1/100 headstage (Axon Instruments, Foster City, CA,USA). Pipettes had a resistance of 2.2±0.30 MΩ when filled with pipette solution. Junction potentials were zeroed prior to seal formation. Pipette capacitance (9.3±0.2 pF) was compensated after formation of a GΩ seal. Mean series resistance was 6.5±0.3 MΩ. Membrane capacitance (31.2±0.8 pF, N=76) was measured using the calibrated capacity compensation circuit of the Axopatch amplifier. Signals were low-pass filtered using the 4-pole lowpass Bessel filter on the Axopatch-1D amplifier at a frequency of 2 kHz and were then analyzed off-line using pClamp 6.0 software (Axon Instruments).

In a separate series of experiments, we recorded APs from rainbow trout atrial myocytes (Fig. 1). Cells were stimulated to elicit APs at 7°C, 14°C or 21°C and at a frequency that corresponds to the resting heart rate of rainbow trout in vivo at each of these temperatures. The temperature/frequency pairs were as follows: 0.6 Hz at 7°C, 1.0 Hz at 14°C and 1.4 Hz at 21°C(Tuurala et al., 1982; Farrell et al., 1996; Aho and Vornanen, 2001). APs were elicited by the minimum voltage pulse able to trigger a self-sustained AP(approximately 1 ms, 0.8 nA). The resultant AP waveforms were subsequently used to provide a more physiologically relevant stimulus under which to evaluate ICa and [Ca2+]i during temperature change. In all experiments, the capacitive transients resulting from the AP waveform were compensated for by the amplifier.

Fig. 1.

Action potential (AP) waveforms used to elicit L-type Ca2+channel current (ICa) and intracellular Ca2+concentration ([Ca2+]i). The APs were recorded by current-clamp in a separate series of experiments(Shiels et al., 2002a). AP waveforms were obtained by averaging recordings from 6-10 cells at each temperature. The relatively depolarized resting potential is typical for isolated atrial myocytes from rainbow trout(Shiels et al., 2000) and reflects both a low density of the inward rectifier K+ current(IK1) and the lack of cholinergic tone(Vornanen et al., 2002). The inter-pulse holding level during subsequent AP stimulation was -80 mV. The solid line shows the dimensions of the 500 ms square (SQ) pulse.

Fig. 1.

Action potential (AP) waveforms used to elicit L-type Ca2+channel current (ICa) and intracellular Ca2+concentration ([Ca2+]i). The APs were recorded by current-clamp in a separate series of experiments(Shiels et al., 2002a). AP waveforms were obtained by averaging recordings from 6-10 cells at each temperature. The relatively depolarized resting potential is typical for isolated atrial myocytes from rainbow trout(Shiels et al., 2000) and reflects both a low density of the inward rectifier K+ current(IK1) and the lack of cholinergic tone(Vornanen et al., 2002). The inter-pulse holding level during subsequent AP stimulation was -80 mV. The solid line shows the dimensions of the 500 ms square (SQ) pulse.

Peak current amplitude of ICa was calculated as the difference between the peak inward current and the current recorded at the end of the depolarizing pulse. The charge (pC) carried by ICa was calculated from the time integral of the current and then normalized to cell capacitance(pC pF-1).

### Fluorescent recordings

For each cell, after formation of the GΩ seal, and before rupture of the cell membrane, background fluorescence (approximately 40% and 25% of the total signal at 340 nm and 380 nm, respectively) was recorded and then subtracted from all subsequent measurements. Temperature change had no effect on background fluorescence. Myocytes were alternately illuminated (optical chopper, 0.2 kHz, 75 W Xenon arc lamp) at 340 nm and 380 nm with a high-speed dual-wavelength filter-based illuminator (PowerFilter, Photon Technology International, Brunswick, NJ, USA). Emitted light was filtered at 510 nm and detected by a photon-counting photomultiplier tube, the output of which was connected to a computer via an I/O board and saved to disk for later analysis using the FeliX acquisition and analysis software (7-point smoothing and trace averaging, Photon Technology International). The emission aperture window was kept as small as possible and was focused close to, but out of view of, the pipette tip to minimize any effect of cell movement during contraction(cells typically contracted approximately 10-15% of the resting length).

