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Am J Physiol Regul Integr Comp Physiol 292: R1023-R1032, 2007. First published September 28, 2006; doi:10.1152/ajpregu.00432.2006
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COMPARATIVE AND EVOLUTIONARY PHYSIOLOGY

Temperature acclimation modifies sinoatrial pacemaker mechanism of the rainbow trout heart

Jaakko Haverinen and Matti Vornanen

University of Joensuu, Department of Biology, Joensuu, Finland

Submitted 22 June 2006 ; accepted in final form 27 September 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The hypothesis of pacemaker level origin of thermal compensation in heart rate was tested by recording action potentials (AP) in intact sinoatrial tissue and enzymatically isolated pacemaker cells of rainbow trout acclimated at 4°C (cold) and 18°C (warm). With electrophysiological recordings, the primary pacemaker was located at the base of the sinoatrial valve, where a morphologically distinct ring of tissue comprising myocytes and neural elements was found by histological examination. Intrinsic beating rate of this pacemaker was higher in cold-acclimated (46 ± 6 APs/min) than warm-acclimated trout (38 ± 3 APs/min; P < 0.05), and a similar difference was seen in beating rate of isolated pacemaker cells (44 ± 6 vs. 38 ± 6 APs/min; P < 0.05), supporting the hypothesis that thermal acclimation modifies the intrinsic pacemaker mechanism of fish heart. Inhibition of sarcoplasmic reticulum (SR) with 10 µM ryanodine and 1 µM thapsigargin did not affect heart rate in either warm- or cold-acclimated trout at 11°C but reduced heart rate in warm-acclimated trout from 74 ± 2 to 42 ± 6 APs/min (P < 0.05) at 18°C. At 11°C, a half-maximal blockade of the delayed rectifier K+ current (IKr) with 0.1 µM E-4031 reduced heart rate more in warm-acclimated (from 45 ± 1 to 24 ± 5 APs/min) than cold-acclimated trout (56 ± 3 vs. 48 ± 2 APs/min), whereas IKr density was higher and AP duration less in cold-acclimated trout (P > 0.05). Collectively, these findings suggest that a cold-induced increase in AP discharge frequency is at least partly due to higher density of the IKr in the cold-acclimated trout, whereas contribution of SR Ca2+ release to thermal compensation of heart rate is negligible.

cardiac pacemaker; heart rate; sarcoplasmic reticulum; delayed rectifier potassium current

THE VERTEBRATE HEART has an autonomous beating rhythm generated by a relatively small number of specialized cardiac myocytes of the primary pacemaker area (7, 12, 25). The rate of this center is modulated by nervous and humoral factors and determines the working pace of the cardiac pump, and thereby regulates cardiac output under various physiological conditions such as exercise and hypoxia (26, 38). In fish heart, the primary pacemaker area has been located into the sinoatrial junction that fires spontaneous and slowly rising action potentials (AP) (17, 22, 28, 29, 35, 40, 41). Size and exact location of the primary pacemaker area of the fish heart is, however, incompletely defined, and the morphology of the isolated pacemaker cells and their electrical activity has not yet been described. Therefore, the first aim of the present study was to determine the exact location of the primary pacemaker in the rainbow trout heart by direct electrical recordings and histological examination and to characterize pacemaker potentials of enzymatically isolated pacemaker cells.

Atrial and ventricular myocytes have a stable resting membrane potential (RMP) and are therefore unable to elicit spontaneous APs. Unlike working myocytes of the atrium and ventricle, pacemaker cells show a tightly regulated diastolic depolarization that results in regular firing of pacemaker APs. Diastolic depolarization is an outcome from a concerted action of various sarcolemmal K+, Ca2+, and Na+ ion currents, none of which alone is capable of producing it (12). Currents involved in pacemaking include T- and L-type Ca2+ current, slow and fast components of the delayed rectifier K+ current, transient outward K+ current, inward Na+ current, and the pacemaker current carried by both K+ and Na+ ions (9, 25). In addition to ion channels, the pacemaker mechanism seems to involve a close interplay between Ca2+-induced Ca2+ release (CICR) of the sarcoplasmic reticulum (SR) and sarcolemmal Na+/Ca2+ exchange (NCX), since subsarcolemmal SR Ca2+ release can accelerate diastolic depolarization via inward NCX current (5, 11, 13, 16, 20). The multiplicity of mechanisms makes pacemaking robust and unlikely to fail due to malfunction of any single actor.

