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In cardiomyocytes, ryanodine receptors (RYRs) mediate Ca(2+)-induced Ca(2+)-release (CICR) from the sarcoplasmic reticulum (SR) during excitation-contraction (e-c) coupling. In rainbow trout heart, the relative importance of CICR increases with cold-acclimation. Thus, the aim of this study was to investigate the effect of temperature acclimation (4, 11 and 18 degrees C) on RYR intracellular localization and expression density. We used immunocytochemistry to assess intracellular localization in ventricular myocytes and Western blotting to assess RYR expression in both atrial and ventricular tissue. In ventricular myocytes, RYRs were localized peripherally in transverse bands aligning with sarcomeric m-lines and centrally around mitochondria and the nucleus. Localization did not change with temperature acclimation. RYR expression was also unaffected by temperature acclimation. The localization of RYRs at the m-line is similar to neonatal mammalian cardiomyocytes. We suggest this positioning is indicative of myocytes which rely predominantly on transsarcolemmal Ca(2+)-influx, rather than CICR, during e-c coupling.

We measured the temperature sensitivity, adrenergic sensitivity, and dependence on sarcoplasmic reticulum (SR) Ca(2+) of ventricular muscle from pelagic fishes with different vertical mobility patterns: bigeye tuna (Thunnus obesus), yellowfin tuna (Thunnus albacares), and mahimahi (Coryphaena hippurus) and a single specimen from swordfish (Xiphias gladius). Ventricular muscle from the bigeye tuna and mahimahi exhibited a biphasic response to an acute decrease in temperature (from 26 degrees to 7 degrees C); twitch force and kinetic parameters initially increased and then declined. The magnitude of this response was larger in the bigeye tuna than in the mahimahi. Under steady state conditions at 26 degrees C, inhibition of SR Ca(2+) release and reuptake with ryanodine and thapsigargin decreased twitch force and kinetic parameters, respectively, in the bigeye tuna only. However, the initial inotropy associated with decreasing temperature was abolished by SR inhibition in both the bigeye tuna and the mahimahi. Application of adrenaline completely reversed the effects of ryanodine and thapsigargin, but this effect was diminished at cold temperatures. In the yellowfin tuna, temperature and SR inhibition had minor effects on twitch force and kinetics, while adrenaline significantly increased these parameters. Limited data suggest that swordfish ventricular muscle responds to acute temperature reduction, SR inhibition, and adrenergic stimulation in a manner similar to that of bigeye tuna ventricular muscle. In aggregate, our results show that the temperature sensitivity, SR dependence, and adrenergic sensitivity of pelagic fish hearts are species specific and that these differences reflect species-specific vertical mobility patterns.

Most fish regulate cardiac output via changes in stroke volume whereas most mammals regulate cardiac output via changes in heart rate. We hypothesized that this change in mechanism of regulation would coincide with a change in the myocardial response to stretch. This possibility was tested in permeabilized cardiomyocytes prepared from rat and rainbow trout ventricles, where both sarcomere length (SL) and degree of Ca2+ activation could be controlled. Myofilament Ca2+ sensitivity activation was higher in trout than in rat at each SL tested (2.0, 2.3, 2.5 and 2.7 ìm). We also found that permeabilized trout myocytes produce greater passive tension at any given SL than rat. This was surprising as the trout heart is known to be more compliant. Interestingly, addition of phosphatase inhibitors in the permeabilizing solution reduced passive tension in the trout cells suggesting that phosphorylation of titin may be important in determining passive tension in particular in trout heart. In conclusion contractile properties in trout seem highly sensitive to the length-dependent modulation.

Inactivation of L-type calcium current (ICa) is due to two mechanisms: voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). In fish cardiac myocytes, it is unknown whether Ca release from the sarcoplasmic reticulum (SR) participates in CDI of ICa. This study assesses the relative contribution of different inactivation mechanisms of ICa in trout ventricular myocytes. Trout ventricular myocytes were enzymatically isolated. ICa was recorded using whole cell patch clamp with Na- and K-free solutions to avoid contaminating currents. With a low concentration of a slow Ca buffer (EGTA 2mM) in the pipette solution, ICa inactivated slowly (compared to mammalian cardiac myocytes): the time to reach 37% of peak current (T37) was 26.2±2.4 ms (mean±SEM, n=14). When a fast Ca buffer (BAPTA 10 mM) was present in the pipette solution ICa decay was similar to the decay in the presence of EGTA (T37: 25.4±1.5 ms, NS, t-test, n=9). When barium was used as a charge carrier, IBa inactivates mainly via VDI and T37 was significantly increased (43.7±3.1 ms, n=9, p

