Temperature and external K⁺ dependence of electrical excitation in ventricular myocytes of cod-like fishes Free

Electrical excitability (EE) can be defined as the ease with which propagating (all-or-none) action potentials (APs) are triggered in the cell membrane of nerve and muscle cells by a complex interaction of ion currents (Boyett and Jewell, 1980). This narrow definition of EE considers only the stimulus intensity that is needed to trigger an all-or-none AP. In a wider sense, EE could include – in addition to threshold events needed to trigger AP – quantitative changes in AP characteristics such as duration, amplitude and rate of AP upstroke. EE is modulated by, among others, heart rate, temperature and concentration of external K⁺ ([K⁺]_o) (Dominguez and Fozzard, 1970; Buchanan et al., 1985; Shaw and Rudy, 1997; Hund and Rudy, 2000; Badr et al., 2018), which affect the conduction velocity and refractoriness of APs.

The vertebrate heart is a syncytium of several types of cardiac muscle cells (nodal, atrial and ventricular), all of which generate propagating APs (Irisawa, 1978; Vornanen, 2017). In the fish heart, impulse propagation starts from the primary pacemaker centre at the base of the sinoatrial valve and propagates from there through the whole heart, triggering the sequential activation of the atrium and the ventricle (Saito, 1969; Sedmera et al., 2003; Haverinen and Vornanen, 2007; Tessadori et al., 2012). The sinoatrial pacemaker determines the normal rate and rhythm of the heartbeat under various physiological conditions and environmental stresses including acute and chronic temperature changes (Vornanen, 2017).

Heart rate is modulated in a predictable and regular manner by acute temperature changes within the thermal tolerance window, the width of which is characteristic for each fish species (von Skramlik, 1935; Farrell, 1984). However, recent findings show that when temperature approaches the upper critical limit of the fish, the normal heartbeat pattern is distorted and the heart may eventually completely cease beating (Aho and Vornanen, 2001; Casselman et al., 2012; Anttila et al., 2013; Vornanen et al., 2014; Badr et al., 2016). When fish are exposed to temperatures approaching or exceeding their upper thermal tolerance limit, cardiac disturbances appear in the electrocardiogram (ECG) of intact fish as a slower increase in heart rate, occasional missing of QRS complexes (failure of ventricular excitation due to conduction block of the atrioventricular node or the ventricular muscle), overall slowing of heart rate (bradycardia) and complete cessation of heartbeat (asystole) (Vornanen, 2017). However, the rate generator mechanism of pacemaker cells seems to be resistant to high temperatures, as the breakpoint temperature and AP discharge rate of single pacemaker cells are much higher than the in vivo heart rate of the fish (brown trout, Salmo trutta) at the same experimental temperature (Haverinen et al., 2017). This suggests that the thermal failure of heartbeat is due to the interruption of AP propagation, and not caused by the failure of the sinoatrial pacemaker. In particular, ventricular excitation is suggested to fail at high temperatures, as indicated by the missing QRS complexes, which are often the first sign of thermal deterioration of the heartbeat, finally resulting in bradycardia (Vornanen et al., 2014; Badr et al., 2016).

EE of atrial and ventricular myocytes is determined by the antagonism between inward Na⁺ (I_Na) and outward K⁺ (I_K1) currents (Davidenko et al., 1990; Hund and Rudy, 2000; Varghese, 2016). To attain the voltage threshold of AP generation, the density of the inward I_Na must exceed the density of the outward I_K1 (Varghese, 2016). Interestingly, the thermal failure of the fish heart can be mechanistically explained – from the molecular to the organismal level – by temperature-dependent deterioration of EE (TDEE hypothesis) (Vornanen, 2016). At the molecular level, an imbalance between I_Na and I_K1 appears at high temperatures. Initially, when temperature starts to rise, the density of I_Na increases, but at higher temperatures – below the upper critical temperature of the fish – I_Na steeply declines (Vornanen et al., 2014; Badr et al., 2016). In contrast, I_K1 is much more heat tolerant, which means that the density of I_K1 continues to increase, while I_Na is already decreasing (Vornanen et al., 2014; Badr et al., 2016). At high temperatures, the large size and unsurpassed thermal resistance of the ventricular I_K1 cause a mismatch between I_Na and I_K1. This impairs the excitation of ventricular tissue, which appears at the organ level as a failure of ventricular activation and at the organismal level as bradycardia (Vornanen, 2016). Bradycardia results in depression of cardiac output, as stroke volume is insensitive to temperature changes (Gollock et al., 2006). The objective of this study was to test the TDEE hypothesis in cold-adapted Arctic fishes using the current-clamp variety of the single-cell patch-clamp method. Current clamp is a powerful method for examining EE, as it directly measures the concerted outcome of all ion currents involved in cellular excitation (Badr et al., 2018).

