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For some species of ectothermic vertebrates, early exposure to hypoxia during embryonic development improves hypoxia-tolerance later in life. However, the cellular mechanisms underlying this phenomenon are largely unknown. Given that hypoxic survival is critically dependent on the maintenance of cardiac function, we tested the hypothesis that developmental hypoxia alters cardiomyocyte physiology in a manner that protects the heart from hypoxic stress. To test this hypothesis, we studied the common snapping turtle, which routinely experiences chronic developmental hypoxia and exploits hypoxic environments in adulthood. We isolated cardiomyocytes from juvenile turtles that embryonically developed in either normoxia (21% O 2 ) or hypoxia (10% O 2 ), and subjected them to simulated anoxia and reoxygenation, while simultaneously measuring intracellular Ca 2+ , pH and reactive oxygen species (ROS) production. Our results suggest developmental hypoxia improves cardiomyocyte anoxia-tolerance of juvenile turtles, which is supported by enhanced myofilament Ca 2+ -sensitivity and a superior ability to suppress ROS production. Maintenance of low ROS levels during anoxia might limit oxidative damage and a greater sensitivity to Ca 2+ could provide a mechanism to maintain contractile force. Our study suggests developmental hypoxia has long-lasting effects on turtle cardiomyocyte function, which might prime their physiology for exploiting hypoxic environments.

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.)

Embryological stages of oviparous elasmobranch during development can be difficult to identify, requiring magnification and/or fixation of an anaesthetized embryo. These restrictions are poorly suited for monitoring the development of living elasmobranchs inside their egg cases. There are two major aims of this study. The first was to observe elasmobranch embryonic development non-invasively and produce a non-invasive developmental key for identifying the life stages for an elasmobranch inside the egg case. To this end, 7 key developmental stages were identified for the greater spotted catshark, Scyliorhinus stellaris, and are provided here with diagrams from multiple perspectives to demonstrate the key features of each stage. The physiological and ecological relevance of each stage are discussed in terms of structure and function for embryonic survival in the harsh intertidal zone. Also discussed is the importance of the egg case membrane and the protective embryonic jelly. The second aim of the study was to understand the applicability of the 7 developmental stages from S. stellaris to other oviparous elasmobranchs. Thus, changes in embryonic body size and egg yolk volume at each stage were measured and compared with those of the closely related, lesser spotted catshark, Scyliorhinus canicula. We find nearly identical growth patterns and yolk consumption patterns in both species across the 7 developmental stages. Thus, although the 7 developmental stages have been constructed in reference to the greater spotted catshark, we suggest that it can be applied to other oviparous elasmobranch species with only minor modification., Competing Interests: The authors have declared that no competing interests exist.

Numerous pathologies lead to remodelling of the mammalian ventricle, often associated with fibrosis. Recent work in fish has shown that fibrotic remodelling of the ventricle is 'reversible', changing seasonally as temperature-induced changes in blood viscosity alter haemodynamic load on the heart. The atrial response to varying haemodynamic load is less understood in mammals and completely unexplored in non-mammalian vertebrates. To investigate atrial remodelling, rainbow trout were chronically cooled (from 10 ± 1 to 5 ± 1 °C) and chronically warmed (from 10 ± 1 to 18 ± 1 °C) for a minimum of 8 weeks. We assessed the functional effects on compliance using ex vivo heart preparations and atomic force microscopy nano-indentation and found chronic cold increased passive stiffness of the whole atrium and micromechanical stiffness of tissue sections. We then performed histological, biochemical and molecular assays to probe the mechanisms underlying functional remodelling of the atrial tissue. We found cooling resulted in collagen deposition which was associated with an upregulation of collagen-promoting genes, including the fish-specific collagen I alpha 3 chain, and a reduction in gelatinase activity of collagen-degrading matrix metalloproteinases (MMPs). Finally, we found that cooling reduced mRNA expression of cardiac growth factors and hypertrophic markers. Following long-term warming, there was an opposing response to that seen with cooling; however, these changes were more moderate. Our findings suggest that chronic cooling causes atrial dilation and increased myocardial stiffness in trout atria analogous to pathological states defined by changes in preload or afterload of the mammalian atria. The reversal of this phenotype following chronic warming is particularly interesting as it suggests that typically pathological features of mammalian atrial remodelling may oscillate seasonally in the fish, revealing a more dynamic and plastic atrial remodelling response.

Domesticated brown trout Salmo trutta parr were subjected to increased, variable flow under controlled experimental conditions. Using geometric morphometric analyses, K ¯ (a mass-length index) and caudal fin area-body length ratio, this study assessed morphological responses in lateral body depth, growth and robustness and propulsive potential, respectively, of parr over the course of 32 weeks. Geometric morphometric analyses did not reveal an effect of exercise on either lateral body depth or caudal fin area. However, improved overall robustness and growth trajectories in exercised parr showed a positive adaptive response to the enriched habitat. Exercise and habitat heterogeneity thus have the potential to improve survivability of domesticated salmonids in the wild., (© 2018 The Authors/World Fish. Journal of Fish Biology published by John Wiley & Sons Ltd on behalf of The Fisheries Society of the British Isles.)

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.)

