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

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

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.

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

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

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 (0.5−2.0 Hz)—a monophasic negative force–frequency response, independent of temperature.

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

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

This study quantified the cell surface beta-adrenoreceptor density and ligand binding affinity in the ventricular tissue of seven teleost species; skipjack tuna (Katsowonus pelamis), yellowfin tuna (Thunnus albacares), Pacific mackerel (Scomber japonicus), mahimahi (dolphin fish; Coryphaena hippurus), sockeye salmon (Oncorhynchus nerka), rainbow trout (Oncorhynchus mykiss) and an Antarctic nototheniid (Trematomus bernacchii). Beta-Adrenoreceptor density varied by almost fourfold among these species, being highest for the athletic fish: sockeye salmon among the salmonids and skipjack tuna among the scombrids. Beta-Adrenoreceptor density was lowest for the Antarctic icefish. Beta-Adrenoreceptor binding affinity varied by almost threefold. We conclude that there is a significant species-specific variability in myocardial beta-adrenoreceptor density and binding affinity and these interspecific differences cannot be attributed to temperature even though intraspecifically cold temperature can stimulate an increase in myocardial beta-adrenoreceptor density. Instead, we suggest that interspecifically myocardial beta-adrenoreceptor density is highest in fish that inhabit tropical water.

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

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

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

To investigate the cellular mechanisms underlying the negative force-frequency relationship (FFR) in the ventricle of the varanid lizard, Varanus exanthematicus , we measured sarcomere and cell shortening, intracellular Ca2+([Ca2+]i), action potentials (APs), and K+currents in isolated ventricular myocytes. Experiments were conducted between 0.2 and 1.0 Hz, which spans the physiological range of in vivo heart rates at 20–22°C for this species. As stimulation frequency increased, diastolic length, percent change in sarcomere length, and relaxation time all decreased significantly. Shortening velocity was unaffected. These changes corresponded to a faster rate of rise of [Ca2+]i, a decrease in [Ca2+]itransient amplitude, and a seven-fold increase in diastolic [Ca2+]i. The time constant for the decay of the Ca2+transient (τ) decreased at higher frequencies, indicating a frequency-dependent acceleration of relaxation (FDAR) but then reached a plateau at moderate frequencies and did not change above 0.5 Hz. The rate of rise of the AP was unaffected, but the AP duration (APD) decreased with increasing frequency. Peak depolarization tended to decrease, but it was only significant at 1.0 Hz. The decrease in APD was not due to frequency-dependent changes in the delayed inward rectifier ( IKr) or the transient outward ( Ito) current, as neither appeared to be present in varanid ventricular myocytes. Our results suggest that a negative FFR relationship in varanid lizard ventricle is caused by decreased amplitude of the Ca2+transient coupled with an increase in diastolic Ca2+, which leads to incomplete relaxation between beats at high frequencies. This coincides with shortened APD at higher frequencies.

In cardiomyocytes, ryanodine receptors (RYRs) mediate Ca(2+)-induced Ca(2+)-release (CICR) from the sarcoplasmic reticulum (SR) during excitation-contraction (e-c) coupling. In rainbow trout heart, the relative importance of CICR increases with cold-acclimation. Thus, the aim of this study was to investigate the effect of temperature acclimation (4, 11 and 18 degrees C) on RYR intracellular localization and expression density. We used immunocytochemistry to assess intracellular localization in ventricular myocytes and Western blotting to assess RYR expression in both atrial and ventricular tissue. In ventricular myocytes, RYRs were localized peripherally in transverse bands aligning with sarcomeric m-lines and centrally around mitochondria and the nucleus. Localization did not change with temperature acclimation. RYR expression was also unaffected by temperature acclimation. The localization of RYRs at the m-line is similar to neonatal mammalian cardiomyocytes. We suggest this positioning is indicative of myocytes which rely predominantly on transsarcolemmal Ca(2+)-influx, rather than CICR, during e-c coupling.

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

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

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

The purpose of this study was to investigate how the endogenous catecholamine adrenaline protects sarcolemmal Ca(2+) flux through the L-type Ca(2+) channel (I(Ca)) during acute exposure to cold in the fish heart. We examined the response of I(Ca) to adrenergic stimulation at three temperatures (7 degrees, 14 degrees, and 21 degrees C) in atrial myocytes isolated from rainbow trout acclimated to 14 degrees C. We found that I(Ca) amplitude varied directly with test temperature and was increased by adrenergic stimulation (AD; 5 nM and 1 microM) at all temperatures. However, I(Ca) was significantly more sensitive to adrenergic stimulation at the coldest test temperature. In fact, at 7 degrees C in the absence of AD, I(Ca) was extremely low. The addition of 1 microM AD increased peak I(Ca) 7.2-fold at 7 degrees C, 2.6-fold at 14 degrees C, and 1.6-fold at 21 degrees C and ameliorated the temperature-dependent difference in Ca(2+) influx across the cell membrane. We suggest that this increased adrenergic sensitivity is a critical compensatory mechanism that allows the rainbow trout heart to maintain contractility during acute exposure to cold temperatures. In particular, the tonic level of adrenergic stimulation provided by circulating plasma catecholamines (i.e., in the nM concentration range) may be crucial for effective excitation-contraction coupling in the cold cardiomyocyte.

The zebrafish is widely used for human related disease studies. Surprisingly, there is no information about the electrical activity of single myocytes freshly isolated from adult zebrafish ventricle. In this study, we present an enzymatic method to isolate ventricular myocytes from zebrafish heart that yield a large number of calcium tolerant cells. Ventricular myocytes from zebrafish were imaged using light and confocal microscopy. Myocytes were mostly rod shaped and responded by vigorous contraction to field electrical stimulation. Whole cell configuration of the patch clamp technique was used to record electrophysiological characteristics of myocytes. Action potentials present a long duration and a plateau phase and action potential duration decreases when increasing stimulation frequency (as observed in larger mammals). Together these results indicate that zebrafish is a species ideally suited for investigation of ion channels related mutation screening of cardiac alteration important in human.