The basic features of cardiac excitation have been mainly determined using relatively eurythermic temperate fish species such as brown trout (Salmo trutta fario) and roach (Rutilus rutilus), while nothing is known about cellular excitability of the heart in stenothermic Antarctic or Arctic marine fish species (Vornanen et al., 2002a; Haverinen and Vornanen, 2006, 2007; Vornanen et al., 2014; Abramochkin and Vornanen, 2015, 2016; Haverinen et al., 2017). Therefore, we decided to test the TDEE hypothesis on ventricular myocytes of two cod-like fishes of the Arctic Ocean, the polar cod (Boreogadus saida) and navaga (Eleginus nawaga). Results from these marine species were compared with findings from the burbot (Lota lota), the only species of the cod-like fish that has managed to invade the Holarctic freshwater environment above 40°N. In lakes and rivers of temperate latitudes, burbot are exposed to much wider seasonal temperature changes than their relatives in the Arctic Ocean (Hölker et al., 2004). Like their marine relatives, burbot are generally regarded as cold-stenothermic fish (Müllman, 1939). Indeed, burbot share several cold-stenothermic characteristics with the marine cadids, such as spawning in midwinter and early development and optimal growth in cold or cool waters (Pääkkönen and Marjomäki, 2000; Hofmann and Fischer, 2002; Nagel et al., 2011). Larval and juvenile burbot inhabit pelagic and littoral zones, respectively, and are therefore exposed to much higher temperatures than the profundal adult burbot (Hofmann and Fischer, 2002). The differences in evolutionary history and habitat temperatures of these three cod-like fish species provide an interesting setting to compare the temperature dependence of EE among closely related species. We hypothesized that the EE of ventricular myocytes of the Arctic marine species, polar cod and navaga, is more sensitive to high temperatures than that of the temperate burbot, because the latter may have partially adapted to more variable thermal regimes.

Acute thermal challenges of ventricular excitation were conducted at different [K⁺]_o, as temperature-induced increases in heart rate may cause local tissue-level increases in [K⁺]_o (Cohen et al., 1976; Kline and Morad, 1978). Furthermore, strenuous exercise is associated with K⁺ leak from skeletal muscles (Gale et al., 2013; Danylchuk et al., 2014), which results in prolonged increases in [K⁺]_o as a result of the slow clearance of K⁺ ions from the blood plasma of fish (Soivio and Oikari, 1976; Kieffer, 2000). As increases in temperature, heart rate and [K⁺]_o may have synergistic effects on EE (Dominguez and Fozzard, 1970; Kline and Morad, 1978; Gettes, 1992; Lindinger, 1995; Jain and Farrell, 2003; Badr et al., 2018), temperature challenges of ventricular myocytes were made in three different [K⁺]_o at each test temperature. Based on the recent findings on roach ventricular myocytes (Badr et al., 2018), EE was predicted to be depressed by high [K⁺]_o.