Background: Ventricular arrhythmia is a leading cause of cardiac mortality. Most antiarrhythmics present paradoxical proarrhythmic side effects, culminating in a greater risk of sudden death., Methods: We describe a new regulatory mechanism linking mitogen-activated kinase kinase-7 deficiency with increased arrhythmia vulnerability in hypertrophied and failing hearts using mouse models harboring mitogen-activated kinase kinase-7 knockout or overexpression. The human relevance of this arrhythmogenic mechanism is evaluated in human-induced pluripotent stem cell-derived cardiomyocytes. Therapeutic potentials by targeting this mechanism are explored in the mouse models and human-induced pluripotent stem cell-derived cardiomyocytes., Results: Mechanistically, hypertrophic stress dampens expression and phosphorylation of mitogen-activated kinase kinase-7. Such mitogen-activated kinase kinase-7 deficiency leaves histone deacetylase-2 unphosphorylated and filamin-A accumulated in the nucleus to form a complex with Krüppel-like factor-4. This complex leads to Krüppel-like factor-4 disassociation from the promoter regions of multiple key potassium channel genes (Kv4.2, KChIP2, Kv1.5, ERG1, and Kir6.2) and reduction of their transcript levels. Consequent repolarization delays result in ventricular arrhythmias. Therapeutically, targeting the repressive function of the Krüppel-like factor-4/histone deacetylase-2/filamin-A complex with the histone deacetylase-2 inhibitor valproic acid restores K + channel expression and alleviates ventricular arrhythmias in pathologically remodeled hearts., Conclusions: Our findings unveil this new gene regulatory avenue as a new antiarrhythmic target where repurposing of the antiepileptic drug valproic acid as an antiarrhythmic is supported., (© 2016 American Heart Association, Inc.)

The Deepwater Horizon disaster drew global attention to the toxicity of crude oil and the potential for adverse health effects amongst marine life and spill responders in the northern Gulf of Mexico. The blowout released complex mixtures of polycyclic aromatic hydrocarbons (PAHs) into critical pelagic spawning habitats for tunas, billfishes, and other ecologically important top predators. Crude oil disrupts cardiac function and has been associated with heart malformations in developing fish. However, the precise identity of cardiotoxic PAHs, and the mechanisms underlying contractile dysfunction are not known. Here we show that phenanthrene, a PAH with a benzene 3-ring structure, is the key moiety disrupting the physiology of heart muscle cells. Phenanthrene is a ubiquitous pollutant in water and air, and the cellular targets for this compound are highly conserved across vertebrates. Our findings therefore suggest that phenanthrene may be a major worldwide cause of vertebrate cardiac dysfunction., Competing Interests: The authors declare no competing financial interests.

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.)

The effect of hypoxia on cellular metabolism is well documented in adult vertebrates, but information is entirely lacking for embryonic organisms. The effect of hypoxia on embryonic physiology is particularly interesting, as metabolic responses during development may have life-long consequences, due to developmental plasticity. To this end, we investigated the effects of chronic developmental hypoxia on cardiac mitochondrial function in embryonic and juvenile American alligators (Alligator mississippiensis). Alligator eggs were incubated in 21% or 10% oxygen from 20 to 90% of embryonic development. Embryos were either harvested at 90% development or allowed to hatch and then reared in 21% oxygen for 3 yr. Ventricular mitochondria were isolated from embryonic/juvenile alligator hearts. Mitochondrial respiration and enzymatic activities of electron transport chain complexes were measured with a microrespirometer and spectrophotometer, respectively. Developmental hypoxia induced growth restriction and increased relative heart mass, and this phenotype persisted into juvenile life. Embryonic mitochondrial function was not affected by developmental hypoxia, but at the juvenile life stage, animals from hypoxic incubations had lower levels of Leak respiration and higher respiratory control ratios, which is indicative of enhanced mitochondrial efficiency. Our results suggest developmental hypoxia can have life-long consequences for alligator morphology and metabolic function. Further investigations are necessary to reveal the adaptive significance of the enhanced mitochondrial efficiency in the hypoxic phenotype., (Copyright © 2016 the American Physiological Society.)

The state of the art of research on the environmental physiology of marine fishes is reviewed from the perspective of how it can contribute to conservation of biodiversity and fishery resources. A major constraint to application of physiological knowledge for conservation of marine fishes is the limited knowledge base; international collaboration is needed to study the environmental physiology of a wider range of species. Multifactorial field and laboratory studies on biomarkers hold promise to relate ecophysiology directly to habitat quality and population status. The 'Fry paradigm' could have broad applications for conservation physiology research if it provides a universal mechanism to link physiological function with ecological performance and population dynamics of fishes, through effects of abiotic conditions on aerobic metabolic scope. The available data indicate, however, that the paradigm is not universal, so further research is required on a wide diversity of species. Fish physiologists should interact closely with researchers developing ecological models, in order to investigate how integrating physiological information improves confidence in projecting effects of global change; for example, with mechanistic models that define habitat suitability based upon potential for aerobic scope or outputs of a dynamic energy budget. One major challenge to upscaling from physiology of individuals to the level of species and communities is incorporating intraspecific variation, which could be a crucial component of species' resilience to global change. Understanding what fishes do in the wild is also a challenge, but techniques of biotelemetry and biologging are providing novel information towards effective conservation. Overall, fish physiologists must strive to render research outputs more applicable to management and decision-making. There are various potential avenues for information flow, in the shorter term directly through biomarker studies and in the longer term by collaborating with modellers and fishery biologists.

