Dual effect of polyaromatic hydrocarbons on sarco(endo)plasmic reticulum calcium ATPase (SERCA) activity of a teleost fish (Oncorhynchus mykiss).

Polycyclic aromatic hydrocarbons (PAHs) are embryo- and cardiotoxic to fish that might be associated with improper intracellular Ca2+ management. Since sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a major regulator of intracellular Ca2+, the SERCA activity and the contractile properties of rainbow trout (Oncorhynchus mykiss) ventricle were measured in the presence of 3- and 4-cyclic PAHs. In unfractionated ventricular homogenates, acute exposure of SERCA to 0.1-1.0 µM phenanthrene (Phe), retene (Ret), fluoranthene (Flu), or pyrene (Pyr) resulted in concentration-dependent increase in SERCA activity, except for the Flu exposure, with maximal effects of 49.7-83 % at 1 µM. However, PAH mixture did not affect the contractile parameters of trout ventricular strips. Similarly, all PAHs, except Ret, increased the myotomal SERCA activity, but with lower effect (27.8-40.8 % at 1 µM). To investigate the putative chronic effects of PAHs on SERCA, the atp2a2a gene encoding trout cardiac SERCA was expressed in human embryonic kidney (HEK) cells. Culture of HEK cells in the presence of 0.3-1.0 µM Phe, Ret, Flu, and Pyr for 4 days suppressed SERCA expression in a concentration-dependent manner, with maximal inhibition of 49 %, 65 %, 39 % (P < 0.05), and 18 % (P > 0.05), respectively at 1 µM. Current findings indicate divergent effects of submicromolar PAH concentrations on SERCA: stimulation of SERCA activity in acute exposure and inhibition of SERCA expression in chronic exposure. The depressed expression of SERCA is likely to contribute to the embryo- and cardiotoxicity of PAHs by depressing muscle function and altering gene expression.

Cardiac Hypoxia Tolerance in Fish: From Functional Responses to Cell Signals.

Aquatic animals are increasingly challenged by O2 fluctuations as a result of global warming, as well as eutrophication processes. Teleost fish show important species-specific adaptability to O2 deprivation, moving from intolerance to a full tolerance of hypoxia and even anoxia. An example is provided by members of Cyprinidae which includes species that are amongst the most tolerant hypoxia/anoxia teleosts. Living at low water O2 requires the mandatory preservation of the cardiac function to support the metabolic and hemodynamic requirements of organ and tissues which sustain whole organism performance. A number of orchestrated events, from metabolism to behavior, converge to shape the heart response to the restricted availability of the gas, also limiting the potential damages for cells and tissues. In cyprinids, the heart is extraordinarily able to activate peculiar strategies of functional preservation. Accordingly, by using these teleosts as models of tolerance to low O2, we will synthesize and discuss literature data to describe the functional changes, and the major molecular events that allow the heart of these fish to sustain adaptability to O2 deprivation. By crossing the boundaries of basic research and environmental physiology, this information may be of interest also in a translational perspective, and in the context of conservative physiology, in which the output of the research is applicable to environmental management and decision making.

Spatial uniformity of action potentials indicates base-to-apex depolarization and repolarization of rainbow trout (Oncorhynchus mykiss) ventricle.

The spatial pattern of electrical activation is crucial for a full understanding of fish heart function. However, it remains unclear whether there is regional variation in action potential (AP) morphologies and underlying ion currents. Because the direction of depolarization and spatial differences in the durations of ventricular APs set limits to potential patterns of ventricular repolarization, we determined AP morphologies, underlying ion currents and ion channel expression in four different ventricular regions (spongy myocardium; and apex, base and middle of the compact myocardium), and correlated them with in vivo electrocardiograms (ECGs) in rainbow trout (Oncorhynchus mykiss). ECGs recorded from three leads indicated that the depolarization and repolarization of APs propagate from base to apex, and the main depolarization axis of the ventricle is between +90 and +120 deg. AP shape was uniform across the whole ventricle, and little regional differences were found in the density of repolarizing K+ currents or depolarizing Ca2+ and Na+ currents and the underlying transcripts of ion channels, providing compelling evidence for the suggested excitation pattern. The spatial uniformity of AP durations and base-to-apex propagation of activation with a relatively slow velocity of propagation indicates no special ventricular conduction pathway in the trout ventricle such as the His-Purkinje system of mammalian hearts. The sequence of repolarization is solely determined by activation time without being affected by regional differences in AP duration.