SUMMARYThe Frank–Starling law of the heart applies to all classes of vertebrates. It describes how stretch of cardiac muscle, up to an optimum length, increases contractility thereby linking cardiac ejection to cardiac filling. The cellular mechanisms underlying the Frank–Starling response include an increase in myofilament sensitivity for Ca2+, decreased myofilament lattice spacing and increased thin filament cooperativity. Stretching of mammalian, amphibian and fish cardiac myocytes reveal that the functional peak of the sarcomere length (SL)–tension relationship occurs at longer SL in the non-mammalian classes. These findings correlate with in vivo cardiac function as non-mammalian vertebrates, such as fish,vary stroke volume to a relatively larger extent than mammals. Thus, it seems the length-dependent properties of individual myocytes are modified to accommodate differences in organ function, and the high extensibility of certain hearts is matched by the extensibility of their myocytes. Reasons for the differences between classes are still to be elucidated, however, the structure of mammalian ventricular myocytes, with larger widths and higher levels of passive stiffness than those from other vertebrate classes may be implicated.

Certain vertebrates, such as freshwater turtles of the genus Chrysemys and Trachemys and crucian carp (Carassius carassius), have anoxia-tolerant hearts that continue to function throughout prolonged periods of anoxia (up to many months) due to successful balancing of cellular ATP supply and demand. In the present review, we summarize the current and limited understanding of the cellular mechanisms underlying this cardiac anoxia tolerance. What emerges is that cold temperature substantially modifies cardiac electrophysiology to precondition the heart for winter anoxia. Intrinsic heart rate is slowed and density of sarcolemmal ion currents substantially modified to alter cardiac action potential (AP) characteristics. These changes depress cardiac activity and reduce the energetic costs associated with ion pumping. In contrast, anoxia per se results in limited changes to cardiac AP shape or ion current densities in turtle and crucian carp, suggesting that anoxic modifications of cardiac electrophysiology to reduce ATP demand are not extensive. Additionally, as knowledge of cellular physiology in non-mammalian vertebrates is still in its infancy, we briefly discuss the cellular defense mechanisms towards the acidosis that accompanies anoxia as well as mammalian cardiac models of hypoxia/ischemia tolerance. By examining if fundamental cellular mechanisms have been conserved during the evolution of anoxia tolerance we hope to have provided a framework for the design of future experiments investigating cardiac cellular mechanisms of anoxia survival.

Bluefin Tuna (Thunnus orientalis) have the capacity to move from warm surface waters to cooler waters beneath the thermocline where they will be faced with the challenge of a cold heart delivering ...

Here we explored the hypothesis that individual variation in thermal tolerance in the European sea bass is associated with other key performance traits involved in determining individual Darwinian fitness. Our finding that whole animal temperature tolerance positively correlates with cardiac size could suggest selection on the cardiorespiratory system may benefit thermal tolerance., The European sea bass (Dicentrarchus labrax) is an economically important fish native to the Mediterranean and Northern Atlantic. Its complex life cycle involves many migrations through temperature gradients that affect the energetic demands of swimming. Previous studies have shown large intraspecific variation in swimming performance and temperature tolerance, which could include deleterious and advantageous traits under the evolutionary pressure of climate change. However, little is known of the underlying determinants of this individual variation. We investigated individual variation in temperature tolerance in 30 sea bass by exposing them to a warm temperature challenge test. The eight most temperature-tolerant and eight most temperature-sensitive fish were then studied further to determine maximal swimming speed (UCAT), aerobic scope and post-exercise oxygen consumption. Finally, ventricular contractility in each group was determined using isometric muscle preparations. The temperature-tolerant fish showed lower resting oxygen consumption rates, possessed larger hearts and initially recovered from exhaustive exercise faster than the temperature-sensitive fish. Thus, whole-animal temperature tolerance was associated with important performance traits. However, the temperature-tolerant fish also demonstrated poorer maximal swimming capacity (i.e. lower UCAT) than their temperature-sensitive counterparts, which may indicate a trade-off between temperature tolerance and swimming performance. Interestingly, the larger relative ventricular mass of the temperature-tolerant fish did not equate to greater ventricular contractility, suggesting that larger stroke volumes, rather than greater contractile strength, may be associated with thermal tolerance in this species.

