Jaakko Haverinen

Responses of Action Potential and K+ Currents to Temperature Acclimation in Fish Hearts: Phylogeny or Thermal Preferences?

Electrical activity of the heart is assumed to be one of the key factors that set thermal tolerance limits for ectothermic vertebrates. Therefore, we hypothesized that in thermal acclimation—the duration of cardiac action potential and the repolarizing K+ currents that regulate action potential duration (APD)—the rapid component of the delayed rectifier K+ current (IKr) and the inward rectifier K+ current (IK1) are more plastic in eurythermal than in stenothermal fish species. The hypothesis was tested in six freshwater teleosts representing four different fish orders (Cadiformes, Cypriniformes, Perciformes, Salmoniformes) acclimated at +4°C (cold acclimation) or +18°C (warm acclimation). In cold acclimation, a compensatory shortening of APD occurred in all species regardless of thermal tolerances, life styles, or phylogenies of the fish, suggesting that this response is a common characteristic of the teleost heart. The strength of the response did not, however, obey simple eurythermy‐stenothermy gradation but differed among the phylogenetic groups. Salmoniformes fish showed the greatest acclimation capacity of cardiac electrical activity, whereas the weakest response appeared in the perch (Perciformes) heart. The underlying ionic mechanisms were also partly phylogeny dependent. Modification of the IKr current was almost ubiquitously involved in acclimation response of fish cardiac myocytes to temperature, while the ability to change the IK1 current under chronic thermal stress was absent or showed inverse compensation in Salmoniformes species. Thus, in Salmoniformes fish, the thermal plasticity of APD is strongly based on IKr, while other fish groups rely on both IKr and IK1.

Temperature acclimation modifies Na+ current in fish cardiac myocytes.

SUMMARY The present study was designed to test the hypothesis that temperature acclimation modifies sarcolemmal Na+ current (INa) of the fish cardiac myocytes differently depending on the animals lifestyle in the cold. Two eurythermal fish species with different physiological strategies for surviving in the cold, a cold-dormant crucian carp (Carassius carassius L.) and a cold-active rainbow trout (Oncorhynchus mykiss), were used in acclimation experiments. The INa of carp and trout were also compared with INa of a cold stenothermal burbot (Lota lota). In accordance with the hypothesis, cold-acclimation decreased the density of INa in crucian carp and increased it in rainbow trout, suggesting depression of impulse conduction in cold-acclimated carp and positive compensation of impulse propagation in cold-acclimated trout. The steady-state activation curve of trout INa was shifted by 6 mV to more negative voltages by cold acclimation, which probably lowers the stimulus threshold for action potentials and further improves cardiac excitability in the cold. In burbot myocytes, the INa density was high and the position of the steady-state activation curve on the voltage axis was even more negative than in trout or carp myocytes, suggesting that the burbot INa is adapted to maintain high excitability and conductivity in the cold. The INa of the burbot heart differed from those of carp and trout in causing four times larger charge influx per excitation, which suggests that INa may also have a significant role in cardiac excitation–contraction coupling of the burbot heart. In summary, INa of fish cardiac myocytes shows thermal plasticity that is different in several respects in cold-dormant and cold-active species and thus has a physiologically meaningful role in supporting the variable life styles and habitat conditions of each species.

Acute heat tolerance of cardiac excitation in the brown trout (Salmo trutta fario)

The upper thermal tolerance and mechanisms of heat-induced cardiac failure in the brown trout (Salmo trutta fario) was examined. The point above which ion channel function and sinoatrial contractility in vitro, and electrocardiogram (ECG) in vivo, started to fail (break point temperature, BPT) was determined by acute temperature increases. In general, electrical excitation of the heart was most sensitive to heat in the intact animal (electrocardiogram, ECG) and least sensitive in isolated cardiac myocytes (ion currents). BPTs of Ca2+ and K+ currents of cardiac myocytes were much higher (>28°C) than BPT of in vivo heart rate (23.5±0.6°C) (P<0.05). A striking exception among sarcolemmal ion conductances was the Na+ current (INa), which was the most heat-sensitive molecular function, with a BPT of 20.9±0.5°C. The low heat tolerance of INa was reflected as a low BPT for the rate of action potential upstroke in vitro (21.7±1.2°C) and the velocity of impulse transmission in vivo (21.9±2.2°C). These findings from different levels of biological organization strongly suggest that heat-dependent deterioration of Na+ channel function disturbs normal spread of electrical excitation over the heart, leading to progressive variability of cardiac rhythmicity (missed beats, bursts of fast beating), reduction of heart rate and finally cessation of the normal heartbeat. Among the cardiac ion currents INa is ‘the weakest link’ and possibly a limiting factor for upper thermal tolerance of electrical excitation in the brown trout heart. Heat sensitivity of INa may result from functional requirements for very high flux rates and fast gating kinetics of the Na+ channels, i.e. a trade-off between high catalytic activity and thermal stability.

