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Cardiac phenotypic plasticity, the remodelling of heart structure and function, is a response to any sustained (or repeated) stimulus or stressor that results in a change in heart performance. Cardiac plasticity can be either adaptive (beneficial) or maladaptive (pathological), depending on the nature and intensity of the stimulus. Here, we draw on articles published in this Special Issue of Journal of Experimental Biology, and from the broader comparative physiology literature, to highlight the core components that enable cardiac plasticity, including structural remodelling, excitation-contraction coupling remodelling and metabolic rewiring. We discuss when and how these changes occur, with a focus on the underlying molecular mechanisms, from the regulation of gene transcription by epigenetic processes to post-translational modifications of cardiac proteins. Looking to the future, we anticipate that the growing use of -omics technologies in integration with traditional comparative physiology approaches will allow researchers to continue to uncover the vast scope for plasticity in cardiac function across animals., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Oxygen deprivation during embryonic development can permanently remodel the vertebrate heart, often causing cardiovascular abnormalities in adulthood. While this phenomenon is mostly damaging, recent evidence suggests developmental hypoxia produces stress-tolerant phenotypes in some ectothermic vertebrates. Embryonic common snapping turtles (Chelydra serpentina) subjected to chronic hypoxia display improved cardiac anoxia tolerance after hatching, which is associated with altered Ca2+ homeostasis in heart cells (cardiomyocytes). Here, we examined the possibility that changes in Ca2+ cycling, through the sarcoplasmic reticulum (SR), underlie the developmentally programmed cardiac phenotype of snapping turtles. We investigated this hypothesis by isolating cardiomyocytes from juvenile turtles that developed in either normoxia (21% O2; 'N21') or chronic hypoxia (10% O2; 'H10') and subjected the cells to anoxia/reoxygenation, in either the presence or absence of SR Ca2+-cycling inhibitors. We simultaneously measured cellular shortening, intracellular Ca2+ concentration ([Ca2+]i), and intracellular pH (pHi). Under normoxic conditions, N21 and H10 cardiomyocytes shortened equally, but H10 Ca2+ transients (Δ[Ca2+]i) were twofold smaller than those of N21 cells, and SR inhibition only decreased N21 shortening and Δ[Ca2+]i. Anoxia subsequently depressed shortening, Δ[Ca2+]i and pHi in control N21 and H10 cardiomyocytes, yet H10 shortening and Δ[Ca2+]i recovered to pre-anoxic levels, partly due to enhanced myofilament Ca2+ sensitivity. SR blockade abolished the recovery of anoxic H10 cardiomyocytes and potentiated decreases in shortening, Δ[Ca2+]i and pHi. Our novel results provide the first evidence of developmental programming of SR function and demonstrate that developmental hypoxia confers a long-lasting, superior anoxia-tolerant cardiac phenotype in snapping turtles, by modifying SR function and enhancing myofilament Ca2+ sensitivity., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Animals at early life stages are generally more sensitive to environmental stress than adults. This is especially true of oviparous vertebrates that develop in variable environments with little or no parental care. These organisms regularly experience environmental fluctuations as part of their natural development, but climate change is increasing the frequency and intensity of these events. The developmental plasticity of oviparous vertebrates will therefore play a critical role in determining their future fitness and survival. In this Review, we discuss and compare the phenotypic consequences of chronic developmental hypoxia on the cardiovascular system of oviparous vertebrates. In particular, we focus on species-specific responses, critical windows, thresholds for responses and the interactive effects of other stressors, such as temperature and hypercapnia. Although important progress has been made, our Review identifies knowledge gaps that need to be addressed if we are to fully understand the impact of climate change on the developmental plasticity of the oviparous vertebrate cardiovascular system., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Adrenaline and noradrenaline, released as hormones and/or neurotransmitters, exert diverse physiological functions in vertebrates, and teleost fishes are widely used as model organisms to study adrenergic regulation; however, such investigations often rely on receptor subtype-specific pharmacological agents (agonists and antagonists; see Glossary) developed and validated in mammals. Meanwhile, evolutionary (phylogenetic and comparative genomic) studies have begun to unravel the diversification of adrenergic receptors (ARs) and reveal that whole-genome duplications and pseudogenization events in fishes results in notable distinctions from mammals in their genomic repertoire of ARs, while lineage-specific gene losses within teleosts have generated significant interspecific variability. In this Review, we visit the evolutionary history of ARs (including α1-, α2- and β-ARs) to highlight the prominent interspecific differences in teleosts, as well as between teleosts and other vertebrates. We also show that structural modelling of teleost ARs predicts differences in ligand binding affinity compared with mammalian orthologs. To emphasize the difficulty of studying the roles of different AR subtypes in fish, we collate examples from the literature of fish ARs behaving