Hypoxic and Thermal Stress: Many Ways Leading to the NOS/NO System in the Fish Heart.

Teleost fish are often regarded with interest for the remarkable ability of several species to tolerate even dramatic stresses, either internal or external, as in the case of fluctuations in O2 availability and temperature regimes. These events are naturally experienced by many fish species under different time scales, but they are now exacerbated by growing environmental changes. This further challenges the intrinsic ability of animals to cope with stress. The heart is crucial for the stress response, since a proper modulation of the cardiac function allows blood perfusion to the whole organism, particularly to respiratory organs and the brain. In cardiac cells, key signalling pathways are activated for maintaining molecular equilibrium, thus improving stress tolerance. In fish, the nitric oxide synthase (NOS)/nitric oxide (NO) system is fundamental for modulating the basal cardiac performance and is involved in the control of many adaptive responses to stress, including those related to variations in O2 and thermal regimes. In this review, we aim to illustrate, by integrating the classic and novel literature, the current knowledge on the NOS/NO system as a crucial component of the cardiac molecular mechanisms that sustain stress tolerance and adaptation, thus providing some species, such as tolerant cyprinids, with a high resistance to stress.

Cardiac Toxicity of Cadmium Involves Complex Interactions Among Multiple Ion Currents in Rainbow Trout (Oncorhynchus mykiss) Ventricular Myocytes.

Cadmium (Cd2+ ) is cardiotoxic to fish, but its effect on the electrical excitability of cardiac myocytes is largely unknown. To this end, we used the whole-cell patch-clamp method to investigate the effects of Cd2+ on ventricular action potentials (APs) and major ion currents in rainbow trout (Oncorhynchus mykiss) ventricular myocytes. Trout were acclimated to +4 °C, and APs were measured at the acclimated temperature and elevated temperature (+18 °C). Cd2+ (10, 20, and 100 µM) altered the shape of the ventricular AP in a complex manner. The early plateau fell to less positive membrane voltages, and the total duration of AP prolonged. These effects were obvious at both +4 °C and +18 °C. The depression of the early plateau is due to the strong Cd2+ -induced inhibition of the L-type calcium (Ca2+ ) current (ICaL ), whereas the prolongation of the AP is an indirect consequence of the ICaL inhibition: at low voltages of the early plateau, the delayed rectifier potassium (K+ ) current (IKr ) remains small, delaying repolarization of AP. Cd2+ reduced the density and slowed the kinetics of the Na+ current (INa ) but left the inward rectifier K+ current (IK1 ) intact. These altered cellular and molecular functions can explain several Cd2+ -induced changes in impulse conduction of the fish heart, for example, slowed propagation of the AP in atrial and ventricular myocardia (inhibition of INa ), delayed relaxation of the ventricle (prolongation of ventricular AP duration), bradycardia, and atrioventricular block (inhibition of ICaL ). These findings indicate that the cardiotoxicity of Cd2+ in fish involves multiple ion currents that are directly and indirectly altered by Cd2+ . Through these mechanisms, Cd2+ may trigger cardiac arrhythmias and impair myocardial contraction. Elevated temperature (+18 °C) slightly increases Cd2+ toxicity in trout ventricular myocytes. Environ Toxicol Chem 2021;40:2874-2885. © 2021 SETAC.

Ionic basis of atrioventricular conduction: ion channel expression and sarcolemmal ion currents of the atrioventricular canal of the rainbow trout (Oncorhynchus mykiss) heart.