Comparison of sarcoplasmic reticulum calcium content in atrial and ventricular myocytes of three fish species

Ryanodine (Ry) sensitivity of cardiac contraction differs between teleost species, between atrium and ventricle, and according to the thermal history of the fish. The hypothesis that variability in Ry sensitivity of contraction is due to species-specific, chamber-specific, and temperature-related differences in the sarcoplasmic reticulum (SR) Ca(2+) content, was tested by comparing steady-state (SS) and maximal (Max) Ca(2+) loads of the SR in three teleost fish, rainbow trout (Oncorhynchus mykiss), burbot (Lota lota), and crucian carp (Carassius carassius), which differ in the extent of SR contribution to excitation-contraction coupling. Fish were acclimated at 4 degrees C (cold-acclimation, CA) or 18 degrees C (warm-acclimation, WA), and SR Ca(2+) content was released by a rapid application of 10 mM caffeine to single cardiac myocytes; its amount was determined from the Na(+)-Ca(2+) exchange current at 18 degrees C. SS Ca(2+) load was larger in atrial (304-915 micromol/l) than ventricular (224-540 micromol/l) myocytes in all fish species (P < 0.05), and the same was true for Max SR Ca(2+) content: 550-1,522 micromol/l and 438-840 micromol/l for atrial and ventricular myocytes, respectively (P < 0.05). Consistent with the hypothesis, acclimation to cold increased Ca(2+) load of the cardiac SR in the burbot heart, but contrary to the hypothesis, temperature acclimation did not affect SR Ca(2+) content in rainbow trout and crucian carp hearts. Furthermore, there was an inverse relation between SR Ca(2+) content and Ry sensitivity of contraction force: the species with the smallest SR Ca(2+) content (burbot) is most sensitive to Ry. Collectively, these findings show that SR Ca(2+) content of fish cardiac myocytes is several times larger than that in mammalian cardiac SR.

Significance of Na+ current in the excitability of atrial and ventricular myocardium of the fish heart

SUMMARY The present study examines the importance of the Na+ current (INa) in the excitability of atrial and ventricular myocardium of the rainbow trout heart. Whole-cell patch-clamp under reduced sarcolemmal Na+ gradient showed that the density of INa is similar in atrial and ventricular myocytes of the trout heart, and the same result was obtained when INa was elicited by chamber-specific action potentials (AP) in normal physiological saline solution. However, the maximum rate (Vmax) of AP upstroke, measured with microelectrodes in intact trout heart, was 21% larger in atrium than ventricle, and thus in variance with the similar INa density of the two myocyte types. Furthermore, Vmax calculated from the INa was 2.1 and 3.2 times larger for atrium and ventricle, respectively, than the values obtained from the APs. The discrepancy between INa of isolated myocytes and Vmax of intact muscle is only partly explained by the inward rectifier K+ current (IK1), which overlaps INa and decreases the net depolarising current. Clear differences exist in the voltage dependence of steady-state activation and inactivation as well as in the inactivation kinetics of INa between atrial and ventricular myocytes. As a result of a more negative voltage dependence of INa activation, smaller IK1 and higher input resistance of atrial myocytes, the voltage threshold for AP generation is more negative in atrium than ventricle of the trout heart. These findings suggest that atrial muscle is more readily excitable than ventricular muscle, and this difference is partly due to the properties of the atrial INa.