Polar cod, Boreogadus saida (Lepechin 1774), and navaga, Eleginus nawaga (Walbaum 1792), of either sex were caught during the routine expedition of the scientific ship ‘Kartesh’ from Murmansk (Russia) to the Arctic Ocean in October 2017. Fish were caught with demersal trawls or hook-and-line from The Kara Sea (west coast of Yamal peninsula) at depths varying from about 10 m down to 50 m. Water temperature varied between +1°C and +3°C. Fish were kept on board in 100 l tanks filled by a continuous flow of outboard water (10 l min⁻¹) at temperatures between +2 and +3°C. All experiments on polar cod and navaga were carried out within 2 days of capture of the fish on board the ship. Burbot, Lota lota (Linnaeus 1758), of either sex were caught in February 2018 from the ice-covered Lake Pyhäselkä (water temperature 0–4°C) in Central Finland (62°35′N, 21°34′E). The burbot were maintained in the animal facilities of the University of Eastern Finland, in 500 l metal aquaria for a minimum of 3 weeks before the experiments began. Water temperature was regulated at +4±0.5°C (Computec Technologies, Joensuu, Finland) and oxygen saturation was maintained by aeration with compressed air. Ground water was constantly flowing through the aquaria at a rate of 150–200 l day⁻¹. Burbot were fed 3 times per week with fresh (recently killed) crucian carp (Carassius carassius). Body mass and the number of fish used in the experiments are shown in Table 1. Experiments were authorized by the national animal experimental board in Finland (permission ESAVI/2832/04.10.07/2015).

All experiments were conducted in vitro on enzymatically isolated ventricular myocytes. The fish were killed by cranial concussion and pithing, and the heart was rapidly excised. Ventricular myocytes were isolated using the perfusion method and the standard concentrations of hydrolytic enzymes as reported in our original paper and a later study on navaga (Vornanen, 1997; Abramochkin and Vornanen, 2015). Cell isolation was conducted at room temperature (15–17°C). Isolated myocytes were stored at 2°C and used in experiments within 8 h of isolation.

All experiments on polar cod and navaga ventricular myocytes were conducted on board ‘Kartesh’. Experiments on burbot myocytes were carried out at the Department of Environmental and Biological Sciences of the University of Eastern Finland in Joensuu. The whole-cell current-clamp recordings of ventricular APs were made by using either an Axopatch 200B amplifier (Molecular Devices, Saratoga, CA, USA) (polar cod and navaga) or an Axopatch 1D amplifier (Molecular Devices) (burbot). WinWCP software (University of Strathclyde, UK) was used in the Axopatch 200B rig; and Clampex 9.2 software (Molecular Devices) was used in the Axopatch 1D rig for data acquisition. Off-line analysis of the recordings was done using the Clampfit 10.4 (Molecular Devices) software package.

During the experiments, myocytes were continuously superfused with external saline solution at a rate of 1.5–2 ml min⁻¹. This solution contained (mmol l⁻¹): 150 NaCl, 3 (or 5.4 or 8) KCl, 1.2 MgCl₂, 1.8 CaCl₂, 10 Hepes and 10 glucose, and pH was adjusted with NaOH to 7.6 at 15°C. The temperature of the external solution in the recording chamber was regulated at 3, 9 or 15°C by using a Peltier device (CL-100, Warner Instruments, Hamden, CT, USA; or HCC-100A, Dagan, MN, USA), and continuously recorded on the same file with electrophysiological data. This temperature range was selected to cover peak temperatures that polar cod may encounter in the Arctic Ocean currently and in the future. Note, the pH of Hepes increases in alpha-stat manner by 0.014 units when the temperature is decreased by 1°C. Therefore, the pH of the experimental solution was 7.68 and 7.77 at 9 and 3°C, respectively. Patch pipettes were pulled (PIP6, Heka Instruments, Lambrecht, Germany; or PP-83, Narishige, Tokyo, Japan) from borosilicate glass (King Precision, Claremont, CA, USA) and had a resistance of 2–3 MΩ when filled with the internal saline solution (mmol l⁻¹): 140 KCl, 5 Na₂ATP, 1 MgCl₂, 0.03 Tris-GTP, 10 Hepes (pH adjusted with KOH to 7.2 at 15°C). To obtain a giga-ohm seal, the patch pipette was moved as close to the cell as possible, taking into account the rocking of the boat on the sea, which produced slow regular movements of the pipette; then, suction was applied to the pipette. In cases where the suction led to initial contact between the pipette and the cell, and the resistance started to go up, the pipette was quickly raised upwards, lifting the cell from the bottom of the chamber. This prevented further disturbance of recordings in this cell.