The ratio of emitted fluorescence at 340 nm/380 nm (R) was calculated to give an index of [Ca2+]i.[Ca2+]i was calculated using the acquired fluorescent ratios and calibration parameters derived both experimentally and through calculation. To measure Rmax and Rmin,cells were patched-clamped and perfused with an extracellular solution containing metabolic inhibitors (rotenone, 2 μmol l-1; carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), 5 μmol l-1;jodo acetic acid, 5 mmol l-1). Pipette and extracellular solutions were the same as those described above except that nucleosides and sulphates were omitted, respectively. To estimate Rmax, 10 μmol l-1 Ca2+ ionophore (4-bromo A23187) and 1.8 mmol l-1 CaCl2 were added to the perfusate. To estimate Rmin, cells were then perfused with a solution containing 10 mmol l-1 EGTA. The values obtained are as follows: Rmax=9.80±0.67, Rmin=0.99±0.01, β (signal fluorescence at 380 nm/background fluorescence at 380 nm)=4.62±0.44; N=17 (A. Ryökkynen and M. Vornanen, unpublished data). The affinity constant Kd of Fura-2 has been measured in vivo over a range of temperatures (5-37°C) in enterocytes from the Atlantic cod(Gadus morhua; Larsson et al.,1999). We adjusted the Kd values obtained in that study to match our experimental conditions of pH, buffer and ionic strength by calculating the effects of pH, buffer and ionic strength on 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)(Harrison and Bers, 1987)using the MaxChelator program (Bers et al.,1994). The calculated Kd values were as follows: 395 nmol l-1 at 7°C, 366 nmol l-1 at 14°C and 336 nmol l-1 at 21°C. The fluorescence ratios obtained during the experiments were converted into[Ca2+]i using these Kd values and the above measurements of Rmax, Rminand β, as described by Grynkiewicz et al.(1985).

### Experimental procedures

Atrial myocytes were superfused at a rate of 2 ml min-1 with extracellular solution at 7°C, 14°C or 21°C. Each cell was tested at one experimental temperature only. The extracellular solution was heated or chilled by a water bath circuit before emptying into the recording chamber(RC-26, Warner Instrument Corp., Hamden, CT, USA; volume 150 μl). A thermocouple was placed inside the recording chamber and positioned no less than 5 mm from the cell to ensure it was experiencing the desired temperature. At the onset of each experiment, cells were stimulated with 25 physiological APs at a frequency that corresponded to the in vivo heart rate of trout at each temperature (see Fig. 1) to ensure that SR Ca2+ content was similar in all cells (Shiels et al., 2002a). However, the subsequent stimulation protocols may alter SR Ca2+content as the experiment proceeds, which is a limitation of our approach.

Cells were first stimulated to contract with five SQ depolarizing pulses(from -80 mV to +10 mV, 500 ms duration) at a frequency of 0.2 Hz(SQ0.2Hz), and ICa and [Ca2+]iwere simultaneously recorded. The cells were then stimulated to contract with five SQ depolarizing pulses at a frequency that corresponded to the in vivo heart rate for rainbow trout at the given test temperature(SQphysiol). SQ voltage-clamp protocols are vital for characterizing channel kinetics and simplify interpretation of mechanistic relationships, but they may have only limited relevance to the condition in vivo (Linz and Meyer,1998; Puglisi et al.,1999). Therefore, after stimulation under SQphysiolconditions, the cells were stimulated with an AP waveform (see Fig. 1) appropriate for the test temperature and at a frequency appropriate for the test temperature(APphysiol). ICa and [Ca2+]i were simultaneously recorded throughout (see Fig. 4). Although the SQphysiol part of the protocol is of little physiological significance, we believe that comparing the results obtained at physiological stimulation frequencies from both a 500 ms SQ (SQphysiol) and a physiological AP (APphysiol) allows us to evaluate the importance of the AP and the possible limitations of the SQ pulse in assessing physiological processes. The duration of each protocol was approximately 1 min and we did not observe any changes in intracellular buffering by Fura-2 or run-down of ICa over this time period.