Atrioventricular (AV) nodal tissue synchronizes activities of atria and ventricles of the vertebrate heart and is also a potential site of cardiac arrhythmia, e.g., under acute heat stress. Since ion channel composition and ion currents of the fish AV canal have not been previously studied, we measured major cation currents and transcript expression of ion channels in rainbow trout (Oncorhynchus mykiss) AV tissue. Both ion current densities and expression of ion channel transcripts indicate that the fish AV canal has a characteristic electrophysiological phenotype that differs from those of sinoatrial tissue, atrium and ventricle. Two types of cardiomyocytes were distinguished electrophysiologically in trout AV nodal tissue: the one (transitional cell) is functionally intermediate between working atrial/ventricular myocytes and the other (AV nodal cell) has a less negative resting membrane potential than atrial and ventricular myocytes and is a more similar to the sinoatrial nodal cells in ion channel composition. The AV nodal cells are characterized by a small or non-existent inward rectifier potassium current (IK1), low density of fast sodium current (INa) and relatively high expression of T-type calcium channels (CACNA3.1). Pacemaker channel (HCN4 and HCN2) transcripts were expressed in the AV nodal tissue but If current was not found in enzymatically isolated nodal myocytes. The electrophysiological properties of the rainbow trout nodal cells are appropriate for a slow rate of action potential conduction (small INa) and a moderate propensity for pacemaking activity (absence of IK1).

Effects of acute warming on cardiac and myotomal sarco(endo)plasmic reticulum ATPase (SERCA) of thermally acclimated brown trout (Salmo trutta).

At high temperatures, ventricular beating rate collapses and depresses cardiac output in fish. The role of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) in thermal tolerance of ventricular function was examined in brown trout (Salmo trutta) by measuring heart SERCA and comparing it to that of the dorsolateral myotomal muscle. Activity of SERCA was measured from crude homogenates of cold-acclimated (+ 3 °C, c.a.) and warm-acclimated (+ 13 °C, w.a.) brown trout as cyclopiazonic acid (20 µM) sensitive Ca2+-ATPase between + 3 and + 33 °C. Activity of the heart SERCA was significantly higher in c.a. than w.a. trout and increased strongly between + 3 and + 23 °C with linear Arrhenius plots but started to plateau between + 23 and + 33 °C in both acclimation groups. The rate of thermal inactivation of the heart SERCA at + 35 °C was similar in c.a. and w.a. fish. Activity of the muscle SERCA was less temperature dependent and more heat resistant than that of the heart SERCA and showed linear Arrhenius plots between + 3 and + 33 °C in both c.a. and w.a. fish. SERCA activity of the c.a. muscle was slightly higher than that of w.a. muscle. The rate of thermal inactivation at + 40 °C was similar for both c.a. and w.a. muscle SERCA at + 40 °C. Although the heart SERCA is more sensitive to high temperatures than the muscle SERCA, it is unlikely to be a limiting factor for heart rate, because its heat tolerance, unlike that of the ventricular beating rate, was not changed by temperature acclimation.

Reduced ventricular excitability causes atrioventricular block and depression of heart rate in fish at critically high temperatures.

At critically high temperature, cardiac output in fish collapses as a result of depression of heart rate (bradycardia). However, the cause of bradycardia remains unresolved. To investigate this, rainbow trout (Oncorhynchus mykiss; acclimated at 12°C) were exposed to acute warming while electrocardiograms were recorded. From 12°C to 25.3°C, electrical excitation between different parts of the heart was coordinated, but above 25.3°C, atrial and ventricular beating rates became partly dissociated because of 2:1 atrioventricular (AV) block. With further warming, atrial rate increased to a peak value of 188±22 beats min-1 at 27°C, whereas the ventricle rate peaked at 124±10 beats min-1 at 25.3°C and thereafter dropped to 111±15 beats min-1 at 27°C. In single ventricular myocytes, warming from 12°C to 25°C attenuated electrical excitability as evidenced by increases in rheobase current and the size of critical depolarization required to trigger action potential. Depression of excitability was caused by temperature-induced decrease in input resistance (sarcolemmal K+ leak via the outward IK1 current) of resting myocytes and decrease in inward charge transfer by the Na+ current (INa) of active myocytes. Collectively, these findings show that at critically high temperatures AV block causes ventricular bradycardia owing to the increased excitation threshold of the ventricle, which is due to changes in the passive (resting ion leak) and active (inward charge movement) electrical properties of ventricular myocytes. The sequence of events from the level of ion channels to cardiac function in vivo provides a mechanistic explanation for the depression of cardiac output in fish at critically high temperature.

Thermal tolerance and fish heart integrity: fatty acids profiles as predictors of species resilience.

