ABSTRACT

Heat tolerance of heart rate in fish is suggested to be limited by impaired electrical excitation of the ventricle due to the antagonistic effects of high temperature on Na+ (INa) and K+ (IK1) ion currents (INa is depressed at high temperatures while IK1 is resistant to them). To examine the role of Na+ channel proteins in heat tolerance of INa, we compared temperature dependencies of zebrafish (Danio rerio, warm-dwelling subtropical species) and rainbow trout (Oncorhynchus mykiss, cold-active temperate species) ventricular INa, and INa generated by the cloned zebrafish and rainbow trout NaV1.4 and NaV1.5 Na+ channels in human embryonic kidney (HEK) cells. Whole-cell patch-clamp recordings showed that zebrafish ventricular INa has better heat tolerance and slower inactivation kinetics than rainbow trout ventricular INa. In contrast, heat tolerance and inactivation kinetics of zebrafish and rainbow trout NaV1.4 channels are similar when expressed in the identical cellular environment of HEK cells. The same applies to NaV1.5 channels. These findings indicate that thermal adaptation of ventricular INa is largely achieved by differential expression of Na+ channel alpha subunits: zebrafish that tolerate higher temperatures mainly express the slower NaV1.5 isoform, while rainbow trout that prefer cold waters mainly express the faster NaV1.4 isoform. Differences in elasticity (stiffness) of the lipid bilayer and/or accessory protein subunits of the channel assembly may also be involved in thermal adaptation of INa. The results are consistent with the hypothesis that slow Na+ channel kinetics are associated with increased heat tolerance of cardiac excitation.

INTRODUCTION

Temperature is a major environmental factor that has exerted a strong selective pressure on animal life forms. Temperature-driven evolution has led to significant variation in thermal tolerance of ectothermic vertebrates, including fishes (Beitinger, 2000; Johnston and Bennett, 2008). Some fish species are adapted to a relatively narrow range of temperatures such as the stenothermic teleosts of the Antarctic Ocean and the polar cod (Boreogadus saida) of the Arctic Ocean (Somero and DeVries, 1967; Beers and Sidell, 2011; Drost et al., 2016; Abramochkin et al., 2019). In temperate climates, fishes usually have a wider thermal tolerance range, e.g. rainbow trout (Oncorhynchus mykiss) are cold-water fish that survive temperatures between 0 and 28°C (Hokanson et al., 1977; Beitinger, 2000). The zebrafish (Danio rerio), a teleost fish of the lakes and rivers of South-eastern Asia, also tolerates a wide temperature range (7–41°C) but occupies warmer habitats than rainbow trout (Cortemeglia and Beitinger, 2005; López-Olmeda and Sánchez-Vázquez, 2011).

Despite considerable research efforts, the physiological basis for the high temperature tolerance of fishes and other ectotherms remains unresolved. Practically all major processes and functions of the animal body (e.g. circulation, sensory/motor functions, behaviour, metabolism, digestion, immune defence, reproduction) are affected by temperature and therefore more or less adapted to the habitat temperature of the animal (Angilletta et al., 2002; Gracey et al., 2004; Podrabsky and Somero, 2004; Vornanen et al., 2005; MacMillan, 2019). Therefore, it is likely that when approaching the upper temperature tolerance limit of an animal, several processes simultaneously weaken in the animal's body, but the severity of their effect at the organismal level may vary and manifest with varying delays (Vornanen, 2020). Alternatively, different processes/functions may have slightly different thermal optima or failure temperatures, depending for example on the level of biological organization where the process/function appears (Lagerspetz, 1987; Clark et al., 2013). A fundamental question arises: is there a single underlying process or interaction principle that drives the thermal collapse of multiple organ systems? Given that similar thermal limitations appear to apply to unicellular organisms and Metazoa, it has been suggested that the limiting processes of animal life are found at the molecular level (Tattersall et al., 2012).

Electrical excitability is a common process for most tissues of the animal body including nerves, skeletal muscles, heart and smooth muscles. These tissues are responsible for almost all vital functions of the animal body like sensation, learning, behaviour, locomotion, blood circulation, digestion and homeostasis of the body (Hille, 2001). Recently, we have gathered evidence showing that high temperatures can impair electrical excitability and therefore potentially limit the upper thermal tolerance of both ectothermic and endothermic animals (Vornanen, 2020). Electrical excitability or generation of propagating action potentials (