Low temperature slows down heart rate and thereby tends to decrease cardiac output and activity of ectothermic animals in habitats where large temperature changes occur seasonally. Rainbow trout and several other ectotherms of the north temperate latitudes are able to oppose or circumvent the depressive effect of cold temperatures on heart rate by recruiting compensatory mechanisms upon prolonged exposure to low temperature (2, 6, 26, 30, 32). In some fish, thermal compensation occurs as an outcome of the cold-induced decrease in the inhibitory cholinergic control of heart rate (30, 32). That the cold-induced increase in beating frequency persists in the excised heart (2, 6) suggests, however, that it is not solely based on autonomic control of the pacemaker rate but is likely to involve the pacemaker mechanism itself. Considering the complexity of cardiac pacemaking, a number of ionic mechanisms could be involved in temperature acclimation of heart rate. In light of previous knowledge of temperature-related changes in excitation-contraction coupling of the trout heart, two ion transport mechanisms, the delayed rectifier K+ current IKr and CICR, are particularly interesting, since both are enhanced in the cold-acclimated trout heart (15, 37) and both systems are implicated as important in cardiac pacemaking (9). Therefore, the second objective of the study was to test the hypothesis that thermal compensation of the heart rate in rainbow trout involves IKr and/or CICR. To this end, we examined the effect of ryanodine (Ry) + thapsigargin (Tg) and E-4031 (specific inhibitors of SR Ca2+ release channel and delayed rectifier channel, respectively) on heart rate in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Fish. Rainbow trout (Oncorhynchus mykiss, Walbaum) were obtained from a local fish farm (Kontiolahti, Finland). In the laboratory, the fish (body mass 212.68 ± 11.83 g, n = 89) were reared in temperature-controlled 1,000-liter stainless steel tanks with a continuous supply of aerated groundwater at the rate of ~0.5 l/min. Fish were acclimated for at least 4 wk at constant temperature at either 4°C (cold acclimation) or 18°C (warm acclimation) under a 15:9-h light-dark photoperiod before experimentation. Trout were fed commercial fish food (Biomar, Brande, Denmark) to satiation five times a week. All experiments were made with the consent of the local committee for animal experimentation.

Histology. Whole hearts were immersed in Bouin's fixative for 24 h, dehydrated, cleared, and embedded in paraffin (J. T. Baker, UltraPar, Holland). Sections were cut at 10 µm (Leica RM2165 microtome) and stained with either hematoxylin-eosin for light microscopic examination of the general heart structure or picrosirius red to visualize collagenous matrix of the heart (10). Photomicrographs were taken with a Zeiss Axioplan microscope equipped with Axiovision software. Enzymatically isolated myocytes were suspended in low-Na+ solution (for composition, see Current-clamp recording of pacemaker APs from single myocytes) and were photographed in the native state without staining.

Microelectrode recording of APs from intact tissue. For AP measurements, the tissue containing sinus venosus, sinoatrial valve, and an adjacent part of the atrium was dissected free and gently fixed with insect pins on the Sylgard-coated bottom of a 10-ml recording chamber filled with continuously oxygenated (100% O2) physiological saline containing (in mM) 150 NaCl, 3 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 1.8 CaCl2, 10 HEPES, and 10 glucose, with pH adjusted to 7.6 with NaOH. The preparation was allowed to equilibrate for ~1 h to reach a stable beating rate before APs were recorded with sharp microelectrodes. Pipettes were fabricated from borosilicate glass with an internal filament by using a Sutter P-97 puller and had a resistance of ~22 M{Omega} when filled with 3 M KCl. Analog signals were amplified by a high-impedance amplifier (KS-700; WPI) and digitized (Digidata 1200 analog-to-digital/digital-to-analog board; Axon Instruments) with a sampling rate of 2 kHz before being stored on the computer with the aid of Axotape acquisition software (Axon Instruments). AP characteristics (Fig. 1A) were analyzed with Clampfit software (Axon Instruments). Frequency of AP discharge was used as a measure for the heart rate. Accordingly, heart rate and AP rate are used interchangeably in the text.


Figure 1
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  		Fig. 1. A: a schematic presentation indicating the variables that were determined from pacemaker action potentials (APs) at APD50 and ADP90, the duration of AP at 50 and 90% of repolarization, respectively. ET, excitation threshold; MDP, maximum diastolic potential; CL, cycle length; RDD, rate of diastolic depolarization. B: representative recordings (top) of 3 AP types (primary pacemaker AP, secondary pacemaker AP, and atrial AP) found in the intact sinoatrial nodal tissue of the trout heart (recordings from cold-acclimated trout at 4°C). PM, pacemaker. Bottom panel indicates the rate of voltage changes during pacemaker potentials. Note differences in MDP, the rate of AP upstroke, AP duration, AP overshoot, and the shape of the ET (arrows). C: comparison of the primary pacemaker potentials from the sinoatrial nodal tissue of cold (c.a.)- and warm-acclimated (w.a.) trout heart at the acclimation temperatures of the fish and at 11°C.

