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The Alaska blackfish (Dallia pectoralis) is a facultative air-breather endemic to northern latitudes where it remains active in winter under ice cover in cold hypoxic waters. To understand the changes in cellular Ca2+ cycling that allow the heart to function in cold hypoxic water, we acclimated Alaska blackfish to cold (5 °C) normoxia or cold hypoxia (2.1–4.2 kPa; no air access) for 5–8 weeks. We then assessed the impact of the acclimation conditions on intracellular Ca2+ transients (Δ[Ca2+]i) of isolated ventricular myocytes and contractile performance of isometrically-contracting ventricular strips. Measurements were obtained at various contractile frequencies (0.2–0.6 Hz) in normoxia, during acute exposure to hypoxia, and reoxygenation at 5 °C. The results show that hypoxia-acclimated Alaska blackfish compensate against the depressive effects of hypoxia on excitation-contraction coupling by remodelling cellular Δ[Ca2+]i to maintain ventricular contractility. When measured at 0.2 Hz in normoxia, hypoxia-acclimated ventricular myocytes had a 3.8-fold larger Δ[Ca2+]i peak amplitude with a 4.1-fold faster rate of rise, compared to normoxia-acclimated ventricular myocytes. At the tissue level, maximal developed force was 2.1-fold greater in preparations from hypoxia-acclimated animals. However, maximal attainable contraction frequencies in hypoxia were lower in hypoxia-acclimated myocytes and strips than preparations from normoxic animals. Moreover, the inability of hypoxia-acclimated ventricular myocytes and strips to contract at high frequency persisted upon reoxygenation. Overall, the findings indicate that hypoxia alters aspects of Alaska blackfish cardiac myocyte Ca2+ cycling, and that there may be consequences for heart rate elevation during hypoxia, which may impact cardiac output in vivo., Highlights • The air-breathing Alaska blackfish remains active under ice cover in hypoxic waters. • Maintained activity is supported by compensation of intracellular Ca2+ transients. • The compensation permits greater ventricular maximal developed force. • However, maximal attainable contraction frequencies are limited by hypoxia exposure.

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.

Bird cardiomyocytes are long, thin and lack transverse (t)-tubules, which is akin to the cardiomyocyte morphology of ectothermic non-avian reptiles, who are typified by low maximum heart rates and low pressure development. However, birds can achieve greater contractile rates and developed pressures than mammals, whose wide cardiomyocytes contain a dense t-tubular network allowing for uniform excitation–contraction coupling and strong contractile force. To address this apparent paradox, this paper functionally links recent electrophysiological studies on bird cardiomyocytes with decades of ultrastructure measurements. It shows that it is the strong transsarcolemmal Ca 2+ influx via the L-type Ca 2+ current ( I CaL ) and the high gain of Ca 2+ -induced Ca 2+ release from the sarcoplasmic reticulum (SR), coupled with an internal SR Ca 2+ release relay system, that facilitates the strong fast contractions in the long thin bird cardiomyocytes, without the need for t-tubules. The maintenance of an elongated myocyte morphology following the post-hatch transition from ectothermy to endothermy in birds is discussed in relation to cardiac load, myocyte ploidy, and cardiac regeneration potential in adult cardiomyocytes. Overall, the paper shows how little we know about cellular Ca 2+ dynamics in the bird heart and suggests how increased research efforts in this area would provide vital information in our quest to understand the role of myocyte architecture in the evolution of the vertebrate heart. This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’. Please see glossary at the end of the paper for definitions of specialized terms.