The cardiovascular system is a major limiting system in thermal adaptation, but the exact physiological mechanisms underlying responses to thermal stress are still not completely understood. Recent studies have uncovered the possible role of reactive oxygen species production rates of heart mitochondria in determining species' upper thermal limits. The present study examines the relationship between individual response to a thermal challenge test (CTmax), susceptibility to peroxidation of membrane lipids, heart fatty acid profiles and cardiac antioxidant enzyme activities in two salmonid species from different thermal habitats (Salvelinus alpinus, Salvelinus fontinalis) and their hybrids. The susceptibility to peroxidation of membranes in the heart was negatively correlated with individual thermal tolerance. The same relationship was found for arachidonic and eicosapentaenoic acid. Total H2O2 buffering activity of the heart muscle was higher for the group with high thermal resistance. These findings underline a potential general causative relationship between sensitivity to oxidative stress, specific fatty acids, antioxidant activity in the cardiac muscle and thermal tolerance in fish and likely other ectotherms. Heart fatty acid profile could be indicative of species resilience to global change, and more importantly the plasticity of this trait could predict the adaptability of fish species or populations to changes in environmental temperature.

Transcript expression of inward rectifier potassium channels of Kir2 subfamily in Arctic marine and freshwater fish species.

Inward rectifier K+ (Kir2) channels are critical for electrical excitability of cardiac myocytes. Here, we examine expression of Kir2 channels in the heart of three Gadiformes species, polar cod (Boreogadus saida) and navaga (Eleginus nawaga) of the Arctic Ocean and burbot (Lota lota) of the temperate lakes to find out the role of Kir2 channels in cardiac adaptation to cold. Five boreal freshwater species: brown trout (Salmo trutta fario), arctic char (Salvelinus alpinus), roach (Rutilus rutilus), perch (Perca fluviatilis) and pike (Esox lucius), and zebrafish (Danio rerio), were included for comparison. Transcript expression of genes encoding Kir2.1a, - 2.1b, - 2.2a, - 2.2b and - 2.4 was studied from atrium and ventricle of thermally acclimated or acclimatized fish by quantitative PCR. Kir2 composition in the polar cod was more diverse than in other species in that all Kir2 isoforms were relatively highly expressed. Kir2 composition of navaga and burbot differed from that of the polar cod as well as from those of other species. The relative expression of Kir2.2 transcripts, especially Kir2.2b, was higher in both atrium and ventricle of navaga and burbot (56-89% from the total Kir2 pool) than in other species (0.1-11%). Thermal acclimation induced only small changes in cardiac Kir2 transcript expression in Gadiformes species. However, Kir2.2b transcripts were upregulated in cold-acclimated navaga and burbot hearts. All in all, the cardiac Kir2 composition seems to be dependent on both phylogenetic position and thermal preference of the fish.

Electrical excitability of roach ( Rutilus rutilus) ventricular myocytes: effects of extracellular K+, temperature, and pacing frequency.

Exercise, capture, and handling stress in fish can elevate extracellular K+ concentration ([K+]o) with potential impact on heart function in a temperature- and frequency-dependent manner. To this end, the effects of [K+]o on the excitability of ventricular myocytes of winter-acclimatized roach ( Rutilus rutilus) (4 ± 0.5°C) were examined at different test temperatures and varying pacing rates. Frequencies corresponding to in vivo heart rates at 4°C (0.37 Hz), 14°C (1.16 Hz), and 24°C (1.96 Hz) had no significant effect on the excitability of ventricular myocytes. Acute increase of temperature from 4 to 14°C did not affect excitability, but a further rise to 24 markedly decreased excitability: stimulus current and critical depolarization needed to elicit an action potential (AP) were ~25 and 14% higher, respectively, at 24°C than at 4°C and 14°C ( P < 0.05). This depression could be due to temperature-related mismatch between inward Na+ and outward K+ currents. In contrast, an increase of [K+]o from 3 to 5.4 or 8 mM at 24°C reduced the stimulus current needed to trigger AP. However, other aspects of excitability were strongly depressed by high [K+]o: maximum rate of AP upstroke and AP duration were drastically (89 and 50%, respectively) reduced at 8 mM [K+]o in comparison with 3 mM ( P < 0.05). As an extreme case, some myocytes completely failed to elicit all-or-none AP at 8 mM [K+]o at 24°C. Also, amplitude and overshoot of AP were reduced by elevation of [K+]o ( P < 0.05). Although high [K+]o antagonizes the negative effects of high temperature on excitation threshold, the precipitous depression of the rate of AP upstroke and complete loss of excitability in some myocytes suggest that the combination of high temperature and high [K+]o will severely impair ventricular excitability in roach.