 
Current-clamp recording of pacemaker APs from single myocytes. Single myocytes were enzymatically isolated from the sinoatrial tissue of the trout heart by using previously published methods with slight modifications (36). Briefly, the heart was cannulated through the bulbus arteriosus into the ventricle, and the heart was retrogradely perfused first with a nominally Ca2+-free, low-Na+ solution containing (in mM) 100 NaCl, 10 KCl, 1.2 KH2PO4, 4 MgSO4, 50 taurine, 20 glucose, and 10 HEPES at pH 6.9 at 20°C for 10 min, and then with a fresh low-Na+ solution supplemented with 0.75 mg/ml collagenase (type IA; Sigma, St. Louis, MO), 0.5 mg/ml trypsin (type IX; Sigma), and 0.5 mg/ml fatty acid-free bovine serum albumin for 15 min from a height of 50 cm. Both solutions were oxygenated with 100% O2, and the enzyme solution was recycled using a peristaltic pump. After enzymatic digestion, a thin ring (~1 mm) of tissue from the sinoatrial junction was dissected free under the microscope and chopped in small pieces with scissors in fresh low-Na+ solution. Tissue pieces were gently stirred with a small magnetic bar for 20 min at room temperature (22 ± 1°C), and single cells were released by agitating tissue pieces through the opening of a Pasteur pipette. Myocytes were stored in low-Na+ solution at 6°C and used within 8 h after isolation. Pacemaker cells were recognized on the basis of their morphology (see RESULTS) and spontaneous beating.

For current-clamp recording of APs, a small aliquot of myocyte suspension was transferred to a recording chamber (RC-26; Warner Instrument, Brunswick, NJ; volume 150 µl), and cells were allowed to settle on the chamber bottom before being superfused with external saline solution containing (in mM) 150 NaCl, 5.4 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 glucose, and 10 HEPES, with pH adjusted to 7.7 with NaOH. Temperature was adjusted to 4, 11, or 18°C with the use of circulating water baths, and temperature was continuously controlled by a thermistor located close to the myocyte. APs were recorded with the ruptured patch method in current-clamp mode of an Axopatch 1-D amplifier with a CV-4 1/100 head stage (Axon Instruments). Patch pipettes were pulled (L/M-3P-A; List Medical) from borosilicate glass and had a mean resistance of 3.02 ± 0.05 M{Omega} (n = 116) when filled with the pipette solution, which contained (in mM) 140 KCl, 5 Na2ATP, 1 MgCl2, 0.03 Tris-GTP, and 10 HEPES at pH 7.2. Offset potentials were zeroed just before the formation of a gigaohm seal, and the pipette capacitance (7.85 ± 0.07 pF, n = 116) was compensated for after seal formation. After rupture of the patch, capacitive transients were eliminated by adjusting series resistance and cell capacitance compensation circuits before switching into the current-clamp mode. Sampling and filtering frequencies were 10 and 5 kHz, respectively. The digitized signals were stored on the hard drive of the computer for off-line analysis of AP variables (Fig. 1A).

Whole cell patch clamp. The whole cell voltage-clamp recording of the IKr from atrial and pacemaker cells was performed using an Axopatch 1-D amplifier. Myocytes were superfused in a small recording chamber with a precooled (11°C) external saline solution (for composition, see Current clamp recording of pacemaker APs from single myocytes) at the rate of 1.5–2.0 ml/min. Patch pipettes were pulled from borosilicate glass (Garner, Claremont, CA) and filled with K+-based electrode solution (in mM: 140 KCl, 1 MgCl2, 5 EGTA, 4 MgATP, and 10 HEPES with pH adjusted to 7.2 with KOH), giving a mean (±SE) pipette resistance of 2.80 ± 0.12 M{Omega}. Initial steps of patching were made in blocker-free external saline so that pacemaker cells could be recognized on the basis of spontaneous beating. After access to the cell was obtained, pipette capacitance (7.87 ± 0.19 pF, n = 27) and series resistance (9.84 ± 0.14 M{Omega}, n = 27) were compensated, and IKr currents were elicited from the holding potential of –80 mV in the presence of tetrodotoxin (0.5 µM; Tocris Cookson), nifedipine (10 µM; Sigma), and glibenclamide (10 µM; Sigma) to block Na+, Ca2+, and ATP-sensitive K+ current, respectively. Dose-dependent inhibition of the IKr by a specific blocker of the fast component of the delayed rectifier, E-4031 (Alomone Labs) was determined by cumulatively adding concentrations of E-4031 (10–9–10–6 M) to atrial myocytes. The cell was exposed to each E-4031 concentration for 4 min. Dose-response curves were fitted with a Hill equation:

Formula

where Imin is the minimum IKr at the highest E-4031 concentration, Imax is the IKr before the addition of the blocker, IC50 is the drug concentration that causes half-maximal inhibition of the IKr, [E-4031] is the drug concentration, and nH is the Hill slope of the line.

Statistical analyses. A paired t-test was used for evaluating the effects of E-4031 and Ry + Tg on heart rate and AP duration and to compare heart rate and AP duration between cold- and warm-acclimated trout. One-way analysis of variance with Tukey's honestly significant difference post hoc test was used for multiple comparisons between cell types and different temperatures. P < 0.05 was regarded as a limit for a statistically significant difference.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Localization of pacemaker potentials in trout sinoatrial tissue. The border area between sinus venosus and atrium and the sinoatrial valve were impaled in multiple places with sharp microelectrodes to determine the primary pacemaker area of the trout heart and to record pacemaker potentials. Three different types of APs were recognized (Fig. 1B). An atrial AP with a stable negative RMP (–83 ± 2 mV) was the most frequently recorded voltage waveform in both the sinoatrial valve and the junctional tissue at the base of the valve. Although stable impalements of cells generating atrial APs were easily obtained from both cold- and warm-acclimated hearts, finding pacemaker APs was surprisingly difficult, and obtaining stable electrode impalement in a pacemaker cell was extremely tedious. However, two types of pacemaker APs were recorded. A pacemaker potential that closely resembles the pacemaker AP of the central cells of the mammalian sinoatrial node was characterized by a low maximum diastolic potential, a low upstroke rate of AP, a small overshoot, and small AP amplitude. This is assumed to represent the AP of the primary pacemaker cells and is thence hereafter called the primary pacemaker potential. The other type of pacemaker potential of the trout heart was similar to the pacemaker potential of the peripheral cells of the mammalian sinoatrial node with fast and abruptly starting AP upstroke, a more negative maximum diastolic potential, and large AP amplitude (Fig. 1B). According to the mammalian convention, this is called the secondary pacemaker potential.

Temperature-related differences were clearly evident in AP duration and in AP discharge frequency. When recorded at the same experimental temperature (11°C), AP rate (heart rate) was faster and duration of pacemaker AP much shorter in cold- than warm-acclimated trout (P < 0.05) (Fig. 1C and Table 1).


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  		Table 1. Action potential characteristics and action potential rate of the rainbow trout sinoatrial pacemaker cells measured with intracellular microelectrodes from intact sinoatrial preparations of c.a. and w.a. fish

 
Histological localization of the trout primary pacemaker. Microelectrode recordings indicated that pacemaker potentials could be very seldom recorded in the sinoatrial valve, and those few ones obtained were typical secondary pacemaker potentials. Instead, primary pacemaker potentials were exclusively recorded from the base of the sinoatrial valve, suggesting that the location of the primary pacemaker is in the border zone between sinus venosus and atrium. Indeed, histological inspection showed a separate bundle of cardiac tissue circling the base of the sinoatrial valve (Fig. 2). The bundle was separated from the surrounding tissue by a delicate sheet of connective tissue, which also intruded into the nodal tissue, dividing it in several smaller sections. A number of nerve profiles were evident at the periphery of the bundle as well as in the sinoatrial valve. The thickness of the nodal tissue was only ~200 µm in a 200- to 300-g trout, which explains the difficulties of finding and recording pacemaker APs with microelectrodes from the intact pacemaker tissue.


Figure 2
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  		Fig. 2. Light microscopic histology showing structure and location of the sinoatrial nodal tissue of the rainbow trout heart. A: a schematic presentation of the heart structure and the area for histological inspection (within the box). B: a photomicrograph of the trout atrium and the adjacent part of the sinus venosus. The junctional area between sinus venosus and atrium, including the sinoatrial valve, is shown framed in the box. C: a closer look at the nodal tissue at the base of sinoatrial valve. D: an enlarged image of the sinoatrial nodal tissue. The sections were stained with picrosirius red. CT, connective tissue; NT, nervous tissue; PA, pacemaker area.