atypically compared with standard mammalian pharmacology. Thereafter, we focus on specific case studies of the liver, heart and red blood cells, where our understanding of AR expression has benefited from combining pharmacological approaches with molecular genetics. Finally, we briefly discuss the ongoing advances in 'omics' technologies that, alongside classical pharmacology, will provide abundant opportunities to further explore adrenergic signalling in teleosts., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Warming is predicted to have negative consequences for fishes by causing a mismatch between oxygen demand and supply, and a consequent reduction in aerobic scope (AS) and performance. This oxygen and capacity limited thermal tolerance (OCLTT) hypothesis features prominently in the literature but remains controversial. Within the OCLTT framework, we hypothesised that fish would select temperatures that maximise their AS, and thus their performance. We tested this hypothesis using intermittent flow respirometry to measure AS at, above (+2.5°C) and below (-2.5°C) the self-selected, preferred temperature (Tpref) of individual zebrafish (Danio rerio). AS was greatest 2.5°C above Tpref, which was driven by an increase in maximal metabolic rate. This mismatch between Tpref and the optimal temperature for AS suggests that factor(s) aside from AS maximisation influence the thermal preference of zebrafish., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Contraction of atrial smooth muscle in the hearts of semi-aquatic emydid turtles regulates ventricular filling, and it has been proposed that it could regulate stroke volume during characteristic rapid transitions in cardiac output associated with diving. For this hypothesis to be supported, atrial smooth muscle should be widely distributed in diving Testudines. To further understand the putative function and evolutionary significance of endocardial smooth muscle in Testudines, we studied the hearts of loggerhead sea turtles, Caretta caretta (n=7), using immunohistochemistry and histology. Surprisingly, we found no evidence of prominent atrial smooth muscle in C. caretta. However, smooth muscle was readily identified in the sinus venosus. Our results suggest that atrial smooth muscle does not contribute to the diving capabilities of C. caretta, indicating that the possible roles of smooth muscle in emydid turtle hearts require a re-evaluation. In sea turtles, the sinus venosus may instead contribute to regulate cardiac filling., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Birds occupy a unique position in the evolution of cardiac design. Their hearts are capable of cardiac performance on par with, or exceeding that of mammals, and yet the structure of their cardiomyocytes resembles those of reptiles. It has been suggested that birds use intracellular Ca 2+ stored within the sarcoplasmic reticulum (SR) to power contractile function, but neither SR Ca 2+ content nor the cross-talk between channels underlying Ca 2+ -induced Ca 2+ release (CICR) have been studied in adult birds. Here we used voltage clamp to investigate the Ca 2+ storage and refilling capacities of the SR and the degree of trans-sarcolemmal and intracellular Ca 2+ channel interplay in freshly isolated atrial and ventricular myocytes from the heart of the Japanese quail ( Coturnix japonica ). A trans-sarcolemmal Ca 2+ current ( I Ca ) was detectable in both quail atrial and ventricular myocytes, and was mediated only by L-type Ca 2+ channels. The peak density of I Ca was larger in ventricular cells than in atrial cells, and exceeded that reported for mammalian myocardium recorded under similar conditions. Steady-state SR Ca 2+ content of quail myocardium was also larger than that reported for mammals, and reached 750.6±128.2 μmol l -1 in atrial cells and 423.3±47.2 μmol l -1 in ventricular cells at 24°C. We observed SR Ca 2+ -dependent inactivation of I Ca in ventricular myocytes, indicating cross-talk between sarcolemmal Ca 2+ channels and ryanodine receptors in the SR. However, this phenomenon was not observed in atrial myocytes. Taken together, these findings help to explain the high-efficiency avian myocyte excitation-contraction coupling with regard to their reptilian-like cellular ultrastructure., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Seasonal thermal remodelling (acclimatization) and laboratory thermal remodelling (acclimation) can induce different physiological changes in ectothermic animals. As global temperatures are changing at an increasing rate, there is urgency to understand the compensatory abilities of key organs such as the heart to adjust under natural conditions. Thus, the aim of the present study was to directly compare the acclimatization and acclimatory response within a single eurythermal fish species, the European shorthorn sculpin ( Myoxocephalus scorpio ). We used current- and voltage-clamp to measure ionic current densities in both isolated atrial and ventricular myocytes from three groups of fish: (1) summer-caught fish kept at 12°C ('summer-acclimated'); (2) summer-caught fish kept at 3°C ('cold acclimated'); and (3) fish caught in March ('winter-acclimatized'). At a common test temperature of 7.5°C, action potential (AP) was shortened by both winter acclimatization and cold acclimation compared with summer acclimation; however, winter acclimatization caused a greater shortening than did cold acclimation. Shortening of AP was achieved mostly by a significant increase in repolarizing current density ( I Kr and I K1 ) following winter acclimatization, with cold acclimation having only minor effects. Compared with summer