Effects of seasonal acclimatization on thermal tolerance of inward currents in roach (Rutilus rutilus) cardiac myocytes.

To test the hypothesis of temperature-dependent deterioration of electrical excitability (TDEE) (Vornanen, J Exp Biol 219:1941-1952, 2016), the role of sodium (I Na) and calcium (I Ca) currents in heat tolerance of cardiac excitability was examined in a eurythermic fish, the roach (Rutilus rutilus). Densities of cardiac I Ca and I Na and their acute heat tolerance were measured in winter-acclimatized (WiR) and summer-acclimatized (SuR) fish maintained in the laboratory at 4 ± 1 and 18 ± 1 °C, respectively. A robust L-type Ca2+ current (I CaL), but no T-type Ca2+ current, was present in roach atrial and ventricular myocytes. Peak density of I CaL was smaller in atrial (- 1.97 ± 0.14 and - 1.75 ± 0.19 pA/pF for WiR and SuR, respectively) than ventricular myocytes (- 4.00 ± 0.59 and - 2.88 ± 0.47 pA/pF for WiR and SuR, respectively) (p < 0.05), but current density and heat tolerance of I CaL did not change between seasons in either cell type. In contrast to I Ca, marked differences appeared in I Na between WiR and SuR. I Na density was 38% higher in WiR than SuR atrial myocytes (- 80.03 ± 5.92 vs. - 49.77 ± 4.72 pA/pF; p < 0.05) and 48% higher in WiR than SuR ventricular myocytes (- 39.25 ± 3.06 vs. - 20.03 ± 1.79 pA/pF; p < 0.05). The winter increase in I Na density was associated with 55% (1.70 ± 0.27 vs. 0.77 ± 0.12) and 54% (1.08 ± 0.19 vs. 0.50 ± 0.10) up-regulation of the total Na+ channel (scn4 + scn5 + scn8) transcripts in atrium and ventricle, respectively (p < 0.05). Heat tolerance of atrial I Na was lower in WiR with a breakpoint temperature of 20.3 ± 1.2 °C than in SuR (23.8 ± 0.7 °C) (p < 0.05). The response of I Na to seasonal acclimatization conforms to the TDEE hypothesis. The lower heat tolerance of I Na in WiR is consistent with the lower heat tolerance of in vivo heart rate in WiR in comparison to SuR, but the match is not quantitatively perfect, suggesting that other factors in addition to I Na may be involved.

Taurine protects cardiac contractility in killifish, Fundulus heteroclitus, by enhancing sarcoplasmic reticular Ca2+ cycling.

Intracellular taurine is abundant in many animals and it influences an array of physiological processes, including osmoregulation, metabolism, and cardiac contractility. Taurine is an important osmolyte in teleost hearts, but its role in stress tolerance, cardiac metabolism, and contractility has not been assessed. The goal of this study was to determine if ventricular taurine concentration changes in response to environmental stress and to characterize its influence on contractility. Cardiac taurine concentrations varied in killifish (Fundulus heteroclitus) but were generally maintained following acute environmental challenges. In isometrically contracting ventricular strips, supplemental taurine (40 mmol L-1) protected peak tension development (F max) at high stimulation frequencies, an effect abolished by treatment with ryanodine, a blocker of sarcoplasmic reticulum Ca2+ release. In the presence of ryanodine, taurine-treated preparations were also better able to maintain F max at supraphysiological extracellular Ca2+ levels, but a prior anoxia exposure abolished this effect. Taurine had no impact on basal F max during or after anoxia, but it provided additive protection to high-frequency contractility post-anoxia. Tissue oxygen consumption and extracellular glucose utilization were unaffected by taurine in non-contracting preparations, indicating that it does not impact energy metabolism. Overall, the results suggest that cardiac taurine levels are well maintained on acute time scales in this highly stress-tolerant species. Supplemental taurine has no effect on aerobic metabolism in vitro, but it significantly improved cardiac contractility in a manner dependent upon sarcoplasmic reticulum Ca2+ cycling. The data indicate that taurine likely plays an important role in the regulation of cardiac performance in teleosts.