Fig. 4.

This figure is an example of a recording for a single myocyte at 21°C. Part (A) shows intracellular Ca2+ concentration([Ca2+]i) and part (B) shows the corresponding L-type Ca2+ channel current (ICa) at each stimulus change. Each panel is marked according to the frequency and waveform used to elicit currents and transients. The first five transients at each temperature were elicited by square pulses at 0.2 Hz (SQ0.2Hz). The middle five transients were elicited by SQ pulses applied at a physiologically relevant frequency for the test temperature (SQ1.4Hz), but with a long pulse duration (500ms). Together these factors contribute to the dramatic rise in diastolic Ca2+ levels. The last five transients were elicited by temperature- and frequency-dependent action potentials (AP1.4Hz). The dotted line in the lower panel indicates 0mV. Leakage correction was employed at SQ0.2Hz, but not at SQ1.4Hz or AP1.4Hz.

Fig. 4.

This figure is an example of a recording for a single myocyte at 21°C. Part (A) shows intracellular Ca2+ concentration([Ca2+]i) and part (B) shows the corresponding L-type Ca2+ channel current (ICa) at each stimulus change. Each panel is marked according to the frequency and waveform used to elicit currents and transients. The first five transients at each temperature were elicited by square pulses at 0.2 Hz (SQ0.2Hz). The middle five transients were elicited by SQ pulses applied at a physiologically relevant frequency for the test temperature (SQ1.4Hz), but with a long pulse duration (500ms). Together these factors contribute to the dramatic rise in diastolic Ca2+ levels. The last five transients were elicited by temperature- and frequency-dependent action potentials (AP1.4Hz). The dotted line in the lower panel indicates 0mV. Leakage correction was employed at SQ0.2Hz, but not at SQ1.4Hz or AP1.4Hz.

### Statistical analysis

One-way repeated measures analysis of variance (RM ANOVA) were used to compare the effects of changing the frequency and the stimulus waveform on ICa and [Ca2+]i. One-way ANOVAs were used to test the effects of temperature on ICa and[Ca2+]i. Differences were considered significant at P<0.05, as assessed by Student—Newman—Keuls (SNK) post-hoc analysis. The mean values ± S.E.M. and the statistical analysis for [Ca2+]i appear in the tables. Five transients were recorded under each experimental pulse condition (i.e. SQ0.2Hz, SQphysiol and APphysiol; see Fig. 4). The diastolic[Ca2+]i at the end of the 4th transient and the peak[Ca2+]i of the 5th transient were used to represent a steady-state value for each condition (Tables 1,2,3). For future experiments, it would, however, be prudent to provide a longer train of stimulation pulses, especially at warm temperatures, to ensure a steady-state level had been attained. On the other hand, excursions by fish across thermoclines can be very rapid and transitory in nature and therefore ICa may not reach a steady-state in vivo before temperature (and thus heart rate) change again.

Table 1.

Amplitude of intracellular Ca2+ concentration([Ca2+]i) (nmol l-1) in trout atrial myocytes

SQ0.2HzSQphysiolAPphysiol
7°C 474±53a,e 662±65a,f 234±49a,g
14°C 398±40a,e 506±48a,f 122±41b,g
21°C 198±27b,e 278±41b,f 79±12b,g
SQ0.2HzSQphysiolAPphysiol
7°C 474±53a,e 662±65a,f 234±49a,g
14°C 398±40a,e 506±48a,f 122±41b,g
21°C 198±27b,e 278±41b,f 79±12b,g

The effect of temperature and the frequency/stimulus waveform on the amplitude of the stimulus-induced change in [Ca2+]i,calculated as the difference between the diastolic and systolic Ca2+ concentration with each depolarization. Values are means of 4-8 cells at each temperature.

The frequency of stimulation during SQphysiol (square pulses)and APphysiol (action potential) was 0.6 Hz at 7°C, 1.0 Hz at 14°C and 1.4 Hz at 21°C.