Low temperature directly alters cardiovascular physiology in freshwater turtles, causing bradycardia, arterial hypotension, and a reduction in systemic blood pressure. At the same time, blood viscosity and systemic resistance increase, as does sensitivity to cardiac preload (e.g., via the Frank-Starling response). However, the long-term effects of these seasonal responses on the cardiovascular system are unclear. We acclimated red-eared slider turtles to a control temperature (25°C) or to chronic cold (5°C). To differentiate the direct effects of temperature from a cold-induced remodeling response, all measurements were conducted at the control temperature (25°C). In anesthetized turtles, cold acclimation reduced systemic resistance by 1.8-fold and increased systemic blood flow by 1.4-fold, resulting in a 2.3-fold higher right to left (R-L; net systemic) cardiac shunt flow and a 1.8-fold greater shunt fraction. Following a volume load by bolus injection of saline (calculated to increase stroke volume by 5-fold, ∼2.2% of total blood volume), systemic resistance was reduced while pulmonary blood flow and systemic pressure increased. An increased systemic blood flow meant the R-L cardiac shunt was further pronounced. In the isolated ventricle, passive stiffness was increased following cold acclimation with 4.2-fold greater collagen deposition in the myocardium. Histological sections of the major outflow arteries revealed a 1.4-fold higher elastin content in cold-acclimated animals. These results suggest that cold acclimation alters cardiac shunting patterns with an increased R-L shunt flow, achieved through reducing systemic resistance and increasing systemic blood flow. Furthermore, our data suggests that cold-induced cardiac remodeling may reduce the stress of high cardiac preload by increasing compliance of the vasculature and decreasing compliance of the ventricle. Together, these responses could compensate for reduced systolic function at low temperatures in the slider turtle., (Copyright © 2016 the American Physiological Society.)

Climate change and urbanisation of watercourses affect water temperatures and current flow velocities in river systems on a global scale. This represents a particularly critical issue for migratory fish species with complex life histories that use rivers to reproduce. Salmonids are migratory keystone species that provide substantial economical value to ecosystems and human societies. Consequently, a comprehensive understanding of the effects of environmental stressors on their reproductive success is critical in order to ensure their continued abundance during future climatic change. Salmonids are capital breeders, relying entirely on endogenous energy stores to fuel return migration to their natal spawning sites and reproduction upon arrival. Metabolic rates and cost of transport en-route increase with temperature and at extreme temperatures, swimming is increasingly fuelled anaerobically, resulting in an oxygen debt and reduced capacity to recover from exhaustive exercise. Thermally challenged salmonids also produce less viable gametes, which themselves are affected by water temperature after release. Passage through hydrological barriers and temperature changes both affect energy expenditure. As a result, important energetic tradeoffs emerge between extra energy used during migration and that available for other facets of the reproductive cycle, such as reproductive competition and gamete production. However, studies identifying these tradeoffs are extremely sparse. This review focuses on the specific locomotor responses of salmonids to thermal and hydrological challenges, identifying gaps in our knowledge and highlighting the potential implications for key aspects of their reproduction., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Chronic pressure or volume overload can cause the vertebrate heart to remodel. The hearts of fish remodel in response to seasonal temperature change. Here we focus on the passive properties of the fish heart. Building upon our previous work on thermal-remodeling of the rainbow trout ventricle, we hypothesized that chronic cooling would initiate fibrotic cardiac remodeling, with increased myocardial stiffness, similar to that seen with pathological hypertrophy in mammals. We hypothesized that, in contrast to pathological hypertrophy in mammals, the remodeling response in fish would be plastic and the opposite response would occur following chronic warming. Rainbow trout held at 10°C (control group) were chronically (>8 weeks) exposed to cooling (5°C) or warming (18°C). Chronic cold induced hypertrophy in the highly trabeculated inner layer of the fish heart, with a 41% increase in myocyte bundle cross-sectional area, and an up-regulation of hypertrophic marker genes. Cold acclimation also increased collagen deposition by 1.7-fold and caused an up-regulation of collagen promoting genes. In contrast, chronic warming reduced myocyte bundle cross-sectional area, expression of hypertrophic markers and collagen deposition. Functionally, the cold-induced fibrosis and hypertrophy were associated with increased passive stiffness of the whole ventricle and with increased micromechanical stiffness of tissue sections. The opposite occurred with chronic warming. These findings suggest chronic cooling in the trout heart invokes a hypertrophic phenotype with increased cardiac stiffness and fibrosis that are associated with pathological hypertrophy in the mammalian heart. The loss of collagen and increased compliance following warming is particularly interesting as it suggests fibrosis may oscillate seasonally in the fish heart, revealing a more dynamic nature than the fibrosis associated with dysfunction in mammals.

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 (U CAT), 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 U CAT) 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.

Hypoxia is a pervasive problem in coastal environments and is predicted to have enduring impacts on aquatic ecosystems. Intraspecific variation in hypoxia tolerance is well documented in fish; however, the factors underlying this variation remain unknown. Here, we investigate the role of the heart in individual hypoxia tolerance of the European sea bass (Dicentrarchus labrax). We found individual whole-animal hypoxia tolerance is a stable trait in sea bass for more than 18 months (duration of study). We next examined in vitro cardiac performance and found myocardial muscle from hypoxia-tolerant individuals generated greater force, with higher rates of contraction and relaxation, than hypoxic-sensitive individuals during hypoxic exposure. Thus, whole-animal hypoxia tolerance is associated with cardiac hypoxia tolerance. As the occurrence of aquatic hypoxia is expected to increase in marine ecosystems, our experimental data suggest that cardiac performance may influence fish survival and distribution., (© 2016 The Authors.)

