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

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

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

The electrolytic manganese industry produces a large amount of electrolytic manganese residue (EMR). Soluble Mn, NH 4 + -N, and other pollutants may be released from the open-air stacked EMR and transported to the environment along with rainfall or surface runoff. Aqueous EMR solution (AES) generally contains various elements required for plant growth, and phytoremediation can be applied to remove these pollutants from AES. Since the contents of Fe and Co vary greatly in AES depending on the ore sources as well as the pre-treatment processes, the presence of bioavailable Fe and Co at different levels may affect plant growth, the rhizosphere microbes, and pollutant removal. The present study investigated the in-situ removal of Mn(II) and NH 4 + -N from AES solution using free floating aquatic plant Pistia stratiotes, focusing especially on the effects of Fe/Co presence and rhizospheric microbe synergistic involvement on contaminant removal. The results showed that 69.08% of Mn and 94.99% of NH 4 + -N were removed by P. stratiotes in 24 d. Both the presence of Fe(II) and Co(II) facilitated the Mn(II) immobilization and increased Mn(II) removal by 19-31% due to the enhanced peroxidase activity and the increased Mn accumulating in roots The complete removal of Mn from AES was found in the presence of Fe(II) at 2 mg L -1 or Co(II) at 0.5 mg L -1 and more than 51% accumulated Mn in the roots was stored in the vacuole and cytoplasm. BioMnO x was found on the surface of the roots, revealing that rhizofiltration, rhizospheric plaque/biofilm formation, and Mn biogeochemical cycle exert synergic effects on Mn(II) immobilization. The findings of the present study demonstrate the feasibility of using P. stratiotes in the treatment of aqueous EMR solutions and the presence of an appropriate amount of bio-available Fe and Co can promote the removal of Mn(II) and NH 4 + -N., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The impact of ocean warming on fish and fisheries is vigorously debated. Leading theories project limited adaptive capacity of tropical fishes and 14-39% size reductions by 2050 due to mass-scaling limitations of oxygen supply in larger individuals. Using the world's hottest coral reefs in the Persian/Arabian Gulf as a natural laboratory for ocean warming - where species have survived >35.0 °C summer temperatures for over 6000 years and are 14-40% smaller at maximum size compared to cooler locations - we identified two adaptive pathways that enhance survival at elevated temperatures across 10 metabolic and swimming performance metrics. Comparing Lutjanus ehrenbergii and Scolopsis ghanam from reefs both inside and outside the Persian/Arabian Gulf across temperatures of 27.0 °C, 31.5 °C and 35.5 °C, we reveal that these species show a lower-than-expected rise in basal metabolic demands and a right-shifted thermal window, which aids in maintaining oxygen supply and aerobic performance to 35.5 °C. Importantly, our findings challenge traditional oxygen-limitation theories, suggesting a mismatch in energy acquisition and demand as the primary driver of size reductions. Our data support a modified resource-acquisition theory to explain how ocean warming leads to species-specific size reductions and why smaller individuals are evolutionarily favored under elevated temperatures., (© 2024. The Author(s).)