The effect of temperature is indicated by dissimilar letters a, b and c(compare vertically within each frequency/stimulus treatment) [analysis of variance (ANOVA), Student—Newman—Keuls (SNK) test]. Dissimilar letters e, f and g indicate significant differences between frequency/stimulus protocols (compare horizontally within each temperature) [repeated measures ANOVA, SNK].

Table 2.

Time to peak of [Ca2+]i (ms) in trout atrial myocytes

SQ0.2HzSQphysiolAPphysiol
7°C 601±15a,e 483±44a,f 453±30a,f
14°C 540±36a,b,e 425±57a,b,f 394±29a,f
21°C 502±682b,e 291±46b,f 252±24b,f
SQ0.2HzSQphysiolAPphysiol
7°C 601±15a,e 483±44a,f 453±30a,f
14°C 540±36a,b,e 425±57a,b,f 394±29a,f
21°C 502±682b,e 291±46b,f 252±24b,f

The effect of temperature and the frequency/stimulus waveform on the time required to reach the peak amplitude of [Ca2+]i. All other features are as in Table 1.

Table 3.

Diastolic Ca2+ levels (nmol l-1) in trout atrial myocytes

SQ0.2HzSQphysiolAPphysiol
7°C 92±20e 401±63f 158±37e
14°C 76±8e 450±15f 176±32g
21°C 80±5e 444±42f 186±22g
SQ0.2HzSQphysiolAPphysiol
7°C 92±20e 401±63f 158±37e
14°C 76±8e 450±15f 176±32g
21°C 80±5e 444±42f 186±22g

The effect of temperature and the frequency/stimulus waveform on diastolic[Ca2+]i. Diastolic Ca2+ levels were significantly affected by frequency/stimulus changes but not by temperature changes. All other features are as in Table 1.

### Effects of temperature

For both SQ and AP pulses, the amplitude of the stimulus-induced change in[Ca2+]i decreased with increasing temperature(Fig. 2, Table 1). The time required to reach peak [Ca2+]i also decreased with increasing temperature (Table 2), probably due to the reduced amplitude because the rate of rise did not change significantly (0.625±0.33 μmol l-1 s-1 at 7°C and 0.460±0.07 μmol l-1 s-1 at 21°C with SQ pulses at 0.2 Hz).

Fig. 2.

The effect of temperature on intracellular Ca2+ concentration([Ca2+]i) and L-type Ca2+ channel current(ICa) in trout atrial myocytes. (A) The effect of temperature on[Ca2+]i during SQ0.2Hz, SQphysioland APphysiol (the physiological frequencies were: 0.6 Hz at 7°C, 1.0 Hz at 14°C and 1.4 Hz at 21°C). The increase in the resting level of the transient at SQphysiol reflects the increase in diastolic Ca2+ concentration (see Fig. 4, Table 3). (B) Current recordings of ICa under the same conditions. Peak current amplitude was calculated as the difference between the peak inward current and the current recorded at the end of the depolarizing pulse. The currents elicited at SQ0.2Hz were leakage corrected using the P/N procedure of the software (Clampex, Axon Instruments). Leakage correction was not employed at SQphysiol or APphysiol. Mean data and statistical analysis is given in Tables 1,2,3and in Fig. 3. SQ0.2Hz, square pulses at a frequency of 0.2 Hz;SQphysiol, square pulses at physiological frequency;APphysiol, action potential at physiological frequency.

Fig. 2.

The effect of temperature on intracellular Ca2+ concentration([Ca2+]i) and L-type Ca2+ channel current(ICa) in trout atrial myocytes. (A) The effect of temperature on[Ca2+]i during SQ0.2Hz, SQphysioland APphysiol (the physiological frequencies were: 0.6 Hz at 7°C, 1.0 Hz at 14°C and 1.4 Hz at 21°C). The increase in the resting level of the transient at SQphysiol reflects the increase in diastolic Ca2+ concentration (see Fig. 4, Table 3). (B) Current recordings of ICa under the same conditions. Peak current amplitude was calculated as the difference between the peak inward current and the current recorded at the end of the depolarizing pulse. The currents elicited at SQ0.2Hz were leakage corrected using the P/N procedure of the software (Clampex, Axon Instruments). Leakage correction was not employed at SQphysiol or APphysiol. Mean data and statistical analysis is given in Tables 1,2,3and in Fig. 3. SQ0.2Hz, square pulses at a frequency of 0.2 Hz;SQphysiol, square pulses at physiological frequency;APphysiol, action potential at physiological frequency.