This study examined how acute warming of water temperature affects the mechanical efficiency of swimming and aerobic capabilities of the brown trout Salmo trutta. Swimming efficiency was assessed using the relationship between swimming kinematics and forward speed (U), which is thought to converge upon an optimum range of a dimensionless parameter, the Strouhal number (St ). Swim-tunnel intermittent stopped-flow respirometry was used to record kinematics and measure oxygen consumption (ṀO2) of S. trutta during warming and swimming challenges. Salmo trutta maintained St between 0·2 and 0·3 at any given U over a range of temperatures, irrespective of body size. The maintenance of St within the range for maximum efficiency for oscillatory propulsion was achieved through an increase in tail-beat frequency (ftail) and a decrease in tail-beat amplitude (A) as temperature increased. Maintenance of efficient steady-state swimming was fuelled by aerobic metabolism, which increased as temperature increased up to 18° C but declined above this temperature, decreasing the apparent metabolic scope. As St was maintained over the full range of temperatures whilst metabolic scope was not, the results may suggest energetic trade-offs at any given U at temperatures above thermal optima., (© 2015 The Fisheries Society of the British Isles.)

Electrophysiological properties and molecular background of the zebrafish (Danio rerio) cardiac inward rectifier current (IK1) were examined. Ventricular myocytes of zebrafish have a robust (-6.7 ± 1.2 pA pF(-1) at -120 mV) strongly rectifying and Ba(2+)-sensitive (IC50 = 3.8 μM) IK1. Transcripts of six Kir2 channels (drKir2.1a, drKir2.1b, drKir2.2a, drKir2.2b, drKir2.3, and drKir2.4) were expressed in the zebrafish heart. drKir2.4 and drKir2.2a were the dominant isoforms in both the ventricle (92.9 ± 1.5 and 6.3 ± 1.5%) and the atrium (28.9 ± 2.9 and 64.7 ± 3.0%). The remaining four channels comprised together less than 1 and 7 % of the total transcripts in ventricle and atrium, respectively. The four main gene products (drKir2.1a, drKir2.2a, drKir2.2b, drKir2.4) were cloned, sequenced, and expressed in HEK cells for electrophysiological characterization. drKir2.1a was the most weakly rectifying (passed more outward current) and drKir2.2b the most strongly rectifying (passed less outward current) channel, whilst drKir2.2a and drKir2.4 were intermediate between the two. In regard to sensitivity to Ba(2+) block, drKir2.4 was the most sensitive (IC50 = 1.8 μM) and drKir2.1a the least sensitive channel (IC50 = 132 μM). These findings indicate that the Kir2 isoform composition of the zebrafish heart markedly differs from that of mammalian hearts. Furthermore orthologous Kir2 channels (Kir2.1 and Kir2.4) of zebrafish and mammals show striking differences in Ba(2+)-sensitivity. Structural and functional differences needs to be taken into account when zebrafish is used as a model for human cardiac electrophysiology, cardiac diseases, and in screening cardioactive substances.

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.)

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 Ca(2+) 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., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

Cardiomyocyte contraction depends on rapid changes in intracellular Ca(2+). In mammals, Ca(2+) influx as L-type Ca(2+) current (ICa) triggers the release of Ca(2+) from sarcoplasmic reticulum (SR) and Ca(2+)-induced Ca(2+) release (CICR) is critical for excitation-contraction coupling. In fish, the relative contribution of external and internal Ca(2+) is unclear. Here, we characterized the role of ICa to trigger SR Ca(2+) release in rainbow trout ventricular myocytes using ICa regulation by Ca(2+) as an index of CICR. ICa was recorded with a slow (EGTA) or fast (BAPTA) Ca(2+) chelator in control and isoproterenol conditions. In the absence of β-adrenergic stimulation, the rate of ICa inactivation was not significantly different in EGTA and BAPTA (27.1 ± 1.8 vs. 30.3 ± 2.4 ms), whereas with isoproterenol (1 μM), inactivation was significantly faster with EGTA (11.6 ± 1.7 vs. 27.3 ± 1.6 ms). When barium was the charge carrier, inactivation was significantly slower in both conditions (61.9 ± 6.1 vs. 68.0 ± 8.7 ms, control, isoproterenol). Quantification revealed that without isoproterenol, only 39% of ICa inactivation was due to Ca(2+), while with isoproterenol, inactivation was Ca(2+)-dependent (∼65%) and highly reliant on SR Ca(2+) (∼46%). Thus, SR Ca(2+) is not released in basal conditions, and ICa is the main trigger of contraction, whereas during a stress response, SR Ca(2+) is an important source of cytosolic Ca(2+). This was not attributed to differences in SR Ca(2+) load because caffeine-induced transients were not different in both conditions. Therefore, Ca(2+) stored in SR of trout cardiomyocytes may act as a safety mechanism, allowing greater contraction when higher contractility is required, such as stress or exercise., (Copyright © 2014 the American Physiological Society.)