All vertebrate cardiac muscle responds intrinsically to mechanical stimulation which can lead to changes in both the inotropic and chronotropic state of the heart. However the magnitude and physiological relevance of these mechanically-induced responses differ between vertebrate classes. This review will discuss the differences and similarities in the response of vertebrate hearts to stretch. It will focus on responses to mechanical stimulation that have been well characterised in mammals, and discuss what is known about them in non-mammalian vertebrates. Specifically we focus on the Frank- Starling response or length-tension relationship, stretch acceleration of heart rate (the Bainbridge effect) and mechanically-induced effects on cardiac rhythm. Although they have not been categorically studied, these three basic mechanical and electrical responses to stretch are likely present in all vertebrate classes. For example, in a manner similar to mammals, one of the earliest vertebrates, the hagfish (Myxine glutinosa), shows a remarkable increase (up to 150%) in heart rate in response to cardiac stretch and in amphibian hearts, modification of action potential profiles and mechanically triggered action potentials have been observed. These commonalities are interesting given the differences in whole heart, cellular and sub-cellular morphology and working environments between early and later vertebrates. These differences may have led to fundamental differences in cardiovascular design between classes. For instance, the exquisite sensitivity of the Starling response in the heart of the rainbow trout (Oncorhynchus mykiss) may explain why fish increase predominantly stroke volume rather than heart rate when modulating cardiac output. We speculate that despite the variety of vertebrate hearts, mechanosensitivity is fundamentally similar, if subtly different, across classes. Shared mechanisms, such as mechanosensitive channels (possibly TRP), make early vertebrate hearts useful models for the study of cardiac mechanosensitivity. Finally, given that early vertebrates are known to rely on intrinsic regulation to a greater extent than later vertebrates they may provide useful systems in which to study the role of mechanosensitivity in the evolution of cardiac function and cardiac regulation.

Intracellular Na+-concentration, [Na+]imodulates excitation-contraction coupling of cardiac myocytes via the Na+/Ca2+exchanger (NCX). In cardiomyocytes from rainbow trout ( Oncorhyncus mykiss), whole cell patch-clamp studies have shown that Ca2+influx via reverse-mode NCX contributes significantly to contraction when [Na+]iis 16 mM but not 10 mM. However, physiological [Na+]ihas never been measured. We recorded [Na+]iusing the fluorescent indicator sodium-binding benzofuran isophthalate in freshly isolated atrial and ventricular myocytes from rainbow trout. We examined [Na+]iat rest and during increases in contraction frequency across three temperatures that span those trout experience in nature (7, 14, and 21°C). Surprisingly, we found that [Na+]iwas not different between atrial and ventricular cells. Furthermore, acute temperature changes did not affect [Na+]iin resting cells. Thus, we report a resting in vivo [Na+]iof 13.4 mM for rainbow trout cardiomyocytes. [Na+]iincreased from rest with increases in contraction frequency by 3.2, 4.7, and 6.5% at 0.2, 0.5, and 0.8 Hz, respectively. This corresponds to an increase of 0.4, 0.6, and 0.9 mM at 0.2, 0.5, and 0.8 Hz, respectively. Acute temperature change did not significantly affect the contraction-induced increase in [Na+]i. Our results provide the first measurement of [Na+]iin rainbow trout cardiomyocytes. This surprisingly high [Na+]iis likely to result in physiologically significant Ca2+influx via reverse-mode NCX during excitation-contraction coupling. We calculate that this Ca2+-source will decrease with the action potential duration as temperature and contraction frequency increases.