After gaining a giga-ohm seal, the membrane under the pipette tip was ruptured by a short voltage pulse (zap) to gain access to the cell, transients due to series resistance (4–8 MΩ) and pipette capacitance were cancelled, and the capacitive size of ventricular myocytes was determined. The cell membrane capacitance for each species is given in Table 1. To elicit APs, ventricular myocytes were stimulated with current pulses of constant duration (2 ms) and with increasing amplitude. The initial stimulus strength was 200 pA and it was raised in 20 pA increments until an all-or-none AP was elicited.

Effects of temperature and [K⁺]_o on the excitability of ventricular myocytes were studied in current-clamp mode of the whole-cell patch clamp. Experiments were started at the highest test temperature (15°C) and using 3 mmol l⁻¹ K⁺ in the external saline solution. Pacing frequency was 0.92 Hz, which corresponds to the resting heart rate of the polar cod at this temperature (Drost et al., 2014). Stimulus current was increased in 20 pA steps until a current threshold for all-or-none ventricular APs was attained. The parameters of the first full AP were analysed. Next, the same procedures were repeated in 5.4 or 8.0 mmol l⁻¹ [K⁺]_o (otherwise, the composition of the external solution was constant) (Badr et al., 2018). Then, the temperature was lowered to 9°C and the above protocols were conducted, but this time using a temperature-specific pacing frequency of 0.75 Hz. Finally, the temperature was lowered to 3°C and the same experimental protocols were run using a pacing frequency of 0.5 Hz. The experiments were carried out using the same protocols for all species to obtain comparable results.

The following AP parameters were analysed off-line: resting membrane potential (V_rest, mV), threshold voltage (also called take-off potential) of AP (V_th, mV), threshold current (I_th, pA), critical depolarization (CD=V_th−V_rest, mV), AP overshoot (mV), AP amplitude (mV), AP duration at 50% repolarization level (APD₅₀, ms), maximum rate of AP upstroke (+dV/dt, mV ms⁻¹) and the maximum rate of AP repolarization (−dV/dt, mV ms⁻¹).

Data are presented as means±s.e.m. from n cells. The number of animals from which cells were obtained is specified in the figure legends. According to our previous experience in patch-clamp experiments with fish cardiomyocytes (Abramochkin and Vornanen, 2015, 2016; Badr et al., 2018), the sample size was n≥9. No animals were excluded from the analysis. Cells were excluded from any analysis in the case of a low-quality giga-ohm seal leading to unstable V_rest. After checking normality of distribution and equality of variances, one-way ANOVA (with Tukey's or Dunnett's T3 post hoc test), non-parametric test for several related samples (with Friedman's test) and non-parametric test for several independent samples (with Kruskal–Wallis H-test) were used to assess the statistically significant differences between AP variables and K⁺ concentrations, as indicated in the figure legends. Differences between mean values were deemed statistically significant if P<0.05.

In the experimental conditions of low temperature and [K⁺]_o (3°C, 3 mmol l⁻¹ [K⁺]_o and 0.5 Hz), three of the AP parameters (AP overshoot, APD₅₀, AP amplitude) were not different among the studied species (P>0.05) (Fig. 1, Table 2). Polar cod and navaga APs did not differ with regard to all nine AP parameters (P<0.05). Clear differences were noticed in the maximum rates of depolarization (+dV/dt) and repolarization (−dV/dt) of ventricular AP: +dV/dt was much faster and −dV/dt was much slower in burbot myocytes than in myocytes of the marine species (P<0.05) (Table 2). CD and I_th were smaller in burbot than in navaga (P<0.05). V_rest was more negative in burbot than in polar cod ventricular myocytes (P<0.05). V_th was more negative in burbot than in both polar cod and navaga ventricular myocytes (P<0.05) (Table 2).

