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

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

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

We measured the temperature sensitivity, adrenergic sensitivity, and dependence on sarcoplasmic reticulum (SR) Ca(2+) of ventricular muscle from pelagic fishes with different vertical mobility patterns: bigeye tuna (Thunnus obesus), yellowfin tuna (Thunnus albacares), and mahimahi (Coryphaena hippurus) and a single specimen from swordfish (Xiphias gladius). Ventricular muscle from the bigeye tuna and mahimahi exhibited a biphasic response to an acute decrease in temperature (from 26 degrees to 7 degrees C); twitch force and kinetic parameters initially increased and then declined. The magnitude of this response was larger in the bigeye tuna than in the mahimahi. Under steady state conditions at 26 degrees C, inhibition of SR Ca(2+) release and reuptake with ryanodine and thapsigargin decreased twitch force and kinetic parameters, respectively, in the bigeye tuna only. However, the initial inotropy associated with decreasing temperature was abolished by SR inhibition in both the bigeye tuna and the mahimahi. Application of adrenaline completely reversed the effects of ryanodine and thapsigargin, but this effect was diminished at cold temperatures. In the yellowfin tuna, temperature and SR inhibition had minor effects on twitch force and kinetics, while adrenaline significantly increased these parameters. Limited data suggest that swordfish ventricular muscle responds to acute temperature reduction, SR inhibition, and adrenergic stimulation in a manner similar to that of bigeye tuna ventricular muscle. In aggregate, our results show that the temperature sensitivity, SR dependence, and adrenergic sensitivity of pelagic fish hearts are species specific and that these differences reflect species-specific vertical mobility patterns.

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

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

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 2 0 2 production were measured at 10 °C in isolated brain homogenates with an Oroboros respirometer. Cold acclimation and chronic hypoxia had no effects on mitochondrial aerobic capacity or ADP affinity. However, cold acclimation in normoxia led to a suppression of brain mitochondrial H 2 0 2 production, which persisted and became more pronounced in the cold hypoxic fish. Overall, our study suggests cold acclimation supresses ROS production in Alaska blackfish, which may protect the fish from oxidative stress when oxygen becomes limited during winter. [Display omitted] • Alaska blackfish overwinter under ice-covered lakes in severely hypoxic conditions • We hypothesized survival of these winter conditions would require metabolic readjustments • Cold acclimation and chronic hypoxia suppressed brain reactive oxygen species (ROS) production • Suppression of ROS production may protect the fish from oxidative stress during winter [ABSTRACT FROM AUTHOR]

Abstract: Mammalian cardiac muscle exhibits a number of intrinsic response systems which adjust function to changing conditions. These include the Frank–Starling response and the slow force response which are activated upon myocardial stretch, and the force–frequency response, which is evident upon changes in cardiac frequency. In this study we have examined the effect of myocardial stretch and changes in pacing frequency on isolated ventricular muscle preparations from the ectothermic amphibian, the axolotl (Ambystoma mexicanum). We preformed these studies at two physiologically relevant temperatures 17°C and 21°C. We found that increasing the length of the muscle increased active force via the Frank–Starling response at both temperatures, which is consistent with cardiac muscle preparations in other vertebrates. We found no evidence for the slow force response at either temperature suggesting that in axolotl, unlike mammals but similar to fish, the slow force response is not associated with the Frank–Starling response. Increasing contraction frequency caused a decrease in active force across all frequencies tested (0.5−2.0Hz)—a monophasic negative force–frequency response, independent of temperature. [Copyright &y& Elsevier]

Specialisations in excitation-contraction coupling may have played an important role in the evolution of endothermy and high cardiac performance in scombrid fishes. We examined aspects of Ca2+ handling in cardiomyocytes from Pacific bonito (Sarda chiliensis), Pacific mackerel (Scomber japonicus), yellowfin tuna (Thunnus albacares) and Pacific bluefin tuna (Thunnus orientalis). The whole-cell voltage-clamp technique was used to measure the temperature sensitivity of the L-type Ca2+ channel current (ICa), density, and steady-state and maximal sarcoplasmic reticulum (SR) Ca2+ content (SSSRload and maxSRload). Current-voltage relations, peak ICa density and charge density of ICa were greatest in mackerel and yellowfin at all temperatures tested. ICa density and kinetics were temperature sensitive in all species studied, and the magnitude of this response was not related to the thermal preference of the species. SRload was greater in atrial than in ventricular myocytes in the Pacific bluefin tuna, and in species that are more cold tolerant (bluefin tuna and mackerel). ICa and SRload were particularly small in bonito, suggesting the NaiCa2+ exchanger plays a more pivotal role in Ca2+ entry into cardiomyocytes of this species. Our comparative approach reveals that the SR of cold-tolerant scombrid fishes has a greater capacity for Ca2+ storage. This specialisation may contribute to the temperature tolerance and thermal niche expansion of the bluefin tuna and mackerel. [ABSTRACT FROM AUTHOR]