The sarcoplasmic reticulum (SR) is crucial for contraction and relaxation of the mammalian cardiomyocyte, but its role in other vertebrate classes is equivocal. Recent evidence suggests differences in SR function across species may have an underlying structural basis. Here, we discuss how SR recruitment relates to the structural organization of the cardiomyocyte to provide new insight into the evolution of cardiac design and function in vertebrates., (©2014 Int. Union Physiol. Sci./Am. Physiol. Soc.)

Sturgeon (family Acipenseridae) are regarded as living fossils due to their ancient origin and exceptionally slow evolution. To extend our knowledge of fish cardiac excitability to a Chondrostei fish, we examined electrophysiological phenotype of the Siberian sturgeon (Acipenser baerii) heart with recordings of epicardial ECG, intracellular action potentials (APs), and sarcolemmal ion currents. Epicardial ECG of A. baerii had the typical waveform of the vertebrate ECG with Q-T interval (average duration of ventricular AP) of 650±30 ms and an intrinsic heart rate of 45.5±5 beats min(-1) at 20°C. Similar to other fish species, atrial AP was shorter in duration (402±33 ms) than ventricular AP (585±40) (P<0.05) at 20°C. Densities of atrial and ventricular Na+ currents were similar (-47.6±4.5 and -53.2±5.1 pA/pF, respectively) and close to the typical values of teleost hearts. Two major K+ currents, the inward rectifier K+ current (IK1), and the delayed rectifier K+ current (IKr) were found under basal conditions in sturgeon cardiomyocytes. The atrial IKr (3.3±0.2 pA/pF) was about twice as large as the ventricular IKr (1.3±0.4 pA/pF) (P<0.05) conforming to the typical pattern of teleost cardiac IKr. Divergent from other fishes, the ventricular IK1 was remarkably small (-2.5±0.07 pA/pF) and not different from that of the atrial myocytes (-1.9±0.06 pA/pF) (P>0.05). Two ligand-gated K+ currents were also found: ACh-activated inward rectifier (IKACh) was present only in atrial cells, while ATP-sensitive K+ current (IKATP) was activated by a mitochondrial blocker, CCCP, in both atrial and ventricular cells. The most striking difference to other fishes appeared in Ca2+ currents (ICa). In atrial myocytes, ICa was predominated by nickel-sensitive and nifedipine-resistant T-type ICa, while ventricular myocytes had mainly nifedipine-sensitive and nickel-resistant L-type ICa. ICaT/ICaL ratio of the sturgeon atrial myocytes (2.42) is the highest value ever measured for a vertebrate species. In ventricular myocytes, ICaT/ICaL ratio was 0.09. With the exception of the large atrial ICaT and small ventricular IK1, electrical excitability of A. baerii heart is similar to that of teleost hearts., (Copyright © 2014 the American Physiological Society.)

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.

Monophasic action potentials (MAPs) were recorded from the spongy and compact layers of the yellowfin tuna Thunnus albacares ventricle as stimulation frequency was increased. MAP duration decreased with increase in stimulation frequency in both the spongy and compact myocardial layers, but no significant difference in MAP duration was observed between the layers., (© 2011 The Authors. Journal of Fish Biology © 2011 The Fisheries Society of the British Isles.)

Bluefin tuna have a unique physiology. Elevated metabolic rates coupled with heat exchangers enable bluefin tunas to conserve heat in their locomotory muscle, viscera, eyes and brain, yet their hearts operate at ambient water temperature. This arrangement of a warm fish with a cold heart is unique among vertebrates and can result in a reduction in cardiac function in the cold despite the elevated metabolic demands of endothermic tissues. In this study, we used laser scanning confocal microscopy and electron microscopy to investigate how acute and chronic temperature change affects tuna cardiac function. We examined the temporal and spatial properties of the intracellular Ca2+ transient (Δ[Ca2+]i) in Pacific bluefin tuna (Thunnus orientalis) ventricular myocytes at the acclimation temperatures of 14°C and 24°C and at a common test temperature of 19°C. Acute (less than 5 min) warming and cooling accelerated and slowed the kinetics of Δ[Ca2+]i, indicating that temperature change limits cardiac myocyte performance. Importantly, we show that thermal acclimation offered partial compensation for these direct effects of temperature. Prolonged cold exposure (more than four weeks) increased the amplitude and kinetics of Δ[Ca2+]i by increasing intracellular Ca2+ cycling through the sarcoplasmic reticulum (SR). These functional findings are supported by electron microscopy, which revealed a greater volume fraction of ventricular SR in cold-acclimated tuna myocytes. The results indicate that SR function is crucial to the performance of the bluefin tuna heart in the cold. We suggest that SR Ca2+ cycling is the malleable unit of cellular Ca2+ flux, offering a mechanism for thermal plasticity in fish hearts. These findings have implications beyond endothermic fish and may help to delineate the key steps required to protect vertebrate cardiac function in the cold.