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

Elevated plasma potassium levels (hyperkalemia), reduced plasma pH (acidosis), reduced blood oxygen content, and elevated temperatures are associated with species-specific rates of at-vessel and post-release mortality in elasmobranch fishes. The mechanism linking these physiological disturbances to mortality remains undetermined however, and we hypothesize that the proximate cause is reduced myocardial function. We measured changes in the functional properties of isolated ventricular myocardial strips from clearnose skate (Rostroraja eglanteria), smooth dogfish (Mustelus canis), and sandbar shark (Carcharhinus plumbeus) when subjected to the following stressors (both in isolation and in combination): hyperkalemia (7.4 mM K+), acidosis (from 7.9 to 7.1), and reduced oxygen (to 31% O2 saturation) applied at temperatures 5 °C above and below holding temperatures. We selected these species based on phylogenetic distance, diverse routine activity levels, and their tolerance to capture and transport. Stressors had a few significant species-specific detrimental impacts on myocardial function (e.g., a 33–45% decrease in net force under acidosis + low O2). Net force production of myocardial strips from clearnose skate and smooth dogfish approximately doubled following exposure to isoproterenol, demonstrating that these species possess beta-adrenergic receptors and that their stimulation could provide a mechanism for preservation of cardiac function during stress. Our results suggest that disruption of physiological homeostasis associated with capture may fatally impair cardiac function in some elasmobranch species, although research with more severe stressors is needed.

The troponin (Tn) complex, responsible for the Ca2+ activation of striated muscle, is composed of three interacting protein subunits: TnC, TnI, and TnT, encoded by TNNC, TNNI, and TNNT genes. TNNI and TNNT are sister gene families, and in mammals the three TNNI paralogs (TNNI1, TNNI2, TNNI3), which encode proteins with tissue-specific expression, are each in close genomic proximity with one of the three TNNT paralogs (TNNT2, TNNT3, TNNT1, respectively). It has been widely presumed that all vertebrates broadly possess genes of these same three classes, although earlier work has overlooked jawless fishes (cyclostomes) and cartilaginous fishes (chimaeras, rays and sharks), which are distantly related to other jawed vertebrates. With a new phylogenetic and synteny analysis of a diverse array of vertebrates including these taxonomic groups, we define five distinct TNNI classes (TNNI1-5), with TNNI4 and TNNI5 being only present in non-mammalian vertebrates and typically found in tandem, and four classes of TNNT (TNNT1-4). These genes are located in four genomic loci that were generated by the 2R whole-genome duplication events. TNNI3, encoding ‘cardiac TnI’ in mammals, was independently lost in cartilaginous and ray-finned fishes. Ray-finned fishes predominantly express TNNI1 in the heart. TNNI5 is highly expressed in shark hearts and contains an N-terminal extension similar to that of TNNI3 found in tetrapod hearts. Given that TNNI3 and TNNI5 are distantly related, this supports the hypothesis that the N-terminal extension may be an ancestral feature of vertebrate TNNI and not an innovation unique to TNNI3, as has been commonly believed.

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

Exposure to elevated temperatures during embryogenesis can influence the plasticity of tissues in later-life. Despite these long-term changes in plasticity, few differentially expressed genes are ever identified, suggesting that the developmental programming of later-life plasticity may occur through the modulation of other aspects of the transcriptomic architecture, such as gene network function. Here, we use network modelling approaches to demonstrate that warm temperatures during embryonic development (developmental warming) have consistent effects in later-life on the organisation of transcriptomic networks across four diverse species of fishes:Scyliorhinus canicula, Danio rerio, Dicentrarchus labrax, andGasterosteus aculeatus. The transcriptomes of developmentally warmed fishes are characterised by an increased entropy of their pairwise gene interaction networks, implying a less structured, more ‘random’ set of gene interactions. We also show that, in zebrafish subject to developmental warming, the entropy of an individual gene within a network is associated with that gene’s probability of expression change during temperature acclimation in later-life. However, this association is absent in animals reared under ‘control’ conditions. Thus, the thermal environment experienced during embryogenesis can alter transcriptomic organisation in later-life, and these changes may influence an individual’s responsiveness to future temperature challenges.

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.