We have developed a novel method to quantitatively analyze mitochondrial positioning in three dimensions. Using this method, we compared the relative positioning of mitochondria in adult rat and rainbow trout ( Oncorhynchus mykiss) ventricular myocytes. Energetic data suggest that trout, in contrast to the rat, have two subpopulations of mitochondria in their cardiomyocytes. Therefore, we speculated whether trout cardiomyocytes exhibit two types of mitochondrial patterns. Stacks of confocal images of mitochondria were acquired in live cardiomyocytes. The images were processed and mitochondrial centers were detected automatically. The mitochondrial arrangement was analyzed by calculating the three-dimensional probability density and distribution functions describing the distances between neighboring mitochondrial centers. In the rat (8 cells with a total of 7,546 mitochondrial centers), intermyofibrillar mitochondria are highly ordered and arranged in parallel strands. These strands are separated by ∼1.8 μm and can be found in any transversal direction relative to each other. Neighboring strands exhibit the same mitochondrial periodicity. In contrast to the rat, trout ventricular myocytes (22 cells; 5,528 mitochondrial centers) exhibit a relatively chaotic mitochondrial pattern. Neighboring mitochondria can be found in any direction relative to each other. Thus, two potential subpopulations of mitochondria in trout are not distinguishable by their pattern. The developed method required minor interaction in the filtering of the mitochondrial centers. It is therefore a practical approach to describe intracellular organization and may also be used for analysis of time-dependent organizational changes. The obtained quantitative description of mitochondrial organization is a requisite for accurate mathematical analysis of mitochondrial systems biology.

SUMMARYThe functional significance of the sarcoplasmic reticulum (SR) in the generation of high heart rates and blood pressures was investigated in four species of reptile; the turtle, Trachemys scripta; the python, Python regius, the tegu lizard, Tupinanvis merianae, and the varanid lizard, Varanus exanthematicus. Force-frequency trials and imposed pauses were performed on ventricular and atrial tissue from each species with and without the SR inhibitor ryanodine, and in the absence and presence of adrenaline. In all species, an imposed pause of 1 or 5 min caused a post-rest decay of force, and a negative force-frequency response was observed in all species within their in vivo frequency range of heart rates. These relationships were not affected by either ryanodine or adrenaline. In ventricular strips from varanid lizards and pythons, ryanodine caused significant reductions in twitch force within their physiologically relevant frequency range. In atrial tissue from the tegu and varanid lizards,SR inhibition reduced twitch force across the whole of their physiological frequency range. In contrast, in the more sedentary species, the turtle and the python, SR inhibition only decreased twitch force at stimulation frequencies above maximal in vivo heart rates. Adrenaline caused an increase in twitch force in all species studied. In ventricular tissue, this positive inotropic effect was sufficient to overcome the negative effects of ryanodine. In atrial tissue however, adrenaline could only ameliorate the negative effects of ryanodine at the lower pacing frequencies. Our results indicate that reptiles recruit Ca2+ from the SR for force development in a frequency and tissue dependent manner. This is discussed in the context of the development of high reptilian heart rates and blood pressures.

Confocal microscopy was used to investigate the temporal and spatial properties of Ca2+transients and Ca2+sparks in ventricular myocytes of the rainbow trout ( Oncorhynchus mykiss). Confocal imaging confirmed the absence of T tubules and the long (∼160 μm), thin (∼8 μm) morphology of trout myocytes. Line scan imaging of Ca2+transients evoked by electrical stimulation in cells loaded with fluo 4 revealed spatial inhomogeneities in the temporal properties of Ca2+transients across the width of the myocytes. The Ca2+wavefront initiated faster, rose faster, and reached larger peak amplitudes in the periphery of the myocyte compared with the center. These differences were exacerbated by stimulation with the L-type Ca2+channel agonist (−)BAY K 8644 or by sarcoplasmic reticulum (SR) inhibition with ryanodine and thapsigargin. Results reveal that the shape of the trout myocyte allows for rapid diffusion of Ca2+from the cell periphery to the cell center, with SR Ca2+release contributing to the cytosolic Ca2+rise in a time-dependent manner. Spontaneous Ca2+sparks were exceedingly rare in trout myocytes under control conditions (1 sparking cell from 238 cells examined). This is in marked contrast to the rat where a total of 56 spontaneous Ca2+sparks were observed in 9 of 11 myocytes examined. Ca2+sparklike events were observed in a very small number of trout myocytes (15 sparks from 9 of 378 cells examined) after stimulation with either (−)BAY K 8644 or high Ca2+(6 mM). Reducing temperature to 15°C in intact myocytes or permeabilizing myocytes to adjust intracellular conditions to favor Ca2+spark detection was without significant effects. Possible reasons for the rarity of Ca2+sparks in a cardiac myocyte with an active SR are discussed.