Maximum heart rate in brown trout (Salmo trutta fario) is not limited by firing rate of pacemaker cells.

Temperature-induced changes in cardiac output (Q̇) in fish are largely dependent on thermal modulation of heart rate (fH), and at high temperatures Q̇ collapses due to heat-dependent depression of fH This study tests the hypothesis that firing rate of sinoatrial pacemaker cells sets the upper thermal limit of fH in vivo. To this end, temperature dependence of action potential (AP) frequency of enzymatically isolated pacemaker cells (pacemaker rate, fPM), spontaneous beating rate of isolated sinoatrial preparations (fSA), and in vivo fH of the cold-acclimated (4°C) brown trout (Salmo trutta fario) were compared under acute thermal challenges. With rising temperature, fPM steadily increased because of the acceleration of diastolic depolarization and shortening of AP duration up to the break point temperature (TBP) of 24.0 ± 0.37°C, at which point the electrical activity abruptly ceased. The maximum fPM at TBP was much higher [193 ± 21.0 beats per minute (bpm)] than the peak fSA (94.3 ± 6.0 bpm at 24.1°C) or peak fH (76.7 ± 2.4 at 15.7 ± 0.82°C) (P < 0.05). These findings strongly suggest that the frequency generator of the sinoatrial pacemaker cells does not limit fH at high temperatures in the brown trout in vivo.

Effects of seasonal acclimatization on action potentials and sarcolemmal K+ currents in roach (Rutilus rutilus) cardiac myocytes.

Temperature sensitivity of electrical excitability is a potential limiting factor for high temperature tolerance of ectotherms. The present study examines whether heat resistance of electrical excitability of cardiac myocytes is modified by seasonal thermal acclimatization in roach (Rutilus rutilus), a eurythermal teleost species. To this end, temperature dependencies of ventricular action potentials (APs), and atrial and ventricular K+ currents were measured from winter-acclimatized (WiR) and summer-acclimatized (SuR) roach. Under patch-clamp recording conditions, ventricular APs could be triggered over a wide range of temperatures (4-43°C) with prominent changes in resting membrane potential (RMP), AP duration and amplitude. In general, APs of SuR were slightly more tolerant to high temperatures than those of WiR, e.g. the break point temperature (TBP) of RMP was 37.6±0.4°C in WiR and 41±1°C in SuR (p<0.05). Of the two major cardiac K+ currents, the inward rectifier K+ current (IK1) was particularly heat resistant in both SuR (TBP 39.4±0.4°C) and WiR (TBP 40.0±0.4°C) ventricular myocytes. The delayed rectifier K+ current (IKr) was not as heat resistant as IK1. Surprisingly, IKr of WiR tolerated heat better (TBP 31.9±0.8°C) than IKr of SuR (TBP 24.1±0.5°C) (p<0.05). IKr (Erg2) channel transcripts of both atrial and ventricular myocytes were up-regulated in WiR. IK1 (Kir2) channel transcripts were not affected by seasonal acclimatization, although ventricular IK1 current was up-regulated in summer. Collectively, these findings show that thermal tolerance limits of K+ currents in isolated myocytes between seasonally acclimatized roach are much less pronounced than the heat sensitivity of ECG variables in intact fish.

Effects of prolonged anoxia on electrical activity of the heart in crucian carp (Carassius carassius).