Intracellular Na+-concentration, [Na+]i modulates excitation-contraction coupling of cardiac myocytes via the Na+/Ca2+ exchanger (NCX). In cardiomyocytes from rainbow trout (Oncorhyncus mykiss), whole cell patch-clamp studies have shown that Ca2+ influx via reverse-mode NCX contributes significantly to contraction when [Na+]i is 16 mM but not 10 mM. However, physiological [Na+]i has never been measured. We recorded [Na+]i using the fluorescent indicator sodium-binding benzofuran isophthalate in freshly isolated atrial and ventricular myocytes from rainbow trout. We examined [Na+]i at rest and during increases in contraction frequency across three temperatures that span those trout experience in nature (7, 14, and 21°C). Surprisingly, we found that [Na+]i was not different between atrial and ventricular cells. Furthermore, acute temperature changes did not affect [Na+]i in resting cells. Thus, we report a resting in vivo [Na+]i of 13.4 mM for rainbow trout cardiomyocytes. [Na+]i increased from rest with increases in contraction frequency by 3.2, 4.7, and 6.5% at 0.2, 0.5, and 0.8 Hz, respectively. This corresponds to an increase of 0.4, 0.6, and 0.9 mM at 0.2, 0.5, and 0.8 Hz, respectively. Acute temperature change did not significantly affect the contraction-induced increase in [Na+]i. Our results provide the first measurement of [Na+]i in rainbow trout cardiomyocytes. This surprisingly high [Na+]i is likely to result in physiologically significant Ca2+ influx via reverse-mode NCX during excitation-contraction coupling. We calculate that this Ca2+-source will decrease with the action potential duration as temperature and contraction frequency increases. [ABSTRACT FROM AUTHOR]

The burbot (Lota Iota) is a cold stenothermic fish species whose heart is adapted to function in the cold. In this study we use whole-cell voltage-clamp techniques to characterize the electrophysiological properties of burbot ventricular myocytes and to test the hypothesis that changes in membrane currents and intracellular Ca2+ cycling associated cold-acclimation in other fish species are routine for stenothermic cold-adapted species. Experiments were performed at 4°C, which is the body temperature of burbot for most of the year, and after myocytes were acutely warmed to 11°C, which is in the upper range of temperatures experienced by burbot in nature. Results on K+ channels support our hypothesis as the relative density of K-channel conductances in the burbot heart are similar to those found for cold-acclimated cold-active fish species. IK1 conductance was small (39.2±5.4 pS pF-1 at 4°C and 71.4±1.7 pS pF-1 at 11°C) and IKr was large (199±27 pS pF-1 at 4°C and 320.3±8 pS pF-1 at 11°C) in burbot ventricular myocytes. We found high Na~-Ca2+ exchange (NCX) activity (35.9±6.3 pS pF-1 at 4°C and 58.6±8.4 pS pF-1 at 11°C between -40 and 20 mV), suggesting that it may be the primary pathway for sarcolemmal (SL) Ca2+ influx in this species. In contrast, the density (ICa, 0.81±0.13 pA pF-1 at 4°C, and 1.35±0.18 pA pF-1 at 11°C) and the charge (QCa, 0.24±0.043 pC pF-1 at 4°C and 0.21±0.034 pC pF-1 at 11°C) carried by the L-type Ca2+ current was small. Our results on sarcolemmal ion currents in burbot ventricular myocytes suggest that cold stenothermy and compensative cold-acclimation involve many of the same subcellular adaptations that culminate in enhanced excitability in the cold. [ABSTRACT FROM AUTHOR]

Ultrastructure, molecular composition and electrophysiological properties of cardiac myocytes and functional characteristics of the fish heart suggest that cycling of extracellular Ca2+ is generally more important than intracellular cycling of Ca2+ stores of the sarcoplasmic reticulum (SR) in activating contraction of fish cardiac myocytes. This is especially true for the ventricle. However, prominent species-specific differences exist in cardiac excitation-contraction coupling and in the relative roles of extracellular and intracellular Ca2+ sources among the teleostean fish. In fact, in some fish species (tunas, burbot) the SR of atrial myocytes, under certain circumstances, may act as the major source of systolic Ca2+. These interspecific differences are obviously an outcome of evolutionary adaptation to different habitats and modes of activity in these habitats. There is also substantial intraspecific variation in the SR Ca2+-release-to-SL-Ca2+ influx ratio depending on acute and chronic temperature changes. Consequently excitation-contraction coupling of the fish cardiac myocytes is not a fixed entity, but rather a highly variable and malleable process that enables fish to have an appropriate cardiac scope to exploit a diverse range of environments. [Copyright &y& Elsevier]

Abstract: Certain vertebrates, such as freshwater turtles of the genus Chrysemys and Trachemys and crucian carp (Carassius carassius), have anoxia-tolerant hearts that continue to function throughout prolonged periods of anoxia (up to many months) due to successful balancing of cellular ATP supply and demand. In the present review, we summarize the current and limited understanding of the cellular mechanisms underlying this cardiac anoxia tolerance. What emerges is that cold temperature substantially modifies cardiac electrophysiology to precondition the heart for winter anoxia. Intrinsic heart rate is slowed and density of sarcolemmal ion currents substantially modified to alter cardiac action potential (AP) characteristics. These changes depress cardiac activity and reduce the energetic costs associated with ion pumping. In contrast, anoxia per se results in limited changes to cardiac AP shape or ion current densities in turtle and crucian carp, suggesting that anoxic modifications of cardiac electrophysiology to reduce ATP demand are not extensive. Additionally, as knowledge of cellular physiology in non-mammalian vertebrates is still in its infancy, we briefly discuss the cellular defense mechanisms towards the acidosis that accompanies anoxia as well as mammalian cardiac models of hypoxia/ischemia tolerance. By examining if fundamental cellular mechanisms have been conserved during the evolution of anoxia tolerance we hope to have provided a framework for the design of future experiments investigating cardiac cellular mechanisms of anoxia survival. [Copyright &y& Elsevier]