Ventricular myocytes of polar cod, navaga and burbot were stimulated in current-clamp mode at temperature-specific pacing frequencies and using an ascending amplitude of current pulses to find the minimum stimulus strength for induction of APs in the presence of 3 mmol l⁻¹ [K⁺]_o (Fig. 2B,C). Small current pulses elicited only passive local depolarizations of membrane potential, which decayed back to the resting level when the stimulus pulse was turned off (Figs 2A and 3A). When the stimulus was strong enough to depolarize the membrane to V_th, an all-or-none AP with a fast upstroke was elicited. In myocytes from burbot and polar cod hearts, the minimum strength of stimulus required for AP triggering did not show any clear temperature dependence. In navaga, a significantly stronger stimulus was required for AP activation at 3°C than at 9°C (P<0.05, Fig. 3B).

In both polar cod and navaga, a decrease of experimental temperature from 9°C to 3°C induced a significant positive shift of V_th (Fig. 3C) and V_rest (Figs 2C and 3D), while no significant difference was observed between these parameters at 15 and 9°C. In burbot myocytes, both parameters were independent of experimental temperature. Because V_th and V_rest changed in a similar manner (polar cod and navaga) or remained unaltered (burbot), CD was not affected by an elevation of temperature in any of the species (Fig. 3H). Thus, in ventricular myocytes of polar cod and navaga, warming hyperpolarizes the cell membrane but does not reduce excitability. Burbot myocytes appear to be least sensitive to temperature changes. The amplitude and overshoot of the ventricular AP were generally insensitive to acute temperature changes except the AP overshoot of the navaga, which was slightly depressed at 9 and 15°C (Fig. 3E,F). However, the maximum rate of AP upstroke strongly increased with rising temperature in polar cod and navaga (Fig. 3J), but not in burbot. AP duration was drastically shortened by warming in all studied fish species (Figs 2C and 3G), while the maximum rate of AP repolarization was not significantly affected by temperature changes (Fig. 3G).

In general, increases in [K⁺]_o had a consistent effect on the nine AP parameters throughout the temperature range (Figs 4–7). V_th and V_rest became more depolarized and AP amplitude decreased in all species and at all experimental temperatures (Figs 5–7C–E). The depolarization of V_rest is consistent with temperature dependence of the Nernstian equilibrium potential for K⁺ ions. At 9 and 15°C, CD was elevated by 5.4 and 8 mmol l⁻¹ [K⁺]_o in polar cod and navaga, while in burbot ventricular myocytes it was depressed (Figs 5–7F). At 3°C, CD was statistically significantly changed only in the polar cod. Maximum rate of AP upstroke was depressed at all temperatures and in all species apart from that of the burbot ventricular myocytes at 3°C, where it stayed unaltered (Figs 5–7J). Maximum rate of AP repolarization was not affected by changes in [K⁺]_o in any of the species at any temperature (Figs 5–7I). APD₅₀ got shorter and AP overshoot declined in polar cod (except for AP overshoot in polar cod at 3°C) and navaga, while in burbot ventricular myocytes they were not affected, except for the slight increase of AP overshoot at 15°C (Figs 5–7G,H). In polar cod, I_th increased at 3 and 15°C when [K⁺]_o was elevated, but decreased at 9°C. In burbot, [K⁺]_o had no effect on I_th at any temperature (Figs 5–7B).

According to the TDEE hypothesis, a temperature-dependent mismatch between I_Na and I_K1 results in a failure of ventricular excitation of fish hearts at high temperatures (Vornanen, 2016). The documented atrioventricular block of the fish (Rutilus rutilus) heart is unlikely to be due to thermal failure of the atrioventricular node, as the PQ interval of the ECG is not prolonged by high temperatures (Badr et al., 2016). The present findings show that in three Gadiformes species, acute temperature changes between 3 and 15°C do not impair EE of ventricular myocytes, while small changes in [K⁺]_o strongly depress excitability, in particular in the marine species.

The present findings show that the EE of ventricular myocytes in all three species, acclimatized/acclimated to low temperatures, was relatively resistant to acute changes of temperature in the range between 3 and 15°C. Ventricular myocytes of burbot were clearly the least sensitive to acute temperature changes among the three Gadiformes species. Apart from APD₅₀, none of the AP parameters in burbot was significantly changed by an acute temperature decrease from 15°C to 9°C and further to 3°C. In contrast, in polar cod and navaga, several of the AP characteristics were significantly modified by acute changes of experimental temperature. However, CD and I_th were little affected by stepwise decreases in temperature, suggesting that the requirements for stimulus strength were not significantly altered by acute temperature changes in the range between 3 and 15°C in any of the species.