The effects of sustained anoxia on cardiac electrical excitability were examined in the anoxia-tolerant crucian carp (Carassius carassius). The electrocardiogram (ECG) and expression of excitation-contraction coupling genes were studied in fish acclimatised to normoxia in summer (+18°C) or winter (+2°C), and in winter fish after 1, 3 and 6 weeks of anoxia. Anoxia induced a sustained bradycardia from a heart rate of 10.3±0.77 beats min-1 to 4.1±0.29 beats min-1 (P<0.05) after 5 weeks, and heart rate slowly recovered to control levels when oxygen was restored. Heart rate variability greatly increased under anoxia, and completely recovered under re-oxygenation. The RT interval increased from 2.8±0.34 s in normoxia to 5.8±0.44 s under anoxia (P<0.05), which reflects a doubling of the ventricular action potential (AP) duration. Acclimatisation to winter induced extensive changes in gene expression relative to summer-acclimatised fish, including depression in those genes coding for the sarcoplasmic reticulum calcium pump (Serca2a_q2) and ATP-sensitive K+ channels (Kir6.2) (P<0.05). Genes of delayed rectifier K+ (kcnh6) and Ca2+ channels (cacna1c) were up-regulated in winter fish (P<0.05). In contrast, the additional challenge of anoxia caused only minor changes in gene expression, e.g. depressed expression of Kir2.2b K+ channel gene (kcnj12b), whereas expression of Ca2+ (cacna1a, cacna1c and cacna1g) and Na+ channel genes (scn4a and scn5a) was not affected. These data suggest that low temperature pre-conditions the crucian carp heart for winter anoxia, whereas sustained anoxic bradycardia and prolongation of AP duration are directly induced by oxygen shortage without major changes in gene expression.

Molecular basis and drug sensitivity of the delayed rectifier (IKr) in the fish heart.

Fishes are increasingly used as models for human cardiac diseases, creating a need for a better understanding of the molecular basis of fish cardiac ion currents. To this end we cloned KCNH6 channel of the crucian carp (Carassius carassius) that produces the rapid component of the delayed rectifier K(+) current (IKr), the main repolarising current of the fish heart. KCNH6 (ccErg2) was the main isoform of the Kv11 potassium channel family with relative transcript levels of 98.9% and 99.6% in crucian carp atrium and ventricle, respectively. KCNH2 (ccErg1), an orthologue to human cardiac Erg (Herg) channel, was only slightly expressed in the crucian carp heart. The native atrial IKr and the cloned ccErg2 were inhibited by similar concentrations of verapamil, terfenadine and KB-R7943 (P>0.05), while the atrial IKr was about an order of magnitude more sensitive to E-4031 than ccErg2 (P<0.05) suggesting that some accessory ß-subunits may be involved. Sensitivity of the crucian carp atrial IKr to E-4031, terfenadine and KB-R7943 was similar to what has been reported for the Herg channel. In contrast, the sensitivity of the crucian carp IKr to verapamil was approximately 30 times higher than the previously reported values for the Herg current. In conclusion, the cardiac IKr is produced by non-orthologous gene products in fish (Erg2) and mammalian hearts (Erg1) and some marked differences exist in drug sensitivity between fish and mammalian Erg1/2 which need to be taken into account when using fish heart as a model for human heart.

Electrical excitability of the heart in a Chondrostei fish, the Siberian sturgeon (Acipenser baerii).

Sturgeon (family Acipenseridae) are regarded as living fossils due to their ancient origin and exceptionally slow evolution. To extend our knowledge of fish cardiac excitability to a Chondrostei fish, we examined electrophysiological phenotype of the Siberian sturgeon (Acipenser baerii) heart with recordings of epicardial ECG, intracellular action potentials (APs), and sarcolemmal ion currents. Epicardial ECG of A. baerii had the typical waveform of the vertebrate ECG with Q-T interval (average duration of ventricular AP) of 650±30 ms and an intrinsic heart rate of 45.5±5 beats min(-1) at 20°C. Similar to other fish species, atrial AP was shorter in duration (402±33 ms) than ventricular AP (585±40) (P<0.05) at 20°C. Densities of atrial and ventricular Na+ currents were similar (-47.6±4.5 and -53.2±5.1 pA/pF, respectively) and close to the typical values of teleost hearts. Two major K+ currents, the inward rectifier K+ current (IK1), and the delayed rectifier K+ current (IKr) were found under basal conditions in sturgeon cardiomyocytes. The atrial IKr (3.3±0.2 pA/pF) was about twice as large as the ventricular IKr (1.3±0.4 pA/pF) (P<0.05) conforming to the typical pattern of teleost cardiac IKr. Divergent from other fishes, the ventricular IK1 was remarkably small (-2.5±0.07 pA/pF) and not different from that of the atrial myocytes (-1.9±0.06 pA/pF) (P>0.05). Two ligand-gated K+ currents were also found: ACh-activated inward rectifier (IKACh) was present only in atrial cells, while ATP-sensitive K+ current (IKATP) was activated by a mitochondrial blocker, CCCP, in both atrial and ventricular cells. The most striking difference to other fishes appeared in Ca2+ currents (ICa). In atrial myocytes, ICa was predominated by nickel-sensitive and nifedipine-resistant T-type ICa, while ventricular myocytes had mainly nifedipine-sensitive and nickel-resistant L-type ICa. ICaT/ICaL ratio of the sturgeon atrial myocytes (2.42) is the highest value ever measured for a vertebrate species. In ventricular myocytes, ICaT/ICaL ratio was 0.09. With the exception of the large atrial ICaT and small ventricular IK1, electrical excitability of A. baerii heart is similar to that of teleost hearts.