Tetrodotoxin Sensitivity of the Vertebrate Cardiac Na+ Current

Evolutionary origin and physiological significance of the tetrodotoxin (TTX) resistance of the vertebrate cardiac Na+ current (INa) is still unresolved. To this end, TTX sensitivity of the cardiac INa was examined in cardiac myocytes of a cyclostome (lamprey), three teleost fishes (crucian carp, burbot and rainbow trout), a clawed frog, a snake (viper) and a bird (quail). In lamprey, teleost fishes, frog and bird the cardiac INa was highly TTX-sensitive with EC50-values between 1.4 and 6.6 nmol·L−1. In the snake heart, about 80% of the INa was TTX-resistant with EC50 value of 0.65 μmol·L−1, the rest being TTX-sensitive (EC50 = 0.5 nmol·L−1). Although TTX-resistance of the cardiac INa appears to be limited to mammals and reptiles, the presence of TTX-resistant isoform of Na+ channel in the lamprey heart suggest an early evolutionary origin of the TTX-resistance, perhaps in the common ancestor of all vertebrates.

Sinoatrial tissue of crucian carp heart has only negative contractile responses to autonomic agonists

BackgroundIn the anoxia-tolerant crucian carp (Carassius carassius) cardiac activity varies according to the seasons. To clarify the role of autonomic nervous control in modulation of cardiac activity, responses of atrial contraction and heart rate (HR) to carbacholine (CCh) and isoprenaline (Iso) were determined in fish acclimatized to winter (4°C, cold-acclimated, CA) and summer (18°C, warm-acclimated, WA) temperatures.ResultsInhibitory action of CCh was much stronger on atrial contractility than HR. CCh reduced force of atrial contraction at an order of magnitude lower concentrations (EC50 2.75-3.5·10-8 M) in comparison to its depressive effect on HR (EC50 1.23-2.02·10-7 M) (P < 0.05) without differences between winter and summer acclimatized fish. Inhibition of nitric oxide synthase with 100 μM L-NMMA did not change the response of the sinoatrial tissue to CCh. Reduction of atrial force was associated with a strong shortening of action potential (AP) duration to ~50% (48 ± 10 and 50 ± 6% for CA and WA fish, respectively) and 11% (11 ± 3 and 11 ± 2% for CA and WA fish, respectively) of the control value at 3·10-8 M and 10-7 M CCh, respectively (P < 0.05). In atrial myocytes, CCh induced an inwardly rectifying K+ current, IK,CCh, with an EC50 value of 3-4.5·10-7 M and inhibited Ca2+ current (ICa) by 28 ± 8% and 51 ± 6% at 10-7 M and 10-6 M, respectively. These currents can explain the shortening of AP. Iso did not elicit any responses in crucian carp sinoatrial preparations nor did it have any effect on atrial ICa, probably due to the saturation of the β-adrenergic cascade in the basal state.ConclusionIn the crucian carp, HR and force of atrial contraction show cardio-depressive responses to the cholinergic agonist, but do not have any responses to the β-adrenergic agonist. The scope of inhibitory regulation by CCh is increased by the high basal tone of the adenylate cyclase-cAMP cascade. Higher concentrations of CCh were required to induce IK,CCh and inhibit ICa than was needed for CChs negative inotropic effect on atrial muscle suggesting that neither IK,CCh nor ICa alone can mediate CChs actions but they might synergistically reduce AP duration and atrial force production. Autonomic responses were similar in CA winter fish and WA summer fish indicating that cardiac sensitivity to external modulation by the autonomic nervous system is not involved in seasonal acclimatization of the crucian carp heart to cold and anoxic winter conditions.

Effects of deltamethrin on excitability and contractility of the rainbow trout (Oncorhynchus mykiss) heart

Pyrethroids are extensively used for the control of pest insects and disease vectors. Pyrethroid use is regarded safe due to their selective toxicity: they are effective against insects but relatively harmless to mammals and birds. Unfortunately, pyrethroids are very toxic to fishes. The high toxicity of pyrethroids to fishes is only partly explained by slow elimination rate of toxins, suggesting that high affinity binding to their molecular targets, the Na(+) channels, is involved. This study tests the hypothesis that Na(+) channels of the fish heart are targets to a type II pyrethroid, deltamethrin (DM), and therefore pyrethroids are cardiotoxic to fishes. In ventricular myocytes of the rainbow trout (Oncorhynchus mykiss) heart DM (10(-7)-3·10(-5) M) modified Na(+) current by slowing inactivation and shifting the reversal potential of the current to the left. Maximally 31±2% of the cardiac Na(+) channels were modified by DM and the half-maximal effect occurred at the concentration of 2.1 μM. The effect of DM on trout cardiac Na(+) channels is stronger and occurs about an order of magnitude lower in concentration in comparison to the orthologous mammalian Na(+) channels. In sinoatrial preparations of the trout heart DM (10 μM) caused irregularities in rate, rhythm and force of the heartbeat indicating that DM can be arrhythmogenic for the trout heart. Consistent with this, DM (>0.1 μM) induced spontaneous action potentials in otherwise quiescent ventricular myocytes. DM (10 μM) did not affect calcium current or inward rectifier and delayed rectifier potassium currents. Collectively, these findings indicate that DM exerts cardiotoxic effects in trout, and suggest that the high sensitivity of fishes to pyrethroid toxicity might be partially due to the high affinity of fish Na(+) channels to pyrethroids.