The peak amplitude of ICa increased with temperature (Figs 2B, 3A). The charge carried across the SL via ICa was unchanged by temperature during SQ pulse stimulation and decreased with temperature during AP stimulation(Fig. 3B).

Fig. 3.

The effect of temperature and stimulus change on (A) the peak current density of L-type Ca2+ channel current (ICa) and (B) the charge density of ICa in trout atrial myocytes. Dissimilar letters indicate significant effects of temperature within each stimulus treatment(analysis of variance, Student—Newman—Keuls test). *indicates a significant decrease in ICa during AP stimulation at all temperatures and with each successive stimulus protocol at 21°C. indicates greater ICa charge density at 7°C during AP stimulation compared with SQ stimulation. Values are means ±S.E.M.; N=7 for 7°C and 21°C, and N=8 for 14°C.

Fig. 3.

The effect of temperature and stimulus change on (A) the peak current density of L-type Ca2+ channel current (ICa) and (B) the charge density of ICa in trout atrial myocytes. Dissimilar letters indicate significant effects of temperature within each stimulus treatment(analysis of variance, Student—Newman—Keuls test). *indicates a significant decrease in ICa during AP stimulation at all temperatures and with each successive stimulus protocol at 21°C. indicates greater ICa charge density at 7°C during AP stimulation compared with SQ stimulation. Values are means ±S.E.M.; N=7 for 7°C and 21°C, and N=8 for 14°C.

During SQ pulse stimulation, we observed a temperature-dependent change in the amplitude of [Ca2+]i but no change in the charge density calculated by integrating ICa. This suggests that, in addition to Ca2+ flux across the SL, other Ca2+ flux pathways, most probably Ca2+ release from the SR, must be changing with respect to temperature in these cells. During AP stimulation, the reduced charge density of ICa at warm temperatures(Fig. 3B) may underlie the large percentage reduction in [Ca2+]i observed under these conditions (Fig. 2, Table 1).

### Effects of stimulus change

The frequency and shape of the depolarizing stimulus profoundly altered cellular Ca2+ dynamics. As a detailed example, Fig. 4 illustrates the frequency dependence and the influence of pulse shape on ICa and[Ca2+]i for an experiment conducted at 21°C. During SQ0.2Hz, diastolic Ca2+ concentrations returned to rest between depolarizing pulses (Fig. 4A). When stimulation frequency was increased from SQ0.2Hz to SQ1.4Hz, diastolic Ca2+concentration increased significantly due to the long (500 ms) and high-frequency (1.4 Hz) depolarization of the membrane (also see Table 3). In addition,[Ca2+]i increased by approximately 30%(Fig. 4, Table 1). The rate of rise also increased significantly (from 0.73±0.11 μmol l-1s-1 to 1.16±0.84 μmol l-1 s-1), and the time required to reach the peak of the transient decreased during SQ1.4Hz compared with that during SQ0.2Hz(Table 2). When the stimulus waveform was changed from SQ1.4Hz to AP1.4Hz,[Ca2+]i and diastolic Ca2+ levels both decreased significantly (Tables 1, 2), and the rate of rise of the transient decreased significantly to a value (0.59±0.17 μmol l-1 s-1) that was not significantly different from that for the SQ0.2Hz pulse. Qualitatively similar effects of frequency and stimulus shape were observed at 14°C and 7°C (Tables 1,2,3). Thus, independent of temperature, an increase in stimulation frequency from 0.2 Hz to a physiologically relevant frequency for the experimental temperature (SQphysiol) produced a large increase in both[Ca2+]i and in diastolic Ca2+ levels with 500 ms SQ stimulation pulses. By contrast, the AP stimulus, which is much shorter in duration, reduced [Ca2+]i and diastolic Ca2+ levels compared with SQ pulses at the same frequency.