The purpose of this study was to investigate how the endogenous catecholamine adrenaline protects sarcolemmal Ca(2+) flux through the L-type Ca(2+) channel (I(Ca)) during acute exposure to cold in the fish heart. We examined the response of I(Ca) to adrenergic stimulation at three temperatures (7 degrees, 14 degrees, and 21 degrees C) in atrial myocytes isolated from rainbow trout acclimated to 14 degrees C. We found that I(Ca) amplitude varied directly with test temperature and was increased by adrenergic stimulation (AD; 5 nM and 1 microM) at all temperatures. However, I(Ca) was significantly more sensitive to adrenergic stimulation at the coldest test temperature. In fact, at 7 degrees C in the absence of AD, I(Ca) was extremely low. The addition of 1 microM AD increased peak I(Ca) 7.2-fold at 7 degrees C, 2.6-fold at 14 degrees C, and 1.6-fold at 21 degrees C and ameliorated the temperature-dependent difference in Ca(2+) influx across the cell membrane. We suggest that this increased adrenergic sensitivity is a critical compensatory mechanism that allows the rainbow trout heart to maintain contractility during acute exposure to cold temperatures. In particular, the tonic level of adrenergic stimulation provided by circulating plasma catecholamines (i.e., in the nM concentration range) may be crucial for effective excitation-contraction coupling in the cold cardiomyocyte.

Understanding the physiology of vertebrate thermal tolerance is critical for predicting how animals respond to climate change. Pacific bluefin tuna experience a wide range of ambient sea temperatures and occupy the largest geographical niche of all tunas. Their capacity to endure thermal challenge is due in part to enhanced expression and activity of key proteins involved in cardiac excitation–contraction coupling, which improve cardiomyocyte function and whole animal performance during temperature change. To define the cellular mechanisms that enable bluefin tuna hearts to function during acute temperature change, we investigated the performance of freshly isolated ventricular myocytes using confocal microscopy and electrophysiology. We demonstrate that acute cooling and warming (between 8 and 28°C) modulates the excitability of the cardiomyocyte by altering the action potential (AP) duration and the amplitude and kinetics of the cellular Ca2+transient. We then explored the interactions between temperature, adrenergic stimulation and contraction frequency, and show that when these stressors are combined in a physiologically relevant way, they alter AP characteristics to stabilize excitation–contraction coupling across an acute 20°C temperature range. This allows the tuna heart to maintain consistent contraction and relaxation cycles during acute thermal challenges. We hypothesize that this cardiac capacity plays a key role in the bluefin tunas' niche expansion across a broad thermal and geographical range.

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 Ca(2+) concentration ([Ca(2+)](i); cytosolic Ca(2+) transient) in isolated rainbow trout myocytes at 7 degrees C, 14 degrees C and 21 degrees C. Myocytes were voltage-clamped and loaded with Fura-2 to measure the L-type Ca(2+) channel current (I(Ca)) and [Ca(2+)](i) during physiological action potential (AP) pulses at frequencies that correspond to trout heart rates in vivo at 7 degrees C, 14 degrees C and 21 degrees C. Additionally, [Ca(2+)](i) and I(Ca) were examined with square (SQ) pulses at slow (0.2 Hz) and physiologically relevant contraction frequencies. The amplitude of [Ca(2+)](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, [Ca(2+)](i) decreased from 474+/-53 nmol l(-1) at 7 degrees C to 198+/-21 nmol l(-1) at 21 degrees C, while the decrease in [Ca(2+)](i) with AP pulses was from 234+/-49 nmol l(-1) to 79+/-12 nmol l(-1), respectively. Sarcolemmal Ca(2+) 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 degrees C, 14 degrees C and 21 degrees C, respectively). At all temperatures, cells were better able to maintain diastolic Ca(2+) 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 Ca(2+) regulation during temperature and frequency change in rainbow trout heart.

We used an isometric muscle preparation to test the hypothesis that yellowfin tuna Thunnus albacares utilize the intracellular Ca2+ storage sites of the sarcoplasmic reticulum (SR) during routine contractions. Ryanodine (a blocker of SR Ca2+ release) reduced the force of contraction by approximately 50 % and the rates of contraction and relaxation by 60 % in yellowfin tuna atrium. High levels of adrenaline were unable to ameliorate the effects of ryanodine. We conclude that the SR is active in contributing Ca2+ to force development at physiological contraction frequencies. Further, we suggest that, by using intracellular Ca2+ cycling, the yellowfin tuna is able to increase the maximum contraction frequency of its cardiac muscle beyond that of most other fishes.