The finding that CD was not affected by acute temperature changes in burbot is consistent with the thermal tolerance of this species. Burbot spawn in midwinter, and in summer, adult fish prefer cool bottom waters between 9 and 13°C (Edsall and Kennedy, 1993; Pääkkönen and Marjomäki, 2000). This shows that burbot have retained some of the typical characteristics of the cold-stenothermic marine Gadiformes (Lawler, 1963; Kieckhäfer, 1972). However, burbot are able to acclimate to fairly high temperatures (Pääkkönen and Marjomäki, 2000; Hofmann and Fischer, 2002), which is also evident at the molecular level (sarco/endoplasmic reticulum Ca²⁺-ATPase, SERCA) of the heart (Korajoki and Vornanen, 2013). Indeed, the critical thermal maximum of burbot varies between 26.8 and 31.7°C fish acclimated between 5.9 and 19.6°C (Hofmann and Fischer, 2002). Thus, it is no surprise that the highest experimental temperature of 15°C was well tolerated by burbot ventricular myocytes. Consistent with the present findings, it has previously been shown that under rising temperature regimes, cardiac arrhythmia (due to atrioventricular block) appeared in the perfused hearts of cold-acclimated (2°C) burbot at about 18°C (Tiitu and Vornanen, 2002a).

Navaga are cold adapted and spawn in winter at sub-zero temperatures. Their preferred temperature is 6.9°C (Schurmann and Christiansen, 1994). Although no experimental data exist on the upper thermal tolerance of navaga in the literature, their ability to flourish in the White Sea, where water temperatures in the summer may exceed 15°C, indicates that they are relatively eurythermic fish in comparison to polar cod. Therefore, it is not unexpected that their ventricular myocytes remain excitable at 15°C. In contrast, polar cod are cryopelagic fish, which prefer habitats where ice is available year round. They can be regarded as the most stenothermic among the three species, even though they are reported to occur in areas of the Arctic Ocean where temperature varies from sub-zero up to 9°C (Moulton and Tarbox, 1987; Crawford and Jorgenson, 1996). Under laboratory conditions, the critical thermal maximum of the polar cod varies between 14.9 and 17.1°C depending on acclimation temperature (Hofmann and Fischer, 2002; Drost et al., 2016a,b). Considering their relative stenothermicity, it is slightly surprising that the CD of the polar cod ventricular myocytes was not elevated at 15°C. In anaesthetized adult polar cod, the temperature for cardiac arrhythmia was reported to be 12.4–13.3°C, although the type of cardiac arrhythmia was not mentioned (Drost et al., 2014). Collectively, the present findings suggest that excitation threshold is not markedly affected in any of the species at 15°C.

In polar cod and navaga myocytes, the maximum rate of depolarization (+dV/dt) was markedly higher at warmer temperatures, suggesting that the density of I_Na, and therefore the rate of AP propagation, increases with acute rises of temperature and thereby adjusts heart function to the elevated heart rate (Tiitu and Vornanen, 2002a; Hassinen et al., 2014; Drost et al., 2016a). In navaga myocytes, the AP overshoot was depressed at higher temperatures, which may indicate that in this species the repolarizing K⁺ currents increase more than the depolarizing I_Na. Previous studies have shown that atrial and ventricular K⁺ currents (I_K1 and I_Kr) are strongly enhanced by cold acclimation in navaga (Hassinen et al., 2014; Abramochkin and Vornanen, 2015). Unfortunately, no data are available for thermal responses of I_Na in polar cod and navaga myocytes. Patch-clamp measurement of sarcolemmal ion currents is needed to resolve these issues.