The amplitude of ICa was significantly smaller with an AP stimulus compared with a SQ stimulus at the same frequency (Figs 2,3,4; Table 1). The charge carried by ICa did not differ significantly at 14°C and 21°C but was greater at 7°C (Fig. 3B). This situation probably arose as a result of sustained Ca2+ influx during the AP plateau and because of Ca2+ influx occurring via the window current (see Fig. 2B and below).

This is the first study we are aware of to measure cytosolic free Ca2+ in trout atrial myocytes and examine how it changes with temperature, stimulus shape and frequency. Furthermore, an effort was made to simulate conditions that were more physiologically relevant with respect to temperature, as well as stimulation waveform and frequency. There were three principle findings: (1) the transient change in [Ca2+]iincreases with decreasing temperature, (2) the transient change in[Ca2+]i is smaller in response to physiological AP stimulation than to 500 ms SQ stimulus pulses, and (3) diastolic Ca2+ levels are lower at physiological frequencies when contraction is stimulated with APs compared with 500 ms SQ pulses. Our results indicate that temperature-dependent changes in ICa alone cannot account for the temperature-dependent changes in [Ca2+]i. Thus,although we made no direct measurements of SR Ca2+ flux, our results suggest temperature may modulate SR Ca2+ cycling in trout atrial myocytes.

### Effects of temperature on [Ca2+]i and ICa

With both SQ and AP pulses, [Ca2+]i was slowest and largest at 7°C compared with at 14°C and 21°C. This observation agrees with results from multicellular cardiac muscle studies from rainbow trout, where isometric contraction is slower and stronger as temperature is decreased (Driedzic and Gesser,1994; Shiels and Farrell,1997; Aho and Vornanen,2001). These findings also agree with mammalian studies that demonstrate an increase in the amplitude of the transient change in[Ca2+]i with decreases in temperature(Wang et al., 2000). The small amplitude of [Ca2+]i and the elevated diastolic Ca2+ levels during physiological AP pulses at 21°C is striking and is consistent with the finding that the maximum power output of trout heart preparations in situ falls off at temperatures above 18°C(Farrell et al., 1996). Indeed, the poor maximum cardiac performance at temperatures approaching the upper incipient lethal temperature for salmonids (23-25°C; Black, 1953), coupled with the observed reduction in maximum swimming capabilities of salmonids as temperatures approach 21°C in both lab(Brett, 1971) and field(Tierney, 2000) studies, has led to the suggestion (Farrell,1997) that cardiac failure may be a critical factor when salmon are exposed to temperatures that exceed their preferred temperature(approximately 15°C; Brett,1971). The implication of the present study is that the small amplitude of [Ca2+]i in the cardiac myocyte may be critical to setting the upper thermal regime in fishes. Thus, it follows that data obtained from trout cardiac myocytes at room temperature should be regarded with some caution.

Previous studies with trout atrial(Shiels et al., 2000) and ventricular (Kim et al., 2000)myocytes have shown a reduction in the peak current amplitude of ICa with decreasing temperature, and our present study supports these earlier findings. The similar Ca2+ influx viaICa at 7°C, 14°C and 21°C probably resulted from a combination of higher and faster peak ICa, offset by more rapid ICa inactivation as temperatures increased (see Fig. 2 and Shiels et al., 2000). A similar lack of effect of temperature on the charge density of ICahas been reported for rabbit myocytes at 25°C and 35°C(Puglisi et al., 1999). We observed an increase in the outward current at the end of the stimulus pulse at 21°C compared with at 14°C and 7°C (see Fig. 2B), which is probably attributable to increased Ca2+ influx via reverse-mode sarcolemmal Na+/Ca2+ exchange (NCX; Hove-Madsen et al., 2000). During AP stimulation, the charge density of ICa was reduced at warm temperatures (Fig. 3B),probably due to the shorter AP duration. This will have contributed to the reduction in the transient change in [Ca2+]i observed during stimulation at 21°C. However, certainly during the SQ pulse stimulus protocol, and also during the AP stimulus protocol, the mismatch'between temperature-induced changes in SL Ca2+ influx and[Ca2+]i suggests that temperature must modulate another Ca2+ flux pathway in trout atrial myocytes.