acclimation, the depolarizing L-type calcium current ( I Ca ) was larger following winter acclimatization, but again, there was no effect of cold acclimation on I Ca Interestingly, the other depolarizing current, I Na , was downregulated at low temperatures. Our further analysis shows that ionic current remodelling is primarily due to changes in ion channel density rather than current kinetics. In summary, acclimatization profoundly modified the electrical activity of the sculpin heart while acclimation to the same temperature for >1.5 months produced very limited remodelling effects., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Excitation-contraction coupling in vertebrate hearts is underpinned by calcium (Ca 2+ ) release from Ca 2+ release units (CRUs). CRUs are formed by clusters of channels called ryanodine receptors on the sarcoplasmic reticulum (SR) within the cardiomyocyte. Distances between CRUs influence the diffusion of Ca 2+ , thus influencing the rate and strength of excitation-contraction coupling. Avian myocytes lack T-tubules, so Ca 2+ from surface CRUs (peripheral couplings, PCs) must diffuse to internal CRU sites of the corbular SR (cSR) during centripetal propagation. Despite this, avian hearts achieve higher contractile rates and develop greater contractile strength than many mammalian hearts, which have T-tubules to provide simultaneous activation of the Ca 2+ signal through the myocyte. We used 3D electron tomography to test the hypothesis that the intracellular distribution of CRUs in the avian heart permits faster and stronger contractions despite the absence of T-tubules. Nearest edge-edge distances between PCs and cSR, and geometric information including surface area and volume of individual cSR, were obtained for each cardiac chamber of the white leghorn chicken. Computational modelling was then used to establish a relationship between CRU distance and cell activation time in the avian heart. Our data suggest that cSR clustered close together along the Z-line is vital for rapid propagation of the Ca 2+ signal from the cell periphery to the cell centre, which would aid in the strong and fast contractions of the avian heart., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Climate change alters the thermal habitat of aquatic species on a global scale, generating novel environmental challenges during all life stages, including reproduction. Changes in water temperature profoundly influence the performance of ectothermic aquatic organisms. This is an especially crucial issue for migratory fish, because they traverse multiple environments in order to reproduce. In externally fertilizing migratory fish, gametes are affected by water temperature indirectly, within the reproductive organ in which they are produced during migration, as well as directly, upon release into the surrounding medium at the spawning grounds. Both direct (after release) and indirect (during production) thermal impacts on gamete quality have been investigated, but never in conjunction. Here, we assessed the cumulative influence of temperature on brown trout, Salmo trutta , sperm quality during sperm production (male acclimation temperature) as well as upon release (sperm activation water temperature) on two consecutive dates during the brown trout spawning season. Early in the season, warm acclimation of males reduced their fertilization probability (lower sperm velocity) when compared with cold-acclimated males, especially when the activation water temperature was also increased beyond the thermal optimum (resulting in a lower proportion of motile sperm with lower velocity). Later in the season, sperm quality was unaffected by acclimation temperature and thermal sensitivity of sperm was reduced. These results give novel insights into the complex impacts of climate change on fish sperm, with implications for the reproduction and management of hatchery and wild trout populations in future climate scenarios., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Thermal acclimation causes the heart of some fish species to undergo significant remodelling. This includes changes in electrical activity, energy utilization and structural properties at the gross and molecular level of organization. The purpose of this Review is to summarize the current state of knowledge of temperature-induced structural remodelling in the fish ventricle across different levels of biological organization, and to examine how such changes result in the modification of the functional properties of the heart. The structural remodelling response is thought to be responsible for changes in cardiac stiffness, the Ca 2+ sensitivity of force generation and the rate of force generation by the heart. Such changes to both active and passive properties help to compensate for the loss of cardiac function caused by a decrease in physiological temperature. Hence, temperature-induced cardiac remodelling is common in fish that remain active following seasonal decreases in temperature. This Review is organized around the ventricular phases of the cardiac cycle - specifically diastolic filling, isovolumic pressure generation and ejection - so that the consequences of remodelling can be fully described. We also compare the thermal acclimation-associated modifications of the fish ventricle with those seen in the mammalian ventricle in response to cardiac pathologies and exercise. Finally, we consider how the plasticity of the fish heart may be relevant to survival in a climate change context, where seasonal temperature changes could become more extreme and variable., Competing Interests: The authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