Thermal adaptation of the crucian carp (Carassius carassius) cardiac delayed rectifier current, IKs, by homomeric assembly of Kv7.1 subunits without MinK

Ectothermic vertebrates experience acute and chronic temperature changes which affect cardiac excitability and may threaten electrical stability of the heart. Nevertheless, ectothermic hearts function over wide range of temperatures without cardiac arrhythmias, probably due to special molecular adaptations. We examine function and molecular basis of the slow delayed rectifier K(+) current (I(Ks)) in cardiac myocytes of a eurythermic fish (Carassius carassius L.). I(Ks) is an important repolarizing current that prevents excessive prolongation of cardiac action potential, but it is extremely slowly activating when expressed in typical molecular composition of the endothermic animals. Comparison of the I(Ks) of the crucian carp atrial myocytes with the currents produced by homomeric K(v)7.1 and heteromeric K(v)7.1/MinK channels in Chinese hamster ovary cells indicates that activation kinetics and pharmacological properties of the I(Ks) are similar to those of the homomeric K(v)7.1 channels. Consistently with electrophysiological properties and homomeric K(v)7.1 channel composition, atrial transcript expression of the MinK subunit is only 1.6-1.9% of the expression level of the K(v)7.1 subunit. Since activation kinetics of the homomeric K(v)7.1 channels is much faster than activation of the heteromeric K(v)7.1/MinK channels, the homomeric K(v)7.1 composition of the crucian carp cardiac I(Ks) is thermally adaptive: the slow delayed rectifier channels can open despite low body temperatures and curtail the duration of cardiac action potential in ectothermic crucian carp. We suggest that the homomeric K(v)7.1 channel assembly is an evolutionary thermal adaptation of ectothermic hearts and the heteromeric K(v)7.1/MinK channels evolved later to adapt I(Ks) to high body temperature of endotherms.

Electrical Excitation of the Heart in a Basal Vertebrate, the European River Lamprey (Lampetra fluviatilis)

Hagfishes and lampreys (order Cyclostomata) are living representatives of an ancient group of jawless vertebrates (class Agnatha). Studies on cyclostome hearts may provide insights into the evolution of the vertebrate heart and thereby increase our understanding of cardiac function in higher vertebrates, including mammals. To this end, electrical excitability of the heart in a basal vertebrate, the European river lamprey (Lampetra fluviatilis), was examined. Ion currents of cardiac myocytes, action potentials (APs) of atrial and ventricular muscle, and electrocardiogram (in vivo) were measured using the patch-clamp method, intracellular microelectrodes, and trailing wires, respectively. The characteristic features of fairly high heart rate (28.4 ± 3 beats min−1) and short AP duration (550 ± 44 and 122.1 ± 28.5 for ventricle and atrium, respectively) at low ambient temperature (5°C) are shared with cold-active teleost fishes. However, the ion current basis of the ventricular AP differs from that of other fishes. For inward currents, sodium current density (INa) is lower and calcium current density (ICa) higher than in teleost ventricles, while the kinetics of INa is slow and that of ICa is fast in comparison. Among the ventricular repolarizing currents, the delayed rectifier K+ current is smaller than in myocytes of several teleost species. Unlike mammalian hearts, ATP-sensitive K+ channels are constitutively open under normoxic conditions, thus contributing to negative resting membrane potential and repolarization of APs. Upstroke velocity of AP (5.4 ± 0.9 and 6.3 ± 0.6 V s−1 for ventricular and atrial myocytes, respectively) is slower than in teleost hearts. Excitability of the lamprey heart seems to possess both primitive and advanced characteristics. Short APs are appropriate to support brief and vigorous contractions (in common with higher vertebrates), while relatively low AP upstroke velocities enable only relatively slow propagation of contraction over the heart.