Numerous studies have examined the effect of temperature on in vivo and in situ cardiovascular function in trout. However, little information exists on cardiac function at temperatures near the trout’s upper lethal limit. This study measured routine and maximum in situ cardiac performance in rainbow trout (Oncorhynchus mykiss) following acclimation to 15, 18 and 22 °C, under conditions of tonic (30 nmol l-1), intermediate (60 nmol l-1) and maximal (200 nmol l-1) adrenergic stimulation. Heart rate increased significantly with both temperature and adrenaline concentration. The Q10 values for heart rate ranged from 1.28 at 30 nmol l-1 adrenaline to 1.36 at 200 nmol l-1 adrenaline. In contrast to heart rate, maximum stroke volume declined by approximately 20 % (from 1.0 to 0.8 ml kg-1) as temperature increased from 15 to 22 °C. This decrease was not alleviated by maximally stimulating the heart with 200 nmol l-1 adrenaline. Because of the equal and opposite effects of increasing temperature on heart rate and stroke volume, maximum cardiac output did not increase between 15 and 22 °C. Maximum power output decreased (by approximately 10–15 %) at all adrenaline concentrations as temperature increased. This reduction reflected a poorer pressure-generating ability at temperatures above 15 °C. These results, in combination with earlier work, suggest (1) that peak cardiac performance occurs around the trout’s preferred temperature and well below its upper lethal limit; (2) that the diminished cardiac function concomitant with acclimation to high temperatures was associated with inotropic failure; (3) that Q10 values for cardiac rate functions, other than heart rate per se, have a limited predictive value at temperatures above the trout’s preferred temperature; and (4) that heart rate is a poor indicator of cardiac function at temperatures above 15 °C.

The zebrafish is widely used for human related disease studies. Surprisingly, there is no information about the electrical activity of single myocytes freshly isolated from adult zebrafish ventricle. In this study, we present an enzymatic method to isolate ventricular myocytes from zebrafish heart that yield a large number of calcium tolerant cells. Ventricular myocytes from zebrafish were imaged using light and confocal microscopy. Myocytes were mostly rod shaped and responded by vigorous contraction to field electrical stimulation. Whole cell configuration of the patch clamp technique was used to record electrophysiological characteristics of myocytes. Action potentials present a long duration and a plateau phase and action potential duration decreases when increasing stimulation frequency (as observed in larger mammals). Together these results indicate that zebrafish is a species ideally suited for investigation of ion channels related mutation screening of cardiac alteration important in human.

Rainbow trout, Oncorhynchus mykiss, inhabit eurythermal environments and must therefore be able to cope with changes in environmental temperature. As ectotherms, their heart is required to maintain cardiac function over a range of ambient water temperatures. This raises important questions concerning the temperature-dependence of cardiac ion channel function in fish hearts, in particular, the channels involved in Ca(2+) transport. Thus, we studied the effects of acute, physiologically relevant temperature changes on the density and kinetics of the L-type Ca(2+) channel current (I(Ca)) in rainbow trout atrial myocytes using the whole-cell patch-clamp technique. Myocytes from fish acclimated to 14 degrees C were first tested at 14 degrees C, then at 21 degrees C and finally at 7 degrees C. Using a square-pulse voltage-clamp in the first series of experiments, the peak density of I(Ca) increased (Q(10)=1.9) as temperature was increased from 14 to 21 degrees C and decreased (Q(10)=2.1) as temperature was decreased from 14 to 7 degrees C. In contrast to current density, the charge carried by I(Ca) was inversely related to temperature as a result of changes in the kinetic properties of the channel; both the fast (tau(f)) and slow (tau(s)) components of inactivation were slower at 7 degrees C than at 14 and 21 degrees C. Action potentials were recorded at the three test temperatures and then used as voltage-clamp stimulus waveforms to reassess I(Ca) in a second series of experiments. While the temperature-dependency of I(Ca) was similar to that found with the square-pulse voltage-clamp, the charge carried by I(Ca) was temperature-independent. These results show that the temperature-dependency of I(Ca) in rainbow trout is in the lower range of that reported in mammals and, although this could have profound effects on Ca(2+) delivery to the myofilaments, the temperature-induced modifications in the action potential may help to maintain a fairly constant Ca(2+) delivery during an acute temperature change in rainbow trout.