In all three species, the most prominent temperature-induced change occurred in APD₅₀, which was markedly shorter at higher temperatures. While the strong temperature-dependent shortening of AP allows more time for diastolic filling of the heart under elevated heart rate, it probably has a negative effect on ventricular force generation. In fish hearts, excitation–contraction coupling is strongly dependent on sarcolemmal Ca²⁺ entry via L-type Ca²⁺ channels and Na⁺/Ca²⁺-exchanger. Therefore, the strong shortening of AP duration may limit Ca²⁺ influx and reduce the force of contraction (Tibbits et al., 1991; Vornanen et al., 2002b). Indeed, in the cold-acclimated (1–2°C) burbot, shortening of contraction duration results in a strong reduction of contractile force and pumping capacity, when temperature is acutely elevated (Tiitu and Vornanen, 2002b).

EE is critically dependent on K⁺ ion concentration of the blood plasma (Sperelakis et al., 1970; Dominguez and Fozzard, 1970; Vornanen, 2017; Badr et al., 2018). The present findings show that among the three teleost fish species, ventricular excitation of polar cod and navaga is more strongly affected by [K⁺]_o than that of burbot. At 3°C (3 mmol l⁻¹ [K⁺]_o and 0.5 Hz), EE – defined as the ease with which an all-or-none AP is triggered – was not markedly weakened by acute temperature increases in any species. However, an increase of [K⁺]_o from 3 to 5.4 mmol l⁻¹ drastically depolarized V_th and increased I_th and CD in both polar cod and navaga myocytes, but had a much lesser effect in burbot myocytes. Thus, relatively modest increases in [K⁺]_o can have dramatic effects on the EE of Arctic marine fish hearts, as also recently reported for the roach (Badr et al., 2018). Although depolarization of V_rest, caused by an increase in [K⁺]_o, was similar in ventricular myocytes of all studied species, only in myocytes of the marine fish did depolarization coincide with the loss of excitability. This failure of excitation was due to a [K⁺]_o-induced increase of CD. Surprisingly, in burbot ventricular myocytes, CD decreased and excitability remained untouched even in the presence of 8 mmol l⁻¹ [K⁺]_o and at all test temperatures. One possible explanation for the insensitivity of the burbot ventricular myocytes to [K⁺]_o is the small size of I_K1. The I_K1 density in the ventricular myocytes of the cold-acclimated burbot heart is only about 1/5 the I_K1 density of the winter-acclimatized navaga (Shiels et al., 2006; Abramochkin and Vornanen, 2015); in terms of I_K1 density, burbot ventricular myocytes are more like the atrial myocytes of other teleost fish species. In contrast, the density of ventricular I_Na in burbot is similar to that in other fishes (Haverinen and Vornanen, 2004). Burbot ventricular myocytes have a favourable I_Na/I_K1 ratio: the very small I_K1 may protect burbot myocytes, not only against [K⁺]_o-dependent reduction in excitability but also against acute temperature changes.

In the blood plasma of various teleost species, [K⁺]_o typically varies between 2 and 12 mmol l⁻¹, even though concentrations as high as 20 mmol l⁻¹ have been reported for some species (Holmes and Donaldson, 1969; Prosser et al., 1970). [K⁺]_o concentrations higher than 7–8 mmol l⁻¹ are usually detrimental for excitability of the vertebrate heart as a result of depolarization of V_rest: I_Na is almost completely inactivated and impulse conduction is, therefore, likely to fail (Sperelakis et al., 1970; Dominguez and Fozzard, 1970; Haverinen and Vornanen, 2004). Indeed, it was recently shown that in the space-clamped ventricular myocytes of the roach heart, 8 mmol l⁻¹ [K⁺]_o can totally prevent the generation of all-or-none APs (Badr et al., 2018).