Rainbow trout remain active in waters that seasonally change between 4°C and 20°C. To explore how these fish are able to maintain cardiac function over this temperature range we characterized changes in cardiac morphology, contractile function, and the expression of contractile proteins in trout following acclimation to 4°C (cold), 12°C (control), and 17°C (warm). The relative ventricular mass (RVM) of the cold acclimated male fish was significantly greater than that of males in the control group. In addition, the compact myocardium of the cold acclimated male hearts was thinner compared to controls while the amount of spongy myocardium was found to have increased. Cold acclimation also caused an increase in connective tissue content, as well as muscle bundle area in the spongy myocardium of the male fish. Conversely, warm acclimation of male fish caused an increase in the thickness of the compact myocardium and a decrease in the amount of spongy myocardium. There was also a decrease in connective tissue content in both myocardial layers. In contrast, there was no change in the RVM or connective tissue content in the hearts of female trout with warm or cold acclimation. Cold acclimation also caused a 50% increase in the maximal rate of cardiac AM Mg(2+)-ATPase but did not influence the Ca(2+) sensitivity of this enzyme. To identify a mechanism for this change we utilized two-dimensional difference gel electrophoresis to characterize changes in the cardiac contractile proteins. Cold acclimation caused subtle changes in the phosphorylation state of the slow skeletal isoform of troponin T found in the heart, as well as of myosin binding protein C. These results demonstrate that acclimation of trout to warm and cold temperatures has opposing effects on cardiac morphology and tissue composition and that this results in distinct warm and cold cardiac phenotypes.

Background: Mechanoelectric feedback (MEF) describes the modulation of electrical activity by mechanical activity. This may occur via the activation of mechanosensitive ion channels (MSCs). MEF has not previously been investigated in fish ventricular tissue even though fish can greatly increase ventricular end diastolic volume during exercise which should therefore provide a powerful mechanical stimulus for MEF., Methodology/principal Finding: When the ventricles of extrinsically paced, isolated working trout hearts were dilated by increasing afterload, monophasic action potential (MAP) duration was significantly shortened at 25% repolarisation, unaltered at 50% repolarisation and significantly lengthened at 90% repolarisation. This observation is consistent with the activation of cationic non-selective MSCs (MSC(NS)s). We then cloned the trout ortholog of TRPC1, a candidate MSC(NS) and confirmed its presence in the trout heart., Conclusions/significance: Our results have validated the use of MAP technology for the fish heart and suggest that, in common with amphibians and mammals, MEF operates in fish ventricular myocardium, possibly via the activation of mechanosensitive TRPC1 ion channels.

Fish myocytes continue to develop active tension when stretched to sarcomere lengths (SLs) on the descending limb of the mammalian length-tension relationship. A greater length-dependent activation in fish than mammals could account for this because the increase in Ca(2+) sensitivity may overcome the tendency for force to fall due to reduced cross-bridge availability at SLs above optimal myofilament overlap. We stretched skinned fish and rat ventricular myocytes over a wide range of SLs, including those on the descending limb of the mammalian length-tension relationship. We found that fish myocytes developed greater active tension than rat myocytes at physiological Ca(2+) concentrations at long SLs as a result of a higher Ca(2+) sensitivity and a steeper relationship between Ca(2+) sensitivity and SL. We also investigated the diastolic properties of fish and rat myocytes at long SLs by measuring titin-based passive tension, titin isoform expression and titin phosphorylation. Fish myocytes produced higher titin-based passive tension despite expressing a higher proportion of a long N2BA-like isoform (38.0+/-2% of total vs 0% in rat). However, titin phosphorylation in fish myocytes was lower than in rat, which may explain some of the difference in passive tension between species. The high level of titin-based passive tension and the differential phosphorylation of sarcomeric proteins in fish myocytes may contribute to the enhanced length-dependent activation and underlie the extended range of in vivo stroke volumes found in fish compared with mammals., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

To investigate the cellular mechanisms underlying the negative force-frequency relationship (FFR) in the ventricle of the varanid lizard, Varanus exanthematicus, we measured sarcomere and cell shortening, intracellular Ca(2+) ([Ca(2+)](i)), action potentials (APs), and K(+) currents in isolated ventricular myocytes. Experiments were conducted between 0.2 and 1.0 Hz, which spans the physiological range of in vivo heart rates at 20-22 degrees C for this species. As stimulation frequency increased, diastolic length, percent change in sarcomere length, and relaxation time all decreased significantly. Shortening velocity was unaffected. These changes corresponded to a faster rate of rise of [Ca(2+)](i), a decrease in [Ca(2+)](i) transient amplitude, and a seven-fold increase in diastolic [Ca(2+)](i). The time constant for the decay of the Ca(2+) transient (tau) decreased at higher frequencies, indicating a frequency-dependent acceleration of relaxation (FDAR) but then reached a plateau at moderate frequencies and did not change above 0.5 Hz. The rate of rise of the AP was unaffected, but the AP duration (APD) decreased with increasing frequency. Peak depolarization tended to decrease, but it was only significant at 1.0 Hz. The decrease in APD was not due to frequency-dependent changes in the delayed inward rectifier (I(Kr)) or the transient outward (I(to)) current, as neither appeared to be present in varanid ventricular myocytes. Our results suggest that a negative FFR relationship in varanid lizard ventricle is caused by decreased amplitude of the Ca(2+) transient coupled with an increase in diastolic Ca(2+), which leads to incomplete relaxation between beats at high frequencies. This coincides with shortened APD at higher frequencies.