 
Morphology and electrical activity of isolated pacemaker cells. After the exact location of the pacemaker area was defined, single pacemaker myocytes could be enzymatically isolated. Following the morphological delineation of the rabbit nodal cells by Verheijck et al. (34), the enzymatically isolated cells of the trout sinoatrial nodal tissue were separated into three different types (spindle cells, elongated spindle cells, and spider cells), and their electrical activity was recorded in the current-clamp mode of the whole cell patch-clamp technique (Fig. 3). The spindle cells were smaller than elongated spindle cells and spider cells, and pacemaker cells of the cold-acclimated trout were larger than those of the warm-acclimated trout (P > 0.05) (Table 2). The two different types of pacemaker potentials found in the intact muscle were also observed in enzymatically isolated pacemaker cells. Primary pacemaker potentials were most often recorded in the small spindle cells, suggesting that they might form the primary pacemaker of the trout heart. Statistical examination indicated, however, that there were few differences in AP characteristics between the three types of myocytes, suggesting that primary pacemaker cells cannot be distinguished on morphological grounds. The pacemaking rate of isolated pacemaker cells was higher in cold- (44 ± 6 beats/min) than warm-acclimated fish (38 ± 6 beats/min; means ± SE of all cell types, P < 0.05), thus confirming that at least part of the compensatory increase in heart rate is inherent to the pacemaker cells. Furthermore, the duration of pacemaker AP was significantly shorter in cold- than warm-acclimated trout (P < 0.05) (Table 3).


Figure 3
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  		Fig. 3. Enzymatically isolated pacemaker cells of the trout heart and their electrical activity. Top: photomicrographs of the 3 morphologically different pacemaker cells of the trout sinoatrial tissue: spindle cell, spider cell, and elongated spindle cell. Note the different scale bar for each cell type. Bottom: representative current-clamp recordings of pacemaker APs from the 3 cell types of cold- and warm-acclimated trout at their acclimation temperatures.

 

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  		Table 2. Capacitive cells size of isolated atrial and pacemaker myocytes from thermally acclimated rainbow trout

 

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  		Table 3. Action potential variables and action potential rate of enzymatically isolated pacemaker cells of the rainbow trout heart

 
Effects of E-4031 on AP rate in intact sinoatrial preparations. Putative contribution of the fast component of the delayed rectifier K+ channels to the heart rate difference between warm- and cold-acclimated trout was tested by applying a specific blocker of the IKr, E-4031, to intact sinoatrial preparations. E-4031 inhibited the IKr of the trout atrial myocytes with IC50 values of 0.12 ± 0.22 and 0.12 ± 0.28 µM (P = 0.66) in warm- and cold-acclimated trout, respectively, indicating a similar blocker sensitivity of the current in both acclimation groups (Fig. 4A). Accordingly, E-4031 was used at the concentration of 0.1 µM to attain nearly half-maximal inhibition of the current. Because pacemaker APs could not be routinely recorded, the effectiveness of the blocker on cardiac electrical activity was checked from cells of the sinoatrial valve, which generated atrial-type APs (Fig. 4B). Before the addition of E-4031, AP rate (at 11°C) was higher and AP duration shorter in cold- than warm-acclimated preparations (P < 0.05), indicating compensatory changes in both variables by cold acclimation. E-4031 depressed AP rate in both acclimation groups, but the inhibition was stronger in warm-acclimated (47%) than cold-acclimated animals (15%) (Fig. 4C). A similar quantitative difference was evident in AP duration (Table 4). Moreover, the effect of E-4031 was enhanced at high experimental temperature, since inhibition of AP rate was 60 and 5% at 18 and 4°C for warm- and cold-acclimated fish, respectively.


Figure 4
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  		Fig. 4. Effect of E-4031, a specific blocker of the delayed rectifier K+ current IKr, on AP rate and AP duration of the trout heart. A: concentration dependence of the E-4031 block of the IKr in atrial myocytes of cold- and warm-acclimated trout heart (left). Representative recordings of the IKr are shown (right) in the absence (control) and presence of E-4031. Note the larger current density of the IKr in the cold-acclimated trout. B: voltage dependence of the IKr tail current density in atrial myocytes and pacemaker cells of warm- and cold-acclimated rainbow trout. C: representative recordings of the effect of 0.1 µM E-4031 on atrial APs (heart rate, HR) of warm- and cold-acclimated trout, recorded with microelectrodes from intact sinoatrial tissue. D: a bar graph showing the effect of 0.1 µM E-4031 on the AP rate. Results are means ± SE of 6 or 7 preparations. Statistically significant differences are indicated (*P < 0.05).