### Effects of increasing SQ pulse stimulation frequency on[Ca2+]i

Increasing SQ pulse stimulation frequency (from SQ0.2Hz to SQphysiol) caused an approximately 30% increase in[Ca2+]i and an approximately 75% increase in the diastolic Ca2+ level (Figs 2, 4; Tables 1, 3), indicating that more Ca2+ enters the cytosol than exits via SR uptake or SL efflux. The high-frequency application of long SQ pulses (500 ms) increased Ca2+ influx via reverse-mode NCX, as evidenced by the increased outward current at the end of the stimulation pulse (see Fig. 4B, SQ1.4Hz). In avian (Lee and Clusin,1987) and mammalian cardiomyocytes(Frampton et al., 1991), the frequency-dependent increase in [Ca2+]i has been attributed, in part, to increased SR Ca2+ load and thus an increased SR Ca2+ release. Increased Ca2+ influx via NCX may have resulted in greater SR Ca2+ loading and thus greater Ca2+-induced Ca2+ release, contributing further to the increase in the amplitude of the transient change in[Ca2+]i in the present study. An increase in the frequency of field stimulation from 0.6 Hz to 1.0 Hz at 14°C increased diastolic Ca2+ (approximately 25-50%) without changing the amplitude of [Ca2+]i in trout ventricular myocytes(Harwood et al., 2000). In the same study, the authors found that application of caffeine revealed a greater SR Ca2+ content at 1.0 Hz compared with at 0.6 Hz, indicating that SR Ca2+ content increased with pacing frequency but was not releasable during a field-stimulated twitch(Harwood et al., 2000). The differences between studies may indicate that a SQ pulse is a better trigger for SR Ca2+ release than is field stimulation, where the membrane is depolarized with an AP (see below). Indeed, Hove-Madsen et al.(2001) have demonstrated a similar degree of CICR at 7°C and 21°C with SQ pulses in trout atrial myocytes.

### Effects of action potential stimulation on[Ca2+]i

[Ca2+]i and diastolic Ca2+ levels decreased dramatically when the SQ pulse was changed to an AP pulse at the same frequency. This reflects a reduction in SL Ca2+ influx and also possibly a reduction in CICR with AP pulses, as the smaller amplitude of ICa during AP pulses (see Figs 2, 3) may be a less effective trigger for CICR.

The myocytes were better able to manage diastolic Ca2+ levels at physiological frequencies with AP pulses(Table 3) but not as effectively as at 0.2 Hz. This suggests that the Ca2+ efflux mechanisms are not able to remove all of the Ca2+ effectively during diastole at physiological pacing frequencies. However, it is possible that resting diastolic Ca2+ concentration in the absence of stimulation and at 0.2 Hz is sub-physiological. The true diastolic Ca2+ concentration' for fish hearts remains unknown and is worthy of future investigation. In any case, visual observations indicate that the cells were relaxing between stimulation pulses at physiological frequencies with AP pulses at all temperatures.

It has been estimated from trout ventricular myocytes at room temperature that, when operating maximally (i.e. at or near Vmax), the trout SR Ca2+-ATPase should be able to remove the total intracellular Ca2+ transient between depolarizations at physiological heart rates (Hove-Madsen et al., 1998). However, our study reveals that, at room temperature,the trout intracellular Ca2+ transient is small compared with cooler temperatures and suggests that the SR Ca2+-ATPase is unlikely to be operating near Vmax. Furthermore, estimates of the maximal Ca2+ efflux rate of the rainbow trout NCX at room temperature (Hove-Madsen and Tort,2001) suggest that the NCX can remove a physiological Ca2+ transient from the cytosol in a few hundred milliseconds. Based on those estimations, the NCX should have been able to remove all of the cytosolic Ca2+ during the 700 ms between depolarizations at 1.4 Hz in the present study at 21°C. The observed increase in the diastolic Ca2+ levels in the present study are in contrast to these estimates of Ca2+ efflux and may reflect exogenous buffering by Fura-2. The in vivo buffering capacity of the fish myoplasm and its modulation by temperature is unknown. However, because both contractility and the time course of ICa were maintained in the presence of Fura-2, it is unlikely that exogenous buffering was large relative to the measurements being made. A comparison of the time to 50% relaxation of[Ca2+]i with AP pulses in our study at 14°C(approximately 250 ms) with the ratiometric results from the only other published study of [Ca2+]i in fish hearts, which examined field-stimulated trout ventricular myocytes loaded with Fura-2-AM at 15°C (approximately 230 ms; Harwood et al., 2000), suggests comparable time courses of the transients. Additional studies are needed to resolve the physiological efficacy of trout Ca2+ efflux pathways and the effect of exogenous buffering and temperature change therein.