Alkylated polycyclic aromatic hydrocarbons are abundant in crude oil and are enriched during petroleum refinement but knowledge of their cardiotoxicity remains limited. Polycyclic aromatic hydrocarbons (PAHs) are considered the main hazardous components in crude oil and the tricyclic PAH phenanthrene has been singled out for its direct effects on cardiac tissue in mammals and fish. Here we test the impact of the monomethylated phenanthrene, 3-methylphenanthrene (3-MP), on the contractile and electrical function of the atrium and ventricle of a polar fish, the navaga cod (Eleginus nawaga). Using patch-clamp electrophysiology in atrial and ventricular cardiomyocytes we show that 3-MP is a potent inhibitor of the delayed rectifier current I Kr (IC50 = 0.25 μM) and prolongs ventricular action potential duration. Unlike the parent compound phenanthrene, 3-MP did not reduce the amplitude of the L-type Ca 2+ current (I Ca ) but it accelerated current inactivation thus reducing charge transfer across the myocyte membrane and compromising pressure development of the whole heart. 3-MP was a potent inhibitor (IC50 = 4.7 μM) of the sodium current (I Na ), slowing the upstroke of the action potential in isolated cells, slowing conduction velocity across the atrium measured with optical mapping, and increasing atrio-ventricular delay in a working whole heart preparation. Together, these findings reveal the strong cardiotoxic potential of this phenanthrene derivative on the fish heart. As 3-MP and other alkylated phenanthrenes comprise a large fraction of the PAHs in crude oil mixtures, these findings are worrisome for Arctic species facing increasing incidence of spills and leaks from the petroleum industry. 3-MP is also a major component of polluted air but is not routinely measured. This is also of concern if the hearts of humans and other terrestrial animals respond to this PAH in a similar manner to fish., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Petroleum hydrocarbon (PHC) degrading bacteria have been frequently discovered. However, in practical application, a single species of PHC degrading bacterium with weak competitiveness may face environmental pressure and competitive exclusion due to the interspecific competition between petroleum-degrading bacteria as well as indigenous microbiota in soil, leading to a reduced efficacy or even malfunction. In this study, the diesel degradation ability and environmental robustness of an endophytic strain Pseudomonas aeruginosa WS02, were investigated. The results show that the cell membrane surface of WS02 was highly hydrophobic, and the strain secreted glycolipid surfactants. Genetic analysis results revealed that WS02 contained multiple metabolic systems and PHC degradation-related genes, indicating that this strain theoretically possesses the capability of oxidizing both alkanes and aromatic hydrocarbons. Gene annotation also showed many targets which coded for heavy metal resistant and metal transporter proteins. The gene annotation-based inference was confirmed by the experimental results: GC-MS analysis revealed that short chain PHCs (C10-C14) were completely degraded, and the degradation of PHCs ranging from C15-C22 were above 90% after 14 d in diesel-exposed culture; Heavy metal (Mn 2+ , Pb 2+ and Zn 2+ ) exposure was found to affect the growth of WS02 to some extent, but not its ability to degrade diesel, and the degradation efficiency was still maintained at 39-59%. WS02 also showed a environmental robustness along with PHC-degradation performance in the co-culture system with other bacterial strains as well as in the co-cultured system with the indigenous microbiota in soil fluid extracted from a PHC-contaminated site. It can be concluded that the broad-spectrum diesel degradation efficacy and great environmental robustness give P. aeruginosa WS02 great potential for application in the remediation of PHC-contaminated soil., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: The three-ringed polycyclic aromatic hydrocarbon (PAH) phenanthrene (Phe) has been implicated in the cardiotoxicity of petroleum-based pollution in aquatic systems, where it disrupts the contractile and electrical function of the fish heart. Phe is also found adsorbed to particulate matter and in the gas phase of air pollution, but to date, no studies have investigated the impact of Phe on mammalian cardiac function., Objectives: Our objectives were to determine the arrhythmogenic potential of acute Phe exposure on mammalian cardiac function and define the underlying mechanisms to provide insight into the toxicity risk to humans., Methods: Ex vivo Langendorff-perfused mouse hearts were used to test the arrhythmogenic potential of Phe on myocardial function, and voltage- and current-clamp recordings were used to define underlying cellular mechanisms in isolated cardiomyocytes., Results: Mouse hearts exposed to ∼ 8 μ M Phe for 15-min exhibited a significantly slower heart rate ( p = 0.0006 , N = 10 hearts), a prolonged PR interval ( p = 0.036 , N = 8 hearts), and a slower conduction velocity ( p = 0.0143 , N = 7 hearts). Whole-cell recordings from isolated cardiomyocytes revealed action potential (AP) duration prolongation (at 80% repolarization; p = 0.0408 , n = 9 cells) and inhibition of key murine repolarizing currents-transient outward potassium current ( I to ) and ultrarapid potassium current ( I Kur )-following Phe exposure. A significant reduction in AP upstroke velocity ( p = 0.0445 , n = 9 cells) and inhibition of the fast sodium current ( I Na ; p = 0.001 , n = 8 cells) and calcium current ( I Ca ; p = 0.0001 ) were also observed, explaining the slowed conduction velocity in intact hearts. Finally, acute exposure to ∼ 8 μ M Phe significantly increased susceptibility to arrhythmias ( p = 0.0455 , N = 9 hearts)., Discussion: To the best of our knowledge, this is the first evidence of direct inhibitory effects of Phe on mammalian cardiac electrical activity at both the whole-heart and cell levels. This electrical dysfunction manifested as an increase in arrhythmia susceptibility due to impairment of both conduction and repolarization. Similar effects in humans could have serious health consequences, warranting greater regulatory attention and toxicological investigation into this ubiquitous PAH pollutant generated from fossil-fuel combustion. https://doi.org/10.1289/EHP12775.

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

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 (chimeras, 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-amniote 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 duplications. TNNI3, encoding "cardiac TnI" in tetrapods, was independently lost in cartilaginous and ray-finned fishes. Instead, ray-finned fishes predominantly express TNNI1 in the heart. TNNI5 is highly expressed in shark hearts and contains a 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 Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

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.