 

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  		Table 4. Effect E-4031, a specific blocker of the IKr, on action potential rate and atrial action potential duration of the rainbow trout heart

 
Interestingly, the density of the IKr in cold-acclimated trout was almost double that in warm-acclimated trout (P < 0.05) for both atrial myocytes and pacemaker cells (Fig. 4D). The maximum density of the IKr tail current of pacemaker cells was 1.8 times larger in cold-acclimated (5.01 ± 0.30 pA/pF) than in warm-acclimated animals (2.81 ± 0.32 pA/pF) (P < 0.05). On the other hand, the density of the IKr was 18% less in pacemaker cells than in atrial myocytes (P < 0.05). Together, these results indicate reduced effect of the IKr blocker E-4031 on AP rate and increased density of the IKr in cold-acclimated trout pacemaker cells.

Effects of Ry + Tg on AP rate in intact sinoatrial preparations. In untreated control preparations, the discharge frequency of APs was again higher and AP duration shorter for cold-acclimated than warm-acclimated trout (P < 0.05). The effect of SR Ca2+ cycling on AP rate was examined by inhibiting SR Ca2+ release and SR Ca2+ uptake with 10 µM Ry and 1 µM Tg, respectively (Fig. 5). The inhibition of SR Ca2+ management by simultaneous application of Ry and Tg exerted only a marginal and insignificant (P > 0.05) depressive effect on AP rate at 11°C (14 and 9% in cold-acclimated and warm-acclimated heart, respectively). Similarly, Ry + Tg had only minor effects on AP duration of the sinoatrial valve (Table 5). However, a statistically significant (44%, P < 0.05) drop in AP rate was evident in the warm-acclimated trout heart at 18°C. Collectively, these experiments suggest that SR Ca2+ cycling does not contribute to temperature-induced changes in pacemaking rate of the rainbow trout heart but may participate in heart rate regulation at high temperatures.


Figure 5
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  		Fig. 5. Effect of sarcoplasmic reticulum (SR) blockade with ryanodine and thapsigargin (Tg) on AP rate and AP duration of the trout heart. A: representative recordings of the effect of ryanodine (10 µM) + Tg (1 µM) on atrial APs of warm- and cold-acclimated trout, recorded with microelectrodes from the intact sinoatrial tissue. B: a bar graph showing the effect of ryanodine + Tg on AP rate. Results are means ± SE of 6 or 7 preparations. Statistically significant differences are indicated (*P < 0.05).

 

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  		Table 5. Effect of SR blockade with ryanodine plus thapsigargin on action potential rate and atrial action potential duration of the rainbow trout heart

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In principle, temperature-induced changes in heart rate could be produced either by altering humoral and neural regulation of pacemaker activity or by modifying the pacemaker mechanism proper, or both. The fish heart is normally under tight parasympathetic control so that continuous release of acetylcholine from cholinergic nerve endings keeps in vivo heart rate lower than the intrinsic beating rate of the pacemaker center (17, 29, 35). In some fish species, acclimation to cold produces a compensatory increase in heart rate by a partial relief of the inhibitory cholinergic tone (30, 32). In the eel (Anguilla anguilla), temperature-induced modulation of heart rate was, however, only partly prevented by a cholinergic blocker (30), suggesting that additional mechanisms for thermal compensation must exist. In fish, the stimulatory influence of adrenergic nervous system on heart rate is generally much weaker than the inhibitory cholinergic effect (8, 35). However, chronic exposure to cold enhances adrenergic sensitivity of the rainbow trout heart by increases in the number of beta-receptors (3, 14), which could increase heart rate through the beta-adrenergic cascade by blood-borne or nervous release of adrenaline. Indeed, the effect of adrenaline on heart rate is stronger in cold-acclimated than in warm-acclimated trout (2). Although external regulatory factors seem to be involved in the acclimation process of heart rate, the cold-induced increase in the AP rate of enzymatically isolated pacemaker cells directly indicates that at least part of the response is independent of autonomic neurotransmission, i.e., intrinsic to the pacemaker process proper. The present results confirm the previous finding (2) that acclimation to cold elicits a compensatory increase in basal beating rate of the rainbow trout sinoatrial preparations and extends it by indicating that it resides in single primary pacemaker cells of the sinoatrial node.