To protect the gill capillaries from high systolic pulse pressure, the fish heart contains a compliant non-contractile chamber called the bulbus arteriosus which is part of the outflow tract (OFT) which extends from the ventricle to the ventral aorta. Thermal acclimation alters the form and function of the fish atria and ventricle to ensure appropriate cardiac output at different temperatures, but its impact on the OFT is unknown. Here we used ex vivo pressure–volume curves to demonstrate remodelling of passive stiffness in the rainbow trout ( Oncorhynchus mykiss ) bulbus arteriosus following more than eight weeks of thermal acclimation to 5, 10 and 18°C. We then combined novel, non-biased Fourier transform infrared spectroscopy with classic histological staining to show that changes in compliance were achieved by changes in tissue collagen-to-elastin ratio. In situ gelatin zymography and SDS-PAGE zymography revealed that collagen remodelling was underpinned, at least in part, by changes in activity and abundance of collagen degrading matrix metalloproteinases. Collectively, we provide the first indication of bulbus arteriosus thermal remodelling in a fish and suggest this remodelling ensures optimal blood flow and blood pressure in the OFT during temperature change.

Air pollution is associated with detrimental effects on human health, including decreased cardiovascular function. However, the causative mechanisms behind these effects have yet to be fully elucidated. Here we review the current epidemiological, clinical and experimental evidence linking pollution with cardiovascular dysfunction. Our focus is on particulate matter (PM) and the associated low molecular weight polycyclic aromatic hydrocarbons (PAHs) as key mediators of cardiotoxicity. We begin by reviewing the growing epidemiological evidence linking air pollution to cardiovascular dysfunction in humans. We next address the pollution‐based cardiotoxic mechanisms first identified in fish following the release of large quantities of PAHs into the marine environment from point oil spills (e.g. Deepwater Horizon). We finish by discussing the current state of mechanistic knowledge linking PM and PAH exposure to mammalian cardiovascular patho‐physiologies such as atherosclerosis, cardiac hypertrophy, arrhythmias, contractile dysfunction and the underlying alterations in gene regulation. Our aim is to show conservation of toxicant pathways and cellular targets across vertebrate hearts to allow a broad framework of the global problem of cardiotoxic pollution to be established. AhR; Aryl hydrocarbon receptor. Dark lines indicate topics discussed in this review. Grey lines indicate topics reviewed elsewhere., Polycyclic aromatic hydrocarbons are common in both air and aquatic pollution. Exposure to these pollutants can lead to PAH accumulation in the body and cause cardiovascular dysfunction via a number of direct and indirect mechanisms. PAH exposure is associated with arrhythmias, hypertrophy, atherosclerosis and developmental toxicity (among others). This review discusses current evidence linking PAH‐based pollution and cardiovascular disease in vertebrates.

Deepwater Horizon crude oil is comprised of polycyclic aromatic hydrocarbons that cause a number of cardiotoxic effects in marine fishes across all levels of biological organization and at different life stages. Although cardiotoxic impacts have been widely reported, the mechanisms underlying these impairments in adult fish remain understudied. In this study, we examined the impacts of crude oil on cardiomyocyte contractility and electrophysiological parameters in freshly isolated ventricular cardiomyocytes from adult mahi-mahi (Coryphaena hippurus). Cardiomyocytes directly exposed to oil exhibited reduced contractility over a range of environmentally relevant concentrations (2.8-12.9 μg l-1∑PAH). This reduction in contractility was most pronounced at higher stimulation frequencies, corresponding to the upper limits of previously measured in situ mahi heart rates. To better understand the mechanisms underlying impaired contractile function, electrophysiological studies were performed, which revealed oil exposure prolonged cardiomyocyte action potentials and disrupted potassium cycling (9.9-30.4 μg l-1∑PAH). This study is the first to measure cellular contractility in oil-exposed cardiomyocytes from a pelagic fish. Results from this study contribute to previously observed impairments to heart function and whole-animal exercise performance in mahi, underscoring the advantages of using an integrative approach in examining mechanisms of oil-induced cardiotoxicity in marine fish.

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 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 16.8 ± 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.