The armored catfish, Pterygoplichthys pardalis (formerly Liposarcus pardalis), is a freshwater, facultative air-breathing teleost that experiences seasonal hypercapnia in the water systems of South America. We studied the tolerance of the P. pardalis heart to hypercapnic acidosis using an isolated ventricular muscle strip preparation. Force generation and kinetic variables were examined across a range of contraction frequencies under normocapnic and hypercapnic conditions in the absence and presence of sarcoplasmic reticulum (SR) inhibitors. Pterygoplichthys pardalis ventricle exhibited robust contractile force, on par with athletic fish species such as trout and tuna and a relatively flat force-frequency relationship between 0.2 and 1.5 Hz under normocapnic conditions (1% CO2, pH 7.78 +/- 0.02). Hypercapnic acidosis (7.5% CO2, pH 7.78 +/- 0.03) did not alter the shape of the force-frequency response but reduced force by approximately 50% across all frequencies tested, with only partial recovery upon return to normocapnic conditions. A subsequent and more severe acidotic challenge (15% CO2, pH 6.77 +/- 0.05) caused an additional 20% decrease in force. Force recovered to the level at which it had stablized after the first hypercapnic insult. SR inhibition had no steady state effect on force production at 0.2 Hz but resulted in a negative force-frequency relationship, suggesting that SR Ca2+ is recruited to a greater extent at high contraction frequencies. Surprisingly, SR-inhibited muscle was more resistant to hypercapnic acidosis (force decreased by approximately 40% across all frequencies) and displayed improved recovery upon return to normocapnic conditions. The significance of this latter finding is not clear. In aggregate, our results demonstrate robust contractile force, which extends across a range of frequencies and appears to be supported by SR Ca2+ cycling. Hypercapnic acidosis reduced contractile force but may provide preconditioning-like protection from subsequent insults.

The varanid lizard possesses one of the largest aerobic capacities among reptiles with maximum rates of oxygen consumption that are twice that of other lizards of comparable sizes at the same temperature. To support this aerobic capacity, the varanid heart possesses morphological adaptations that allow the generation of high heart rates and blood pressures. Specializations in excitation-contraction coupling may also contribute to the varanids superior cardiovascular performance. Therefore, we investigated the electrophysiological properties of the l-type Ca(2+) channel and the Na(+)/Ca(2+) exchanger (NCX) and the contribution of the sarcoplasmic reticulum to the intracellular Ca(2+) transient (Delta[Ca(2+)](i)) in varanid lizard ventricular myocytes. Additionally, we used confocal microscopy to visualize myocytes and make morphological measurements. Lizard ventricular myocytes were found to be spindle-shaped, lack T-tubules, and were approximately 190 microm in length and 5-7 microm in width and depth. Cardiomyocytes had a small cell volume ( approximately 2 pL), leading to a large surface area-to-volume ratio (18.5), typical of ectothermic vertebrates. The voltage sensitivity of the l-type Ca(2+) channel current (I(Ca)), steady-state activation and inactivation curves, and the time taken for recovery from inactivation were also similar to those measured in other reptiles and teleosts. However, transsarcolemmal Ca(2+) influx via reverse mode Na(+)/Ca(2+) exchange current was fourfold higher than most other ectotherms. Moreover, pharmacological inhibition of the sarcoplasmic reticulum led to a 40% reduction in the Delta[Ca(2+)](i) amplitude, and slowed the time course of decay. In aggregate, our results suggest varanids have an enhanced capacity to transport Ca(2+) through the Na(+)/Ca(2+) exchanger, and sarcoplasmic reticulum suggesting specializations in excitation-contraction coupling may provide a means to support high cardiovascular performance.

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.

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.

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.

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.

Intracellular Na(+)-concentration, [Na(+)](i) modulates excitation-contraction coupling of cardiac myocytes via the Na(+)/Ca(2+) exchanger (NCX). In cardiomyocytes from rainbow trout (Oncorhyncus mykiss), whole cell patch-clamp studies have shown that Ca(2+) influx via reverse-mode NCX contributes significantly to contraction when [Na(+)](i) is 16 mM but not 10 mM. However, physiological [Na(+)](i) has never been measured. We recorded [Na(+)](i) using the fluorescent indicator sodium-binding benzofuran isophthalate in freshly isolated atrial and ventricular myocytes from rainbow trout. We examined [Na(+)](i) at rest and during increases in contraction frequency across three temperatures that span those trout experience in nature (7, 14, and 21 degrees C). Surprisingly, we found that [Na(+)](i) was not different between atrial and ventricular cells. Furthermore, acute temperature changes did not affect [Na(+)](i) in resting cells. Thus, we report a resting in vivo [Na(+)](i) of 13.4 mM for rainbow trout cardiomyocytes. [Na(+)](i) increased 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(+)](i) in rainbow trout cardiomyocytes. This surprisingly high [Na(+)](i) is likely to result in physiologically significant Ca(2+) influx via reverse-mode NCX during excitation-contraction coupling. We calculate that this Ca(2+)-source will decrease with the action potential duration as temperature and contraction frequency increases.