Pacemaker mechanism of the vertebrate heart is a complicated interaction among multiple ionic mechanisms including various ion channels, NCX, and CICR of the SR, and in principle, acclimation-related differences in heart rate could be produced by any of the ionic mechanisms that contribute to diastolic depolarization of the pacemaker AP. One more alternative mechanism is provided by intercellular coupling. Because each pacemaker cell has its characteristic beating rate, individual cells have to interact electrically such that their activation times are synchronized to produce a stable and regular discharge rate of the intact pacemaker center (24). Indeed, when two or more cells with different firing rates are electrically coupled in cell culture, the firing rate of the syncytium is intermediate between the coupled cells, with each cell contributing to the overall mutual entrainment. On the basis of this mechanism, it is possible that temperature-induced changes in heart rate could be due to changes in the extent of electric coupling between atrial myocytes and pacemaker cells, or in the electrical properties of atrial myocytes coupled to pacemaker cells. That the acclimation effect is present in single pacemaker cells of the disaggregated nodal tissue suggests, however, that temperature-related differences in electrotonic coupling do not exist. Even then, several possible mechanisms for thermal compensation remain. As the first undertaking to clarify the pacemaker mechanism of the fish heart, we decided to examine the significance of CICR of the SR and the delayed rectifier K+ current (IKr), both of which are known to be enhanced by cold acclimation in atrial and ventricular tissue of the rainbow trout heart (1, 15, 37).

The stronger depression (47 vs. 15% at 11°C) of heart rate in warm-acclimated than cold-acclimated trout by a specific blocker of the fast component of the delayed rectifier K+ channels, E-4031, suggests that IKr contributes to temperature-dependent changes in beating rate of the trout heart. This might be related to the more than twice larger current density of the IKr in cold-acclimated than warm-acclimated hearts. Unlike other ion currents, the IKr is vital for impulse generation, since complete blockade of the current in mammalian sinoatrial pacemaker cells ceases their spontaneous firing, probably because of the depolarization of the membrane potential (33, 42). Activation of the outward IKr upon upstroke of the AP initiates repolarization, thus restoring maximum diastolic potential (MDP) and allowing recovery of inward currents from inactivation, whereas subsequent decrease of the outward IKr due to deactivation of the channels permits various inward currents to produce diastolic depolarization. Accordingly, IKr can accelerate heart rate by shortening the duration of the pacemaker AP and by increasing the MDP. In the rabbit sinoatrial node, peripheral pacemaker cells have 6.2-times larger IKr and a significantly faster beating rate than central cells (19). Furthermore, the peripheral cells are more resistant to the cardioinhibitory action of E-4031. Although it may be counterintuitive that tissue having larger IKr is less sensitive to the rate-depressing effect of its blocker, the smaller effect of E-4031 in cold-acclimated compared with warm-acclimated trout is in complete agreement with the mammalian data. In fact, the requirement of higher IKr density for better E-4031 resistance was first predicted by computer simulation and later confirmed experimentally (42). Since pacemaker cells of the cold-acclimated trout have much larger IKr than those of warm-acclimated trout, after the half-blockade of the current with 0.1 µM E-4031, the cold-acclimated heart would still have IKr equally large as that of the warm-acclimated heart before the blockade. Thus the larger IKr of the pacemaker cells in cold-acclimated trout would shorten AP duration, increase intrinsic beating rate, and make them more resistant to blockade by E-4031.

Recent evidence indicates that intracellular Ca2+ release from the SR contributes importantly to pacemaker function in mammalian and amphibian hearts (13, 27, 43). Intracellular Ca2+ release is triggered by sarcolemmal Ca2+ influx through T-type Ca2+ channels and is thought to accelerate diastolic depolarization through activation of the inward NCX current (21). On the basis of this mechanism, interventions that alter SR Ca2+ management are expected to regulate cardiac pacemaker activity. Since participation of SR Ca2+ to contractile activation in trout atrial and ventricular tissue seems to increase under cold acclimation (1, 15), it was anticipated that CICR release would contribute to pacemaker mechanism, especially in the cold-acclimated trout. This hypothesis is not supported by the current findings. CICR of the SR is apparently not involved in the compensatory increase of heart rate in cold-acclimated trout, and CICR does not contribute to pacemaker mechanism of the trout heart at low temperatures (<11°C), since Ry + Tg failed to affect heart rate. Interestingly, at 18°C, inhibition of the SR Ca2+ cycling with Ry + Tg depressed heart rate in warm-acclimated trout by 44%. This suggests that SR might be a factor in heart rate regulation at temperatures that approach the upper thermal tolerance limit of the rainbow trout. This is consistent with the previous findings on excitation-contraction coupling of the trout heart, indicating improved SR function at high experimental temperatures (31). Since SR Ca2+ cycling is a potentially important ionic mechanism at the upper end of physiological temperatures, focused studies about acute temperature effects on SR function are warranted as means to reveal relative significance of surface membrane-limited ionic mechanisms and SR Ca2+ dynamics in the vertebrate cardiac pacemaker (16, 23, 43).