Respirometry is the current gold-standard for measuring metabolic rate. However, there is a growing need for metabolic rate measurements suitable for developmental studies, particularly in Danio rerio, where many important developmental stages occur at < 4 mm. While many metabolic studies rely on respirometry, the cost and complexity of the equipment limits its appeal in non-specialist labs, and background respiration becomes increasingly problematic as the size of the organism reduces. Here, glucose uptake was compared to stop-flow respirometry as an alternative measure of metabolic rate more suitable to the small scale required for developmental studies. A Passing-Bablok regression revealed the rate of glucose uptake can be considered equivalent to oxygen consumption as a measure of metabolic rate in Danio rerio larvae within a 95% limit of agreement. Therefore, glucose uptake is a valid alternative to the gold-standard in small organisms where conventional respirometry is problematic.Summary statementThe rate of glucose uptake is a valid alternative to respirometry for metabolic rate measurements in small larval fish.

Kompella et al. show that the petroleum-derived pollutant phenanthrene blocks the zebrafish native ERG orthologue mediating IKr, thus suppressing outward protective currents initiated by premature beats and increasing the susceptibility to arrhythmia triggers., Air pollution is an environmental hazard that is associated with cardiovascular dysfunction. Phenanthrene is a three-ringed polyaromatic hydrocarbon that is a significant component of air pollution and crude oil and has been shown to cause cardiac dysfunction in marine fishes. We investigated the cardiotoxic effects of phenanthrene in zebrafish (Danio rerio), an animal model relevant to human cardiac electrophysiology, using whole-cell patch-clamp of ventricular cardiomyocytes. First, we show that phenanthrene significantly shortened action potential duration without altering resting membrane potential or upstroke velocity (dV/dt). L-type Ca2+ current was significantly decreased by phenanthrene, consistent with the decrease in action potential duration. Phenanthrene blocked the hERG orthologue (zfERG) native current, IKr, and accelerated IKr deactivation kinetics in a dose-dependent manner. Furthermore, we show that phenanthrene significantly inhibits the protective IKr current envelope, elicited by a paired ventricular AP-like command waveform protocol. Phenanthrene had no effect on other IK. These findings demonstrate that exposure to phenanthrene shortens action potential duration, which may reduce refractoriness and increase susceptibility to certain arrhythmia triggers, such as premature ventricular contractions. These data also reveal a previously unrecognized mechanism of polyaromatic hydrocarbon cardiotoxicity on zfERG by accelerating deactivation and decreasing IKr protective current.

The southern Persian/Arabian Gulf experiences extreme seasonal temperature variation (> 20 °C) making it among the most hostile reef environments on Earth. Previous anecdotal evidence has suggested that seasonal temperature changes may influence regional reef fish assemblages, but to date research has been limited. To examine the influence of temperature on reef fish abundance and composition, we performed visual surveys in summer and in winter over three years at three reefs in the southern Gulf (Dhabiya, Saadiyat and Ras Ghanada). Overall abundance of fishes was 40% higher in summer than in winter, and multivariate analyses showed strong and significant differences in overall seasonal community structure, consistent at all sites and across all years. Seasonal differences were largely driven by nine of the 30 observed species, which together accounted for 70% of the divergence in community structure between summer and winter. Of these nine species, Lutjanus ehrenbergii, Lutjanus fulviflamma, Plectorhinchus sordidus and Abudefduf vaigiensis were significantly more abundant in summer, Parupeneus margaritatus and Acanthopagrus bifasciatus, were significantly more common on reefs in winter. We discuss these changes in terms of seasonal physiological and ecological constraints, and explore the implications of these changes on the functional ecology of reef fishes in this thermally variable and extreme environment.