We describe here a novel, fast and inexpensive method for producing a 3D 'heart' structure that forms spontaneously, in vitro, from larval zebrafish (ZF). We have named these 3D 'heart' structures 'zebrafish heart aggregate(s)' (ZFHAs) and have characterised their basic morphology and structural composition using histology, immunohistochemistry, electron microscopy and mass spectrometry. After 2 days in culture, the ZFHA spontaneously form and become a stable contractile syncytium consisting of cardiac tissue derived by in vitro maturation, which beats rhythmically and consistently for more than 8 days. We propose this model as a platform technology, which can be developed further to study in vitro cardiac maturation, regeneration, tissue engineering and safety pharmacological/toxicology testing., (© 2015. Published by The Company of Biologists Ltd.)

The relationship between tail (or wing) beat frequency (f(tail)), amplitude (A) and forward velocity (U) in animals using oscillatory propulsion, when moving at a constant cruising speed, converges upon an optimum range of the Strouhal number (St = f(tail) · A/U). Previous work, based on observational data and supported by theory, shows St falling within the broad optimum range (0.2

Mammalian myocardial studies reveal a biphasic increase in the force of contraction due to stretch. The first rapid response, known as the Frank-Starling response, occurs within one heartbeat of stretch. A second positive inotropic response occurs over the minutes following the initial stretch and is known as the slow force response (SFR). The SFR has been observed in mammalian isolated whole hearts, muscle preparations and individual myocytes. We present the first direct study into the SFR in the heart of a non-mammalian vertebrate, the rainbow trout (Oncorhynchus mykiss). We stretched ventricular trabecular muscle preparations from 88% to 98% of their optimal length and individual ventricular myocytes by 7% of their slack sarcomere length (SL). Stretch caused an immediate increase in force in both preparations, indicative of the Frank-Starling response. However, we found no significant effect of prolonged stretch on the force of contraction in either the ventricular trabecular preparations or the single myocytes. This indicates that rainbow trout ventricular myocardium does not exhibit a SFR and that, in contrast to mammals, the piscine Frank-Starling response may not be associated with the SFR. We speculate that this is due to the fish myocardium modulating cardiac output via changes in stroke volume to a larger extent than heart rate.

Specialisations in excitation-contraction coupling may have played an important role in the evolution of endothermy and high cardiac performance in scombrid fishes. We examined aspects of Ca(2+) handling in cardiomyocytes from Pacific bonito (Sarda chiliensis), Pacific mackerel (Scomber japonicus), yellowfin tuna (Thunnus albacares) and Pacific bluefin tuna (Thunnus orientalis). The whole-cell voltage-clamp technique was used to measure the temperature sensitivity of the L-type Ca(2+) channel current (I(Ca)), density, and steady-state and maximal sarcoplasmic reticulum (SR) Ca(2+) content (ssSR(load) and maxSR(load)). Current-voltage relations, peak I(Ca) density and charge density of I(Ca) were greatest in mackerel and yellowfin at all temperatures tested. I(Ca) density and kinetics were temperature sensitive in all species studied, and the magnitude of this response was not related to the thermal preference of the species. SR(load) was greater in atrial than in ventricular myocytes in the Pacific bluefin tuna, and in species that are more cold tolerant (bluefin tuna and mackerel). I(Ca) and SR(load) were particularly small in bonito, suggesting the Na(+)/Ca(2+) exchanger plays a more pivotal role in Ca(2+) entry into cardiomyocytes of this species. Our comparative approach reveals that the SR of cold-tolerant scombrid fishes has a greater capacity for Ca(2+) storage. This specialisation may contribute to the temperature tolerance and thermal niche expansion of the bluefin tuna and mackerel.