### Effects of stimulus change on ICa

The frequency-dependent decrease in ICa at 21°C reported here agrees well with previous studies that report a 15-20% decrease in peak current amplitude over a similar frequency range at 21°C in trout myocytes(Hove-Madsen and Tort, 1998; Harwood et al., 2000). These responses are akin to the negative force-frequency response observed independent of temperature in the myocardium of a number of fish species(Driedzic and Gesser, 1985; Keen et al., 1994; Shiels and Farrell, 1997; see Shiels et al., 2002b for a recent review). The smaller amplitude of the peak current during an AP pulse compared with during a SQ pulse is qualitatively similar to what we have found previously in trout atrial myocytes(Shiels et al., 2000) and similar to what has been described in mammals(Linz and Meyer, 1998; Puglisi et al., 1999).

The shape of the trout AP, in combination with the relatively depolarized resting membrane potential (-50 mV), may have led to an overestimation of SL Ca2+ flux via the ICa window current in the present study (Fig. 2B). This is because the late phase depolarization after the initial hyperpolarization(Fig. 1) brings the membrane potential close to the window current voltage range for ICa in trout atrial myocytes (-40 mV to +30 mV, with a peak at -10 mV; see Shiels et al., 2000). Recent measurements in vivo suggest that the resting membrane potential in rainbow trout atrial cells is approximately -65 mV(Vornanen et al., 2002). At this membrane potential, the late phase depolarization would not enter the window voltage range and thus ICa inactivation would be maintained. The relatively depolarized membrane potential in isolated myocytes from our study results from the loss of cholinergic tonus and also reflects the lower density of inward rectifier K+ channel current (IK1) in atrial compared with ventricular cells(Vornanen et al., 2002). Future studies with isolated trout myocytes could consider including tonic levels of acetylcholine when measuring APs to account for this.

### Summary

Collectively, we have characterized some of the temperature-, frequency-and waveform-dependent changes in [Ca2+]i and ICa that exist in rainbow trout atrial myocytes. By calibrating the Fura-2 signal, we were able to make meaningful comparisons of[Ca2+]i at physiologically relevant temperatures for rainbow trout. We have shown that increased temperature causes a decrease in[Ca2+]i. This is the first cellular observation of a temperature-dependent change in [Ca2+]i in fish myocytes and may help to explain the well-known negative inotropic effect of warm temperatures on trout heart function. Our results suggest that the temperature-dependent changes in [Ca2+]i are not solely dependent on temperature-dependent changes in ICa. Therefore,temperature and frequency modulation of SR Ca2+ accumulation and release may be involved, and future studies should be directed at understanding these mechanisms. Finally, we have shown that the amplitude of[Ca2+]i and the level of the diastolic Ca2+concentration is dependent on the shape and the rate of stimulation. At all temperatures, cells were better able to maintain diastolic Ca2+levels at physiological frequencies with AP pulses compared with 500 ms SQ pulses. This suggests that temperature-dependent modulation of the shape of the AP is important for cellular Ca2+ regulation during temperature and frequency changes in rainbow trout heart.

We acknowledge Kontiolahti fish farm for donating the trout and Ari Ryökkynen for his efforts with the Fura-2 calibrations. We also thank Dr Kerry Delaney for his technical advice and Dr Leif Hove-Madsen for his constructive comments on earlier versions of this manuscript. The study was supported by NSERC to A.P.F., Simon Fraser University Steele Memorial Fellowship to H.A.S., and the Academy of Finland (project #63090) to M.V.

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