In mammals and fish, the concentration of plasma [K⁺]_o increases in exercise as a consequence of K⁺ efflux from the skeletal muscles (Prosser et al., 1970; Soivio and Oikari, 1976; Turner et al., 1983; Wells et al., 1986; Nielsen and Lykkeboe, 1992; Kieffer, 2000; Jain and Farrell, 2003; Danylchuk et al., 2014). Furthermore, exercise- and temperature-induced increase in heart rate may result in elevation of the paracellular K⁺ concentration in the myocardium by similar mechanisms to those in skeletal muscles (Cohen et al., 1976; Kline and Morad, 1978). In fish body, the clearance of [K⁺]_o is a slow process and therefore plasma [K⁺]_o stays elevated for several hours after the exercise bout (Soivio and Oikari, 1976; Wood et al., 1983). For this reason, high temperature and high [K⁺]_o may synergistically depress cardiac excitability in exercising fish (Badr et al., 2018). EE may not fail in resting fish, but exercise- and heart rate-related increases in [K⁺]_o may compromise AP generation and conduction of the heart at high temperatures. Interestingly, the recovery of polar cod from maximum exercise is reported to be poor. Polar cod acclimated to 6.5°C were unable to recover from a maximum swimming test at 8.5°C (Drost et al., 2016b). It remains to be shown whether exercise and poor recovery from it are associated with increases in plasma [K⁺]_o (Nielsen and Lykkeboe, 1992). In this respect, it would be useful to examine [K⁺]_o in blood plasma of these species and how it is affected by species-specific mode of locomotion and exercise stress.

The effect of high [K⁺]_o was particularly strong on the maximum rate of AP upstroke (+dV/dt), which was drastically depressed in polar cod and navaga ventricular myocytes. A modest increase of [K⁺]_o from 3 mmol l⁻¹ to 5.4 mmol l⁻¹ caused 79.2%, 83.9% and 87.4% depression of the maximum rate of AP upstroke at 3, 9 and 15°C, respectively, in the polar cod myocytes. An equally strong effect was present in navaga ventricular myocytes. As the maximum rate of AP upstroke is determined by the density of I_Na, these findings suggest that the availability of Na⁺ channels for opening is almost completely abolished in polar cod and navaga myocytes by the depolarization of V_rest. The precipitous depression of the maximum rate of AP upstroke could impair excitability of the intact ventricle. In the absence of sufficient depolarizing I_Na, membrane potential is unlikely to reach the value of V_th. In space-clamped myocytes, excitability persists because unlimited (artificial) depolarizing stimulus current can be applied. Notably, the burbot ventricular myocytes were less strongly affect by high [K⁺]_o. This may be related to the much more favourable I_Na/I_K1 ratio of the burbot ventricular myocytes (Haverinen and Vornanen, 2004, 2009; Shiels et al., 2006).

The warming rate in the Arctic is about 3 times faster than the average global warming rate: the increase in surface water temperature in the Arctic Ocean was about 0.6°C per decade during the period 1981–2012. Simultaneously, sea ice content has been decreasing (Comiso and Hall, 2014). The mean year-round temperature of temperate lakes is projected to increase by about 0.3 deg per decade (Kirillin, 2010). These progressive changes form a potential threat to the key species of the arctic ecosystem like the stenothermic and cryopelagic polar cod. The present finding that the EE of ventricular myocytes of the adult polar cod in the range 3–15°C is temperature resistant suggests that the anticipated warming of Arctic polar waters in the main habitat areas of the polar cod may not threaten electrical activity of the heart in adult individuals of this species. In contrast, only a modest 2.4 mmol l⁻¹ increase in [K⁺]_o strongly depressed EE of ventricular myocytes in polar cod and navaga. The high sensitivity to [K⁺]_o may be indicative of a poor tolerance of cardiac function in polar cod and navaga to exercise stress, assuming that they have slow K⁺ clearance (Soivio and Oikari, 1976). Among the three Gadiformes species, the freshwater burbot appears to be the most tolerant of elevations of temperature and [K⁺]_o. This may be mechanistically explained by the very low density of the ventricular I_K1 in this species. This suggests that after invading temperate freshwaters more than 5 million years ago (Van Houdt et al., 2003), burbot have acquired physiological characteristics that make them able to resist higher temperatures than their marine relatives.

Anita Kervinen is acknowledged for taking care of the burbot and preparing solutions for experiments. We are grateful to the executive director of MSU Marine Research Center Nikolay V. Shabalin, to the captain of ‘Kartesh’ scientific vessel Vitaly T. Lozinsky and to the staff of the vessel for invaluable help in organizing the on-board electrophysiological experiments.