Pacemaker tissue occurs in multiple places in the fish heart, and in various fish species the primary cardiac pacemaker has been located in sinus venosus, sinoatrial valve, or at the border zone between sinus venosus and atrium (28, 35). There is, however, quite general agreement that specialized myocardial tissue exists at the junction between sinus venosus and atrium, which is assumed to be the nodal pacemaker tissue in many fish species (18). Yamauchi and Burnstock (40) provided histological evidence for this in the heart of brown trout (Salmo trutta). In the present study, the primary pacemaker of the rainbow trout heart was located by electrophysiological and histological observations at the sinoatrial border. The circular nodal tissue was only ~0.2 mm in width (diameter) and ~3 mm in length in a 200- to 300-g fish. From the volume (0.094 mm3) of this nodal tissue and average myocyte size (2,180 µm3; calculated from cell capacitance), and assuming 65% of the tissue comprises cells, it was calculated that the nodal tissue of the trout heart might contain ~28,000 myocytes. This is the same order of magnitude as in mammalian hearts (4). Similar to mammalian sinoatrial nodal tissue, three morphologically different cell types were recognized in trout sinoatrial node. We could not find any significant differences in electrical properties among spindle cells, elongated spindle cells, and spider cells of the trout heart. This agrees with the findings of Verheijck et al. (34) on rabbit sinoatrial myocytes but is at odds with the results of Wu et al. (39), who found distinct electrophysiological features between spider cells and spindles cells of the rabbit heart. Examination of the IKr and other ion currents in different cell types of the trout nodal tissue is needed to clarify possible electrophysiological differences and functional roles of the three nodal cell types.

Limitations and Perspectives

This study examines the effect of temperature acclimation on intrinsic heart rate by measuring pacemaker potentials from intact nodal tissue and isolated pacemaker cells of the rainbow trout heart. The conclusions are based on the specificity of pharmacological blockers E-4031 and Ry + Tg to inhibit IKr and SR Ca2+ cycling, respectively. The results strongly suggest that IKr, but not SR, is involved in temperature-induced changes in heart rate. Since pacemaker function of the vertebrate heart consists of many other ionic mechanisms in addition to IKr and SR Ca2+ release, the present findings cannot exclude the possibility that other ion currents, exchangers, and pumps are involved, too. In particular, several inward currents, including the pacemaker current, T-type Ca2+ current, and L-type Ca2+ current, are suggested to be crucial for diastolic depolarization and could be modified by chronic temperature changes (9). It should be noted, however, that a substantial increase of inward currents would be required to produce compensatory increase in heart rate in the cold. This would tend to increase the duration of pacemaker potential, whereas strong shortening was found. Furthermore, it is probably biologically meaningful to achieve the increase in heart rate by enhancing repolarizing currents, since these will not only limit the cold-dependent lengthening of AP duration but also will indirectly increase the density of inward currents by allowing more time for recovery from inactivation. The present findings cannot, however, exclude the possibility that a small increase in inward currents is masked under a much larger increase in IKr. Clearly, a detailed characterization of various inward currents and the complete ionic basis of the fish cardiac pacemaker are worth pursuing, since environmental perturbation of the underlying processes could reveal important aspects of molecular and cellular mechanism of the vertebrate cardiac pacemaker and their physiological significance.

In conclusion, we have shown that thermal acclimation modifies cardiac pacemaker mechanism of the trout heart by increasing the intrinsic beating rate of enzymatically isolated primary pacemaker cells in the cold. This is associated with shorter duration of pacemaker AP and is likely to involve increased density of the delayed rectifier K+ current IKr, which may allow a higher rate of diastolic depolarization. In contrast, CICR of the SR is not involved in acclimation-induced changes in heart rate but may participate in the pacemaker mechanism at high temperatures, at least in the warm-acclimated trout.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was financed by a research grant from the Academy of Finland to M. Vornanen (Project No. 210400).


   ACKNOWLEDGMENTS
 
A. Kervinen is appreciated for technical assistance. The Kontiolahti fish farm is acknowledged for donating the trout.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Vornanen, Univ. of Joensuu, Dept. of Biology, PO Box 111, 80101 Joensuu, Finland (e-mail: matti.vornanen{at}joensuu.fi)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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