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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.

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 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 requires a re-evaluation. In sea turtles, the sinus venosus may instead contribute to regulate cardiac filling.

The Alaska blackfish (Dallia pectoralis) is the only air-breathing fish in the Arctic. In the summer, a modified esophagus allows the fish to extract oxygen from the air, but this behavior is not possible in the winter because of ice and snow cover. The lack of oxygen (hypoxia) and near freezing temperatures in winter is expected to severely compromise metabolism, and yet remarkably, overwintering Alaska blackfish remain active. To maintain energy balance in the brain and limit the accumulation of reactive oxygen species (ROS), we hypothesized that cold hypoxic conditions would trigger brain mitochondrial remodeling in the Alaska blackfish. To address this hypothesis, fish were acclimated to warm (15 °C) normoxia, cold (5 °C) normoxia or cold hypoxia (5 °C, 2.1-4.2 kPa; no air access) for 5-8 weeks. Mitochondrial respiration, ADP affinity and H

The need for change One of the many great things about being a developmental biologist is our strong sense of community. Conferences of all shapes and sizes reinforce these bonds between us and yet, even before the pandemic, scientists were becoming increasingly reluctant to travel to conferences due to concerns about climate change: ‘Don’t think I will travel to conferences as delegate again. Cutting down as speaker a lot.’Prof. Michael Stumpf (@theosysbio). Twitter; December 2019.Younger scientists in particular expressed concerns about the environmental impact of their travel, but also worried about how not travelling might affect their career: ‘I know many senior scientists who are reducing attendance, but for someone like me without that recognition, how do I get my name out there without physically attending? Would love other options! ’Dr Louise Stephen (@ciliaNcilia). Twitter; December 2019

The lipophilic polycyclic aromatic hydrocarbon (PAH) phenanthrene is relatively abundant in polluted air and water and can access and accumulate in human tissue. Phenanthrene has been reported to interact with cardiac ion channels in several fish species. This study was undertaken to investigate the ability of phenanthrene to interact with hERG (human Ether-à-go-go-Related Gene) encoded Kv11.1 K+ channels, which play a central role in human ventricular repolarization. Pharmacological inhibition of hERG can be proarrhythmic. Whole-cell patch clamp recordings of hERG current (IhERG) were made from HEK293 cells expressing wild-type (WT) and mutant hERG channels. WT IhERG1a was inhibited by phenanthrene with an IC50 of 17.6 ± 1.7 µM, whilst IhERG1a/1b exhibited an IC50 of 1.8 ± 0.3 µM. WT IhERG block showed marked voltage and time dependence, indicative of dependence of inhibition on channel gating. The inhibitory effect of phenanthrene was markedly impaired by the attenuated inactivation N588K mutation. Remarkably, mutations of S6 domain aromatic amino acids (Y652, F656) in the canonical drug binding site did not impair the inhibitory action of phenanthrene; the Y652A mutation augmented IhERG block. In contrast, the F557L (S5) and M651A (S6) mutations impaired the ability of phenanthrene to inhibit IhERG, as did the S624A mutation below the selectivity filter region. Computational docking using a cryo-EM derived hERG structure supported the mutagenesis data. Thus, phenanthrene acts as an inhibitor of the hERG K+ channel by directly interacting with the channel, binding to a distinct site in the channel pore domain.

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 Ca2+ stored within the sarcoplasmic reticulum (SR) to power contractile function, but neither SR Ca2+ content nor the cross-talk between channels underlying Ca2+-induced Ca2+ release (CICR) have been studied in adult birds. Here we used voltage clamp to investigate the Ca2+ storage and refilling capacities of the SR and the degree of trans-sarcolemmal and intracellular Ca2+ channel interplay in freshly isolated atrial and ventricular myocytes from the heart of the Japanese quail (Coturnix japonica). A trans-sarcolemmal Ca2+ current (ICa) was detectable in both quail atrial and ventricular myocytes, and was mediated only by L-type Ca2+ channels. The peak density of ICa was larger in ventricular cells than in atrial cells, and exceeded that reported for mammalian myocardium recorded under similar conditions. Steadystate SR Ca2+ content of quail myocardium was also larger than that reported for mammals, and reached 750.6±128.2 μmol lâ'1 in atrial cells and 423.3±47.2 μmol lâ'1 in ventricular cells at 24°C. We observed SR Ca2+-dependent inactivation of ICa in ventricular myocytes, indicating cross-talk between sarcolemmal Ca2+ 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. © 2020 Company of Biologists Ltd. All rights reserved. The study was supported by the Russian Foundation for Basic Research (19-34-90142 to D.V.A.).