The relative contribution of the sarcoplasmic reticulum (SR), the L-type Ca(2+) channel and the Na(+)/Ca(2+) exchanger (NCX) were assessed in turtle ventricular myocytes using epifluorescent microscopy and electrophysiology. Confocal microscopy images of turtle myocytes revealed spindle-shaped cells, which lacked T-tubules and had a large surface area-to-volume ratio. Myocytes loaded with the fluorescent Ca(2+)-sensitive dye Fura-2 elicited Ca(2+) transients, which were insensitive to ryanodine and thapsigargin, indicating the SR plays a small role in the regulation of contraction and relaxation in the turtle ventricle. Sarcolemmal Ca(2+) currents were measured using the perforated-patch voltage-clamp technique. Depolarizing voltage steps to 0 mV elicited an inward current that could be blocked by nifedipine, indicating the presence of Ca(2+) currents originating from L-type Ca(2+) channels (I(Ca)). The density of I(Ca) was 3.2 +/- 0.5 pA/pF, which led to an overall total Ca(2+) influx of 64.1 +/- 9.3 microM/l. NCX activity was measured as the Ni(+)-sensitive current at two concentrations of intracellular Na(+) (7 and 14 mM). Total Ca(2+) influx through the NCX during depolarizing voltage steps to 0 mV was 58.5 +/- 7.7 micromol/l and 26.7 +/- 3.2 micromol/l at 14 and 7 mM intracellular Na(+), respectively. In the absence of the SR and L-type Ca(2+) channels, the NCX is able to support myocyte contraction independently. Our results indicate turtle ventricular myocytes are primed for sarcolemmal Ca(2+) transport, and most of the Ca(2+) used for contraction originates from the L-type Ca(2+) channel.

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 approximately 1.8 mum 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.

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.

During vertebrate evolution there has been a shift in the way in which the heart varies cardiac output (the product of heart rate and stroke volume). While mammals, birds, and amphibians increase cardiac output through large increases in heart rate and only modest increases (approximately 30%) in stroke volume, fish and some reptiles use modest increases in heart rate and very large increases in stroke volume (up to 300%). The cellular mechanisms underlying these fundamentally different approaches to cardiac output modulation are unknown. We hypothesized that the divergence between volume modulation and frequency modulation lies in the response of different vertebrate myocardium to stretch. We tested this by progressively stretching individual cardiac myocytes from the fish heart while measuring sarcomere length (SL), developed tension, and intracellular Ca2+ ([Ca2+]i) transients. We show that in fish cardiac myocytes, active tension increases at SLs greater than those previously demonstrated for intact mammalian myocytes, representing a twofold increase in the functional ascending limb of the length-tension relationship. The mechanism of action is a length-dependent increase in myofilament Ca2+ sensitivity, rather than changes in the [Ca2+]i transient or actin filament length in the fish cell. The capacity for greater sarcomere extension in fish myocardium may be linked to the low resting tension that is developed during stretch. These adaptations allow the fish heart to volume modulate and thus underpin the fundamental difference between the way fish and higher vertebrates vary cardiac output.

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.

Confocal microscopy was used to investigate the temporal and spatial properties of Ca(2+) transients and Ca(2+) sparks in ventricular myocytes of the rainbow trout (Oncorhynchus mykiss). Confocal imaging confirmed the absence of T tubules and the long ( approximately 160 microm), thin ( approximately 8 microm) morphology of trout myocytes. Line scan imaging of Ca(2+) transients evoked by electrical stimulation in cells loaded with fluo 4 revealed spatial inhomogeneities in the temporal properties of Ca(2+) transients across the width of the myocytes. The Ca(2+) 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 Ca(2+) 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 Ca(2+) from the cell periphery to the cell center, with SR Ca(2+) release contributing to the cytosolic Ca(2+) rise in a time-dependent manner. Spontaneous Ca(2+) 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 Ca(2+) sparks were observed in 9 of 11 myocytes examined. Ca(2+) 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 Ca(2+) (6 mM). Reducing temperature to 15 degrees C in intact myocytes or permeabilizing myocytes to adjust intracellular conditions to favor Ca(2+) spark detection was without significant effects. Possible reasons for the rarity of Ca(2+) sparks in a cardiac myocyte with an active SR are discussed.

Tunas are capable of exceptionally high maximum metabolic rates; such capability requires rapid delivery of oxygen and metabolic substrate to the tissues. This requirement is met, in part, by exceptionally high maximum cardiac outputs, opening the possibility that myocardial Ca(2+) delivery is enhanced in myocytes from tuna compared with those from other fish. In this study, we investigated the electrophysiological properties of the cardiac L-type Ca(2+) channel current (I(Ca)) to test the hypothesis that Ca(2+) influx would be large and have faster kinetics in cardiomyocytes from Pacific bluefin tuna (Thunnus orientalis) than in those from its sister taxon, the Pacific mackerel (Scombe japonicus). In accordance with this hypothesis, I(Ca) in atrial myocytes from bluefin tuna had significantly greater peak current amplitudes and faster fast inactivation kinetics (-4.4 +/- 0.2 pA/pF and 25.9 +/- 1.6 ms, respectively) than those from mackerel (-2.7 +/- 0.5 pA/pF and 32.3 +/- 3.8 ms, respectively). Steady-state activation, inactivation, and recovery from inactivation were also faster in atrial myocytes from tuna than from mackerel. In ventricular myocytes, current amplitude and activation and inactivation rates were similar in both species but elevated compared with those of other teleosts. These results indicate enhanced I(Ca) in atrial myocytes from bluefin tuna compared with Pacific mackerel; this enhanced I(Ca) may be associated with elevated cardiac performance, because I(Ca) delivers the majority of Ca(2+) involved in excitation-contraction coupling in most fish hearts. Similarly, I(Ca) is enhanced in the ventricle of both species compared with other teleosts and may play a role in the robust cardiac performance of fishes of the family Scombridae.

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.

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.

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.