The 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.

The burbot (Lota lota) is a cold stenothermic fish species whose heart is adapted to function in the cold. In this study we use whole-cell voltage-clamp techniques to characterize the electrophysiological properties of burbot ventricular myocytes and to test the hypothesis that changes in membrane currents and intracellular Ca2+ cycling associated cold-acclimation in other fish species are routine for stenothermic cold-adapted species. Experiments were performed at 4 degrees C, which is the body temperature of burbot for most of the year, and after myocytes were acutely warmed to 11 degrees C, which is in the upper range of temperatures experienced by burbot in nature. Results on K+ channels support our hypothesis as the relative density of K-channel conductances in the burbot heart are similar to those found for cold-acclimated cold-active fish species. I(K1) conductance was small (39.2+/-5.4 pS pF(-1) at 4 degrees C and 71.4+/-1.7 pS pF(-1) at 11 degrees C) and I(Kr) was large (199+/-27 pS pF(-1) at 4 degrees C and 320.3+/-8 pS pF(-1) at 11 degrees C) in burbot ventricular myocytes. We found high Na+-Ca2+ exchange (NCX) activity (35.9+/-6.3 pS pF(-1) at 4 degrees C and 58.6+/-8.4 pS pF(-1) at 11 degrees C between -40 and 20 mV), suggesting that it may be the primary pathway for sarcolemmal (SL) Ca2+ influx in this species. In contrast, the density (I(Ca), 0.81+/-0.13 pA pF(-1) at 4 degrees C, and 1.35+/-0.18 pA pF(-1) at 11 degrees C) and the charge (Q(Ca), 0.24+/-0.043 pC pF(-1) at 4 degrees C and 0.21+/-0.034 pC pF(-1) at 11 degrees C) carried by the L-type Ca2+ current was small. Our results on sarcolemmal ion currents in burbot ventricular myocytes suggest that cold stenothermy and compensative cold-acclimation involve many of the same subcellular adaptations that culminate in enhanced excitability in the cold.

The 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.

To explore how the cardiac sarcoplasmic reticulum (SR) functions over a range of temperatures, we used whole-cell voltage clamp combined with rapid caffeine application to study SR Ca(2+) accumulation, release and steady-state content in atrial myocytes from rainbow trout. Myocytes were isolated from rainbow trout acclimated to 14 degrees C, and the effect of varying stimulation pulse number, frequency and experimental temperature (7 degrees C, 14 degrees C and 21 degrees C) on SR function was studied. To add physiological relevance, in addition to 200 ms square (SQ) voltage pulses, myocytes were stimulated with temperature-specific action potentials (AP) applied at relevant frequencies for each test temperature. We found that the SR accumulated Ca(2+) more rapidly and to a greater concentration (1043+/-189 micromol l(-1) Ca(2+), 1138+/-173 micromol l(-1) Ca(2+), and 1095+/-142 micromol l(-1) Ca(2+) at 7 degrees C, 14 degrees C and 21 degrees C, respectively) when stimulated with physiological AP waveforms at physiological frequencies compared with 200 ms SQ pulses at the same frequencies (664+/-180 micromol l(-1) Ca(2+), 474+/-75 micromol l(-1) Ca(2+) and 367+/-42 micromol l(-1) Ca(2+) at 7 degrees C, 14 degrees C and 21 degrees C, respectively). Also, and in contrast to 200 ms SQ pulse stimulation, temperature had little effect on steady-state SR Ca(2+) accumulation during AP stimulation. Furthermore, we observed SR-Ca(2+)-dependent inactivation of the L-type Ca(2+) channel current (I(Ca)) at 7 degrees C, 14 degrees C and 21 degrees C, providing additional evidence of maintained SR function in fish hearts over an acute range of temperatures. We conclude that the waveform of the AP may be critical in ensuring adequate SR Ca(2+) cycling during temperature change in rainbow trout in vivo.

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.