Catecholamines mediate the 'fight or flight' response in a wide variety of vertebrates. The endogenous catecholamine adrenaline increases heart rate and contractile strength to raise cardiac output. The increase in contractile force is driven in large part by an increase in myocyte Ca

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 (IKr and IK1) following winter acclimatization, with cold acclimation having only minor effects. Compared with summer acclimation, the depolarizing L-type calcium current (ICa) was larger following winter acclimatization, but again, there was no effect of cold acclimation on ICa. Interestingly, the other depolarizing current, INa, 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. © 2019. Published by The Company of Biologists Ltd. Russian Foundation for Basic Research, RFBR: 18-315-20049 The study was supported by the Russian Foundation for Basic Research (18-315-20049 to D.V.A.).

Excitation-contraction coupling in vertebrate hearts is underpinned by calcium (Ca2+) release from Ca2+ 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 Ca2+, thus influencing the rate and strength of excitation-contraction coupling. Avian myocytes lack T-tubules, thus Ca2+ 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 Ca2+ 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 T-tubules. Nearest edge-edge distances between PCs and cSR, and geometric information including surface area and volumes of individual cSR, were obtained for each cardiac chamber of the White Leghorn chicken. Computational modelling was then used to establish a relationship between CRUs distances 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 Ca2+ signal from the cell periphery to the cell centre which would aid in the strong and fast contractions of the avian heart.

The relationship between tail (or wing) beat frequency (ftail), 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=ftail·A/U). Previous work, based on observational data and supported by theory, shows St falling within the broad optimum range (0.2

Recent and future 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 upon arrival at their 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 to cold acclimated males, especially when 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.

SUMMARY 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.

SUMMARY 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 Ca2+ 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 Ca2+ channel current (ICa), density, and steady-state and maximal sarcoplasmic reticulum (SR) Ca2+ content (ssSRload and maxSRload). Current–voltage relations, peak ICa density and charge density of ICa were greatest in mackerel and yellowfin at all temperatures tested. ICa 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. SRload 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). ICa and SRload were particularly small in bonito, suggesting the Na+/Ca2+ exchanger plays a more pivotal role in Ca2+ entry into cardiomyocytes of this species. Our comparative approach reveals that the SR of cold-tolerant scombrid fishes has a greater capacity for Ca2+ storage. This specialisation may contribute to the temperature tolerance and thermal niche expansion of the bluefin tuna and mackerel.

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.

Cardiac microtubule stability is increased in the streptozotocin (STZ) model of type 1 diabetes. Here, we investigate the reason for increased microtubule stability, and the functional consequences of stable microtubule disruption. Ventricular myocytes were isolated from rats at 8–12 weeks after injection of STZ. A 10% increase in microtubule density, but no difference in the ratio of microtubule-associated protein 4 (MAP4) to tubulin was seen in myocytes from STZ rats. Functionally, STZ myocytes showed a tendency for reduced shortening and intracellular Ca2+ ([Ca2+] i ) transient amplitude, and a significant prolongation of time to peak (ttp) shortening and [Ca2+] i . Although microtubules in STZ myocytes were less sensitive to the microtubule disruptor nocodazole (NOC; 33 μM) than control myocytes, we only saw marked functional consequences of microtubule disruption by NOC in myocytes from diabetic animals. NOC increased shortening and [Ca2+] i transient amplitude in STZ myocytes by 45 and 24%, respectively (compared with 4 and 6% in controls). Likewise, NOC decreased ttp shortening and [Ca2+] i only in STZ myocytes, such that these parameters were no longer different between the two groups. In conclusion, stable microtubules in diabetes are not associated with an increase in MAP4, but are functionally relevant to cardiac dysfunction in diabetes, regulating both [Ca2+] i and shortening.

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.

1. IntroductionThe relationship between the strength of con-traction and the interval between contractions wasfirst recognised as an important intrinsic controlsystem of the heart when Bowditch (1871 ) notedthat ‘the interval between a contraction of the heartand the proceeding beat is of such importance forthe strength of the contraction that the study of thiseffect is a prime necessity’. This relationship, oftenreferred to as the ‘force–frequency relationship’,the ‘force–interval relationship’ or the ‘staircase-effect’, is studied in multicellular and single cellpreparations to understand a fundamental propertyof the heart, namely, its ability to develop force atdifferent frequencies. Thus, with the aid of phar-macological agents and by extrapolation of theresults to in vivo contraction frequencies, theforce–frequency relationship continues to provideuseful information to cardiac physiologists.In most fish hearts, contractile force decreasesas contraction frequency increases, resulting in a

Ultrastructure, molecular composition and electrophysiological properties of cardiac myocytes and functional characteristics of the fish heart suggest that cycling of extracellular Ca 2+ is generally more important than intracellular cycling of Ca 2+ stores of the sarcoplasmic reticulum (SR) in activating contraction of fish cardiac myocytes. This is especially true for the ventricle. However, prominent species-specific differences exist in cardiac excitation-contraction coupling and in the relative roles of extracellular and intracellular Ca 2+ sources among the teleostean fish. In fact, in some fish species (tunas, burbot) the SR of atrial myocytes, under certain circumstances, may act as the major source of systolic Ca 2+ . These interspecific differences are obviously an outcome of evolutionary adaptation to different habitats and modes of activity in these habitats. There is also substantial intraspecific variation in the SR Ca 2+ -release-to-SL-Ca 2+ influx ratio depending on acute and chronic temperature changes. Consequently excitation-contraction coupling of the fish cardiac myocytes is not a fixed entity, but rather a highly variable and malleable process that enables fish to have an appropriate cardiac scope to exploit a diverse range of environments.