Inhibition of the cardiac ATP-dependent potassium current by KB-R7943.

KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea) was developed as a specific inhibitor of the sarcolemmal sodium-calcium exchanger (NCX) with potential experimental and therapeutic use. However, in cardiomyocytes KB-R7943 also effectively blocks several K(+) currents including the delayed rectifier, IKr, and background inward rectifier, IK1. In the present study we analyze the effects of KB-R7943 on the ATP-dependent potassium current (IKATP) recorded by whole-cell patch-clamp in ventricular cardiomyocytes from a mammal (mouse) and a fish (crucian carp). IKATP was induced by external application of a mitochondrial uncoupler CCCP (3×10(-7) M) and internal perfusion of the cell with ATP-free pipette solution. A weakly inwardly rectifying current with a large outward component, recorded in the presence of CCCP, was blocked with 10(-5) M glibenclamide by 56.1±4.6% and 56.9±3.6% in crucian carp and mouse ventricular myocytes, respectively. In fish cardiomyocytes IKATP was blocked by KB-R7943 with an IC50 value of 3.14×10(-7) M, while in mammalian cells IC50 was 2.8×10(-6) M (P<0.05). 10(-5) M KB-R7943 inhibited CCCP-induced IKATP by 99.9±0.13% and 97.5±1.2% in crucian carp and mouse ventricular myocytes, respectively. In crucian carp the IKATP is about an order of magnitude more sensitive to KB-R7943 than the background IK1, but in mammals IKATP and IK1 are almost equally sensitive to KB-R7943. Therefore, the ability of KB-R7943 to block IKATP should be taken into account together with INCX inhibition when investigating possible cardioprotective effects of this compound.

A new potassium ion current induced by stimulation of M2 cholinoreceptors in fish atrial myocytes.

A novel potassium ion current induced by muscarinic stimulation (IKACh2) is characterized in atrial cardiomyocytes of teleost fishes (crucian carp, Carassius carassius; rainbow trout, Oncorhynchus mykiss) by means of the whole-cell patch-clamp technique. The current is elicited in atrial, but not ventricular, cells by application of carbamylcholine (CCh) in moderate to high concentrations (10(-7)-10(-4) mol l(-1)). It can be distinguished from the classic IKACh, activated by the ßγ-subunit of the Gi-protein, because of its low sensitivity to Ba(2+) ions and distinct current-voltage relationship with a very small inward current component. Ni(2+) ions (5 mmol l(-1)) and KB-R7943 (10(-5) mol l(-1)), non-selective blockers of the sodium-calcium exchange current (INCX), strongly reduced and completely abolished, respectively, the IKACh2. Therefore, IKACh2 was initially regarded as a CCh-induced outward component of the INCX. However, the current is not affected by either exclusion of intracellular Na(+) or extracellular Ca(2+), but is completely abolished by intracellular perfusion with K(+)-free solution. Atropine (10(-6) mol l(-1)), a non-selective muscarinic blocker, completely eliminated the IKACh2. A selective antagonist of M2 cholinoreceptors, AF-DX 116 (2×10(-7) mol l(-1)) and an M3 antagonist, 4-DAMP (10(-9) mol l(-1)), decreased IKACh2 by 84.4% and 16.6%, respectively. Pertussis toxin, which irreversibly inhibits Gi-protein coupled to M2 receptors, reduced the current by 95%, when applied into the pipette solution. It is concluded that IKACh2, induced by stimulation of M2 cholinoceptors and subsequent Gi-protein activation, represents a new molecular target for the cardiac parasympathetic innervation.