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

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

Respirometry, based on oxygen uptake, is commonly employed for measuring metabolic rate. 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. However, respirometry becomes more challenging as the size of the organism reduces. Additionally, respirometry can be costly and require significant experience and technical knowledge which may prohibit uptake in non-specialist/non-physiology labs. Thus, using equipment routine in most developmental/molecular biology laboratories, we measured glucose uptake in 96-h post fertilisation (hpf) zebrafish larvae and compared it to stop-flow respirometry measures of oxygen uptake to test whether glucose uptake was a suitable alternative measure of metabolic rate. A Passing-Bablok regression revealed that within a 95% limit of agreement, the rate of glucose uptake and the rate of oxygen uptake were equivalent as measures of metabolic rate in 96 hpf Danio rerio larvae. Thus, the methodology we outline here may be a useful alternative or a complementary method for assessing metabolic rate in small organisms., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

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 Ca 2+ 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 Ca 2+ transients (Δ[Ca 2+ ] 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[Ca 2+ ] i to maintain ventricular contractility. When measured at 0.2 Hz in normoxia, hypoxia-acclimated ventricular myocytes had a 3.8-fold larger Δ[Ca 2+ ] 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 Ca 2+ cycling, and that there may be consequences for heart rate elevation during hypoxia, which may impact cardiac output in vivo ., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jonathan Stecyk reports financial support was provided by 10.13039/100000001National Science Foundation. Diarmid Hall, Shannon Royal reports financial support was provided by 10.13039/100000002National Institutes of Health., (© 2022 The Authors.)

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 (I hERG ) were made from HEK293 cells expressing wild-type (WT) and mutant hERG channels. WT I hERG1a was inhibited by phenanthrene with an IC 50 of 17.6 ± 1.7 µM, whilst I hERG1a/1b exhibited an IC 50 of 1.8 ± 0.3 µM. WT I hERG 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 I hERG block. In contrast, the F557L (S5) and M651A (S6) mutations impaired the ability of phenanthrene to inhibit I hERG , 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., (© 2021. The Author(s).)

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.

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., (© The Author(s) 2021. Published by Oxford University Press and the Society for Experimental Biology.)

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, I Kr , with an IC 50 value of ~2 µM. We also show a potent inhibitory effect (~55%) of 1 µM phenanthrene on the transient I Kr currents that protects the heart from early-after-depolarizations and arrhythmias. These data, along with more minor effects on inward sodium (I Na ) (~17% inhibition at 10 µM) and calcium (I Ca ) (~17% inhibition at 30 µM) currents, and no effects on inward rectifier (I K1 and I KAch ) 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., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Soluble adenylyl cyclase (sAC) is a HC O 3   - -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 HC O 3   - 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., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2021 Kompella et al.)