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.

An isometric muscle preparation was used to study the inhibitory effect of ryanodine on contractile function in isolated ventricular trabeculae of the Pacific mackerel (Scomber japonicus). Ryanodine (an inhibitor of sarcoplasmic reticulum (SR) function) caused a 20% reduction in peak tension at 20 degrees C, but not 15 degrees C, over the range of frequencies (0.2-3.0 Hz) tested. This indicates that in the absence of a functional SR, the mackerel ventricle can maintain most of its contractile strength utilizing other modes of Ca(2+) delivery to the myofilaments. Ca(2+) flux through the sarcolemmal (SL) L-type Ca(2+)-channels is most likely the predominant pathway for Ca(2+) activation of the myofilaments, although reverse mode Na(+)/Ca(2+) exchange could potentially contribute to a significant extent. High levels of adrenergic stimulation overwhelmed the negative inotropy caused by ryanodine, returning tension to pre-ryanodine levels, further suggesting that the mackerel ventricle can maintain contractile function without Ca(2+) contribution from the SR. These results are discussed within the context of what is known about SR Ca(2+) utilization in rainbow trout and tuna hearts.

This study is the first to examine the contractility of teleost ventricular muscle in an oscillating muscle preparation. The experiments were designed to test the relative importance of Ca2+ released from the sarcoplasmic reticulum (SR) and Ca2+ influx across the sarcolemma (SL) to cardiac performance in rainbow trout Oncorhynchus mykiss. Adrenaline and ryanodine were used to modulate Ca2+ flux through the SL and SR, respectively. Experiments were conducted at two temperatures (12 °C and 22 °C) (1) to investigate the effect of an acute temperature change (from 12 °C to 22 °C) on power production, and (2) to test the effects of acute temperature change on the relative contributions of the SR and SL Ca2+ flux to power production. Concordant with isometric studies, the results showed that trans-sarcolemmal influx was the major source of Ca2+ (approximately 90 %) for cardiac power production at all temperatures. This SL Ca2+ influx was increased with adrenergic stimulation. The power curves generated in this study suggest an optimum frequency for power production of approximately 1.0 Hz at 12 °C, which corresponds well to typical in vivo heart rates for rainbow trout at that temperature. Further, this study indicated that temperature-induced changes in power output cannot be predicted from temperature-induced changes in isometric tension because the temperature-sensitivity of work and power proved to be greater than that of isometric tension. This finding is important because many previous studies have assessed cardiac contractility using only isometric tension.

To investigate the effects of acute temperature change on cardiac excitation-contraction in fish heart, atrial and ventricular myocytes were isolated from rainbow trout acclimated to 12°C and studied using voltage clamp recording while simultaneously recording [Ca2+]i at 7°C, 14°C, and 21°C. Cells were stimulated in the presence or absence of sarcoplasmic reticulum (SR) inhibitors and SR Ca2+ load and intracellular Ca2+ buffering were determined with caffeine. Atrial and ventricular cells showed increased ICa density, faster ICa inactivation, and smaller gain at 14°C and 21°C compared to 7°C. SR inhibition was most effective at 7°C, where it decreased [Ca2+]i rise slope and amplitude by 58% and 44%, respectively, and prolonged recovery of [Ca2+]i by 1.6-fold and decreased gain by 40%. An opposite trend emerged in ventricular cells in which SR inhibition had its largest effect at 21°C, where it decreased [Ca2+]i rise slope and amplitude by 60% and 75%, respectively, resulting in a 56% decrease in gain. Bma...

International audience; Length-tension relationship Skinned cardiac myocytes Phosphorylation Myosin binding protein C (MyBPC) Myosin light chain-2 (MLC-2) TnI TnT 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.

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 Ca2+([Ca2+]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°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 [Ca2+]i, a decrease in [Ca2+]itransient amplitude, and a seven-fold increase in diastolic [Ca2+]i. The time constant for the decay of the Ca2+transient (τ) 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 ( IKr) or the transient outward ( Ito) 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 Ca2+transient coupled with an increase in diastolic Ca2+, which leads to incomplete relaxation between beats at high frequencies. This coincides with shortened APD at higher frequencies.

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

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

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

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

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

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