Oil and gas exploration in the Arctic can result in the release of polycyclic aromatic hydrocarbons (PAHs) into relatively pristine environments. Following the recent spill of approximately 17 500 tonnes of diesel fuel in Norilsk, Russia, May 2020, our study focussed on the effects of phenanthrene, a low molecular weight PAH found in diesel and crude oil, on the isolated atrial and ventricular myocytes from the heart of the polar teleost, the Navaga cod (Eleginus nawaga). Acute exposure to phenanthrene in navaga cardiomyocytes caused significant action potential (AP) prolongation, confirming the proarrhythmic effects of this pollutant. We show AP prolongation was due to potent inhibition of the main repolarising current, IKr, with an IC50 value of ~2 µM. We also show a potent inhibitory effect (~55%) of 1 µM phenanthrene on the transient IKr currents that protects the heart from early-after-depolarizations and arrhythmias. These data, along with more minor effects on inward sodium (INa) (~17% inhibition at 10 µM) and calcium (ICa) (~17% inhibition at 30 µM) currents, and no effects on inward rectifier (IK1 and IKAch) currents, demonstrate the cardiotoxic effects exerted by phenanthrene on the atrium and ventricle of navaga cod. Moreover, we report the first data that we are aware of on the impact of phenanthrene on atrial myocyte function in any fish species. © 2021 The Authors This work is supported by British Heart Foundation Grant PG/17/77/33125 to HAS and SNK and by the Interdisciplinary Scientific and Educational School of Moscow University «Molecular Technologies of the Living Systems and Synthetic Biology» as part of the Scientific Project of the State Order of the Government of Russian Federation to Lomonosov Moscow State University No.121032300071-8 to DVA. We thank Prof Jules Hancox, University of Bristol, UK, and Dr Fabien Brette, Université de Bordeaux for insightful comments. We are grateful to White Sea Biological Station director, Professor Alexander Tzetlin for supporting the study. We thank Valo and Valentina Sivonen for collecting the fish.

Lay Summary Shark embryos use a freezing behaviour to avoid being detected by predators. Here, we show that the duration an individual can perform the freezing behaviour is reduced by ocean warming, potentially increasing the vulnerability of embryonic sharks to predation., Embryogenesis is a vulnerable stage in elasmobranch development due in part to high predation mortality. Embryonic elasmobranchs respond to potential predators by displaying a freezing behaviour, characterized by the cessation of pharyngeal respiration followed immediately by coiling of the tail around the body. We hypothesized that the duration of this freeze response is limited by the embryo’s requirement for oxygen. Here, Scyliorhinus canicula embryos were incubated at either 15°C or 20°C during embryogenesis and tested for the duration of, and metabolic consequence of, the freeze response at their respective incubation temperature. Freeze response duration was negatively impacted by routine metabolic rate; embryos at 20°C had 7-fold shorter freeze duration than those at 15°C, potentially increasing their susceptibility to predation. These data demonstrate the capacity for climate change stressors to affect animal behaviour and suggest that this may occur by eliciting changes in the organism’s metabolism. We suggest altered predator avoidance behaviour is a new factor to consider when assessing the impact of climate change on the conservation and management of oviparous elasmobranch species.

Soluble adenylyl cyclase (sAC) is a [Formula: see text]-stimulated enzyme that produces the ubiquitous signalling molecule cAMP, and deemed an evolutionarily conserved acid–base sensor. However, its presence is not yet confirmed in bony fishes, the most abundant and diverse of vertebrates. Here, we identified sAC genes in various cartilaginous, ray-finned and lobe-finned fish species. Next, we focused on rainbow trout sAC (rtsAC) and identified 20 potential alternative spliced mRNAs coding for protein isoforms ranging in size from 28 to 186 kDa. Biochemical and kinetic analyses on purified recombinant rtsAC protein determined stimulation by [Formula: see text] at physiologically relevant levels for fish internal fluids (EC(50) ∼ 7 mM). rtsAC activity was sensitive to KH7, LRE1, and DIDS (established inhibitors of sAC from other organisms), and insensitive to forskolin and 2,5-dideoxyadenosine (modulators of transmembrane adenylyl cyclases). Western blot and immunocytochemistry revealed high rtsAC expression in gill ion-transporting cells, hepatocytes, red blood cells, myocytes and cardiomyocytes. Analyses in the cell line RTgill-W1 suggested that some of the longer rtsAC isoforms may be preferentially localized in the nucleus, the Golgi apparatus and podosomes. These results indicate that sAC is poised to mediate multiple acid–base homeostatic responses in bony fishes, and provide cues about potential novel functions in mammals.

Embryonic growth trajectory is a risk factor for chronic metabolic and cardiovascular disorder. Grb10 is a negative regulator of the main pathways driving embryonic growth. This study investigates the long-term cardiometabolic consequences and transcriptomic profiles of transient disruption of grb10a expression in Danio rerio. Knockdown was associated with increased embryonic growth (+7%) and metabolic rate (+25%), and decreased heart rate (- 50%) in early life. Juvenile growth and respiratory rate were also elevated (+30% and 7-fold increase respectively). The transcriptome was permanently remodelled by this transient disruption, with dysregulation of multiple growth, cardiac, and metabolic pathways. Phenotypic alteration persisted into adulthood, resulting in a leaner body with elevated skeletal and cardiac muscle content and aerobic scope (43%). This study not only confirms for the first time that transient disruption of a single gene can result in permanent transcriptomic remodelling but correlates this remodelling with persistent alterations to the adult cardiometabolic phenotype.

Crustaceans are important ecosystem bio-indicators but their response to pollutants such as polyaromatic hydrocarbons (PAHs) remains understudied, particularly in freshwater habitats. Here we investigated the effect of phenanthrene (at 0.5, 1.0 and 1.5 mg L−1), a 3-ringed PAH associated with petroleum-based aquatic pollution on survival, in vivo and in situ cardiac performance, the oxidative stress response and the tissue burden in the signal crayfish (Pacifastacus leniusculus). Non-invasive sensors were used to monitor heart rate during exposure. Phenanthrene reduced maximum attainable heart rate in the latter half (days 8–15) of the exposure period but had no impact on routine heart rate. At the end of the 15-day exposure period, the electrical activity of the semi-isolated in situ crayfish heart was assessed and significant prolongation of the QT interval of the electrocardiogram was observed. Enzyme pathways associated with oxidative stress (superoxide dismutase and total oxyradical scavenging capacity) were also assessed after 15 days of phenanthrene exposure in gill, hepatopancreas and skeletal muscle; the results suggest limited induction of protective antioxidant pathways. Lastly, we report that 15 days exposure caused a dose-dependent increase in phenanthrene in hepatopancreas and heart tissues which was associated with reduced survivability. To our knowledge, this study is the first to provide such a thorough understanding of the impact of phenanthrene on a crustacean.

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.).

Elevated plasma potassium levels (hyperkalemia), reduced plasma pH (acidosis), reduced blood oxygen content, and elevated temperatures are associated with species-specific rates of at-vessel and post-release mortality in elasmobranch fishes. The mechanism linking these physiological disturbances to mortality remains undetermined however, and we hypothesize that the proximate cause is reduced myocardial function. We measured changes in the functional properties of isolated ventricular myocardial strips from clearnose skate (Rostroraja eglanteria), smooth dogfish (Mustelus canis), and sandbar shark (Carcharhinus plumbeus) when subjected to the following stressors (both in isolation and in combination): hyperkalemia (7.4 mM K

Birds occupy a unique position in the evolution of cardiac design. Their hearts are capable of cardiac performance on par with, or exceeding that of mammals, and yet the structure of their cardiomyocytes resembles those of reptiles. It has been suggested that birds use intracellular Ca

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

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. Electronic supplementary material The online version of this article (10.1007/s00424-018-2140-1) contains supplementary material, which is available to authorized users.

Tuna species support some of the world’s largest commercial and recreational fisheries. Their extensive migratory patterns expose them to multiple national and international fisheries and fishery management regimes. Several prized species have become the focus of global conservation efforts and there is a growing worldwide interest in establishing optimal strategies for sustainable tuna fisheries. Although this task has proven to be very challenging, it has taken on a new sense of urgency in the face of the potential effects of global climate change. A better understanding of the interactions between environmental conditions and tuna physiology and how they affect tuna behavior will offer population and stock assessment modelers and fisheries biologists a more mechanistic understanding of tuna distribution patterns and may help predict changes in both geographic and depth-related movement patterns. Indeed, physiological data comprise a growing component of multi-trait analysis approaches to species conservation. Our review aims to summarize what is known about differences among tuna species in distribution patterns, tolerances to environmental conditions, and physiological characteristics that correlate with the capacity to inhabit cooler (deeper, higher latitude) and even hypoxic waters. To achieve this goal, we discuss how these physiological traits are associated with habitat partitioning within the three-dimensional oceanic environment and with niche expansion into cooler and hypoxic waters. We also point out areas where additional research is needed to predict more accurately how future changes in oceanographic conditions will affect the distributions and movement patterns of tunas and their availability to fisheries.

Climate change is predicted to impact freshwater aquatic environments through changes to water temperature (Twater), river flow and eutrophication. Riverine habitats contain many economically and ecologically important fishes. One such group is the migratory salmonids, which are sensitive to warm Twater and low O2 (hypoxia). While several studies have investigated the independent effects of Twater and hypoxia on fish physiology, the combined effects of these stressors is less well known. Furthermore, no study has investigated the effects of Twater and O2 saturation levels within the range currently experienced by a salmonid species. Thus, the aim of this study was to investigate the simultaneous effects of Twater and O2 saturation level on the energetics and kinematics of steady-state swimming in brown trout, Salmo trutta. No effect of O2 saturation level (70 and 100% air saturation) on tail-beat kinematics was detected. Conversely, Twater (10, 14, 18 and 22°C) did affect tail-beat kinematics, but a trade-off between frequency (ftail) and amplitude (A, maximum tail excursion) maintained the Strouhal number (St = ftail• A/U, where U is swimming speed) within the theoretically most mechanically efficient range. Swimming oxygen consumption rate (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}${\dot{M}}_{{\mathsf{O}}_{\mathsf{2}}}$\end{document}) and cost of transport increased with both U and Twater. The only effect of O2 saturation level was observed at the highest Twater (22°C) and fastest swimming speed (two speeds were used—0.6 and 0.8 m s−1). As the extremes of this study are consistent with current summer conditions in parts of UK waterways, our findings may indicate that S. trutta will be negatively impacted by the increased Twater and reduced O2 levels likely presented by anthropogenic climate change.

Elasmobranchs are key to a healthy marine ecosystem but are under threat from human activities, such as destructive fisheries and shark finning. Embryos of oviparous elasmobranchs may be further challenged during development by rising temperatures and falling dissolved oxygen concentrations in their intertidal environment. However, the impact of climate change on survival and growth of oviparous elasmobranchs is still poorly understood. Here, we investigate the effects of temperature and hypoxia on the growth and survival of small‐spotted catshark (Scyliorhinus canicula) embryos by incubating eggs in either: normoxia 15 °C, normoxia 20 °C, hypoxia 15 °C, or hypoxia 20 °C. Incubation under the elevated temperature increased the embryonic growth rate, yolk consumption rate, and Fulton's condition factor at hatching, whilst decreasing the total length and body mass of newly hatched sharks. Under low oxygen conditions (50 % air saturation) the survival rate of S. canicula embryos dropped significantly, and the temperature induced increase in Fulton's condition factor was reversed. Together, these data demonstrate both the individual and compound effects of elevated temperature and hypoxia on the survival and growth during early ontogeny of a ubiquitous, coastal elasmobranch, S. canicula.