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

Climate change is predicted to impact freshwater aquatic environments through changes to water temperature ( T water ), 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 T water and low O 2 (hypoxia). While several studies have investigated the independent effects of T water and hypoxia on fish physiology, the combined effects of these stressors is less well known. Furthermore, no study has investigated the effects of T water and O 2 saturation levels within the range currently experienced by a salmonid species. Thus, the aim of this study was to investigate the simultaneous effects of T water and O 2 saturation level on the energetics and kinematics of steady-state swimming in brown trout, Salmo trutta . No effect of O 2 saturation level (70 and 100% air saturation) on tail-beat kinematics was detected. Conversely, T water (10, 14, 18 and 22°C) did affect tail-beat kinematics, but a trade-off between frequency ( f tail ) and amplitude ( A , maximum tail excursion) maintained the Strouhal number (St =  f tail • A / U , where U is swimming speed) within the theoretically most mechanically efficient range. Swimming oxygen consumption rate ([Formula: see text]) and cost of transport increased with both U and T water . The only effect of O 2 saturation level was observed at the highest T water (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 T water and reduced O 2 levels likely presented by anthropogenic climate change., (© The Author(s) 2020. Published by Oxford University Press and the Society for Experimental Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© 2019 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Freshwater systems are faced with a myriad of stressors including geomorphological alterations, nutrient overloading and pollution. Previous studies in marine fish showed polyaromatic hydrocarbons (PAHs) to be cardiotoxic. However, the cardiotoxicity of anthropogenic pollutants in freshwater fishes is unclear and has not been examined across multiple levels of cardiac organization. Here we investigated the effect of phenanthrene (Phe), a pervasive anthropogenic pollutant on a sentinel freshwater species, the brown trout (Salmo trutta). We first examined the electrical activity of the whole heart and found prolongation (∼8.6%) of the QT interval (time between ventricular depolarization and repolarization) of the electrocardiogram (ECG) and prolongation (∼13.2%) of the monophasic action potential duration (MAPD) following ascending doses of Phe. At the tissue level, Phe significantly reduced trabecular force generation by ∼24% at concentration 15 μM and above, suggesting Phe reduces cellular calcium cycling. This finding was supported by florescent microscopy showing a reduction (∼39%) in the intracellular calcium transient amplitude following Phe exposure in isolated brown trout ventricular myocytes. Single-cell electrophysiology was used to reveal the mechanism underlying contractile and electrical dysfunction following Phe exposure. A Phe-dependent reduction (∼38%) in the L-type Ca 2+ current accounts, at least in part, for the lowered Ca 2+ transient and force production. Prolongation of the MAPD and QT interval was explained by a reduction (∼70%) in the repolarising delayed rectifier K + current following Phe exposure. Taken together, our study shows a direct impact of Phe across multiple levels of cardiac organization in a key freshwater salmonid., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Elasmobranchs display various reproductive modes, which have been key to their evolutionary success. In recent decades there has been a rise in the number of reported cases of foetal abnormalities including fertilised, double-embryos held within one egg capsule, hereafter referred to as twins. Previously, the occurrences of twin egg cases have been reported in two batoid and one shark species. We report the first cases of twins in three species of oviparous elasmobranchs: the undulate ray (Raja undulata), the nursehound (Scyliorhinus stellaris), and the small-spotted catshark (Scyliorhinus canicula). We investigated the genetic relationships between the twins in S. stellaris, and S. canicula using microsatellite markers. Whilst the S. stellaris twins displayed the same genotypes, we found that the S. canicula twin individuals arose through heteropaternal superfecundation. This is the first reported incidence of such a paternity in elasmobranchs. The relationship between environmental change and reproductive strategy in elasmobranchs is unclear and further research is needed to determine its effect on the prevalence and mechanisms of formation of elasmobranch twins., Competing Interests: The project was funded by the Higher Education Innovation Fund through The University of Manchester’s Knowledge and Innovation Hub for Environmental Stability (HAS), as part of the Natural Environmental Research Council Doctoral Training Program (SAH) and by the Ministry of Higher Education (KPT, Malaysia) (SMM). Merlin Entertainments, Sea Life, Weymouth Adventures Park provided support in the form of salaries for author JDH but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. All authors declare that there are no significant competing professional or personal interests that might have influenced the performance or presentation of the work described in this manuscript. Merlin Entertainments, Sea Life, Weymouth Adventures Park that provided support in the form of salaries for author JDH does not alter our adherence to PLOS ONE policies on sharing data and materials.

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

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

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.

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.)

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.)

New Findings: What is the topic of this review? Excitation-contraction coupling in fish hearts is maintained over a range of temperatures that would be cardioplegic to most mammals. Here, we review what is known about the fish cardiac ryanodine receptor, and consider how it may be regulated in a different manner from the mammalian cardiac isoforms of this channel. What advances does it highlight? We highlight how a better understanding of the basic gating and conducting properties of fish cardiac ryanodine receptors could provide considerable insight into mechanisms underlying sarcoplasmic reticulum calcium release in fish hearts and the role of the sarcoplasmic reticulum in the evolution of the heart. The fish cardiac sarcoplasmic reticulum (SR) holds large quantities of Ca(2+), but calcium-induced calcium release (CICR) is weak in these myocytes, and contraction and relaxation are largely determined by transsarcolemmal Ca(2+) flux. Many fish species live in a cold and seasonally variable thermal habitat, which could provide challenges to regulation of excitation-contraction coupling. Here, we focus on the cardiac SR Ca(2+)-release channel (RyR2) in fish and ask whether it may be regulated in a different manner from the mammalian RyR2. We review data indicating that fish cardiac RyR are present at lower density, are more spatially separated within the SR membrane and are less responsive to cytosolic Ca(2+) than mammalian RyR2 channels. All of these features would contribute to the weak CICR evident from functional studies. We also consider how CICR can be enhanced in fish myocytes following β-adrenergic stimulation and application of low levels of caffeine, and how acute and chronic temperature change may affect the gating properties of fish RyR2s. It is clear that a lack of insight into the fundamental gating and conductance properties of fish RyR2 channels is hindering our understanding of the role of the SR in fish cardiac excitation-contraction coupling. We conclude by reflecting on how studies that probe the biophysical properties of fish RyR2 channel gating in response to various ligands and temperatures would be very instructive for our understanding of the role of the SR in the evolution of the heart., (© 2015 The Authors. Experimental Physiology © 2015 The Physiological Society.)

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.)