In mammalian cardiomyocytes, Ca

In mammalian cardiomyocytes, Ca2+ influx through L-type voltage-gated Ca2+ channels (VGCCs) is amplified by release of Ca2+ via type 2 ryanodine receptors (RyR2) in the sarcoplasmic reticulum (SR): a process termed Ca2+-induced Ca2+-release (CICR). In mammalian skeletal muscles, VGCCs play a distinct role as voltage-sensors, physically interacting with RyR1 channels to initiate Ca2+ release in a mechanism termed depolarisation-induced Ca2+-release (DICR). In the current study, we surveyed the genomes of animals and their close relatives, to explore the evolutionary history of genes encoding three proteins pivotal for ECC: L-type VGCCs; RyRs; and a protein family that anchors intracellular organelles to plasma membranes, namely junctophilins (JPHs). In agreement with earlier studies, we find that non-vertebrate eukaryotes either lack VGCCs, RyRs and JPHs; or contain a single homologue of each protein. Furthermore, the molecular features of these proteins thought to be essential for DICR are only detectable within vertebrates and not in any other taxonomic group. Consistent with earlier physiological and ultrastructural observations, this suggests that CICR is the most basal form of ECC and that DICR is a vertebrate innovation. This development was accompanied by the appearance of multiple homologues of RyRs, VGCCs and junctophilins in vertebrates, thought to have arisen by 'whole genome replication' mechanisms. Subsequent gene duplications and losses have resulted in distinct assemblies of ECC components in different vertebrate clades, with striking examples being the apparent absence of RyR2 from amphibians, and additional duplication events for all three ECC proteins in teleost fish. This is consistent with teleosts possessing the most derived mode of DICR, with their Cav1.1 VGCCs completely lacking in Ca2+ channel activity.

Soluble adenylyl cyclase (sAC) is aHCO3 −-stimulated enzyme that produces the ubiquitous signalling molecule cAMP, and deemed an evolutionarily conserved acid–base sensor. However, its presence is not yet confirmed in bony fishes, the most abundant and diverse of vertebrates. Here, we identified sAC genes in various cartilaginous, ray-finned and lobe-finned fish species. Next, we focused on rainbow trout sAC (rtsAC) and identified 20 potential alternative spliced mRNAs coding for protein isoforms ranging in size from 28 to 186 kDa. Biochemical and kinetic analyses on purified recombinant rtsAC protein determined stimulation byHCO3 −at physiologically relevant levels for fish internal fluids (EC50∼ 7 mM). rtsAC activity was sensitive to KH7, LRE1, and DIDS (established inhibitors of sAC from other organisms), and insensitive to forskolin and 2,5-dideoxyadenosine (modulators of transmembrane adenylyl cyclases). Western blot and immunocytochemistry revealed high rtsAC expression in gill ion-transporting cells, hepatocytes, red blood cells, myocytes and cardiomyocytes. Analyses in the cell line RTgill-W1 suggested that some of the longer rtsAC isoforms may be preferentially localized in the nucleus, the Golgi apparatus and podosomes. These results indicate that sAC is poised to mediate multiple acid–base homeostatic responses in bony fishes, and provide cues about potential novel functions in mammals.

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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.

Temperature is a ubiquitous environmental factor affecting physiological processes of ectotherms. Due to the effects of climate change on global air and water temperatures, predicting the impacts of changes in environmental thermal conditions on ecosystems is becoming increasingly important. This is especially crucial for migratory fish, such as the ecologically and economically vital salmonids, because their complex life histories make them particularly vulnerable. Here, we addressed the question whether temperature affects the morphology of brown trout, Salmo trutta L. spermatozoa. The fertilising ability of spermatozoa is commonly attributed to their morphological dimensions, thus implying direct impacts on the reproductive success of the male producing the cells. We show that absolute lengths of spermatozoa are not affected by temperature, but spermatozoa from warm acclimated S. trutta males have longer flagella relative to their head size compared to their cold acclimated counterparts. This did not directly affect sperm swimming speed, although spermatozoa from warm acclimated males may have experienced a hydrodynamic advantage at warmer temperatures, as suggested by our calculations of drag based on head size and sperm swimming speed. The results presented here highlight the importance of increasing our knowledge of the effects of temperature on all aspects of salmonid reproduction in order to secure their continued abundance., Summary: Two different acclimation temperatures cause divergence in brown trout sperm morphometry through changes in the relative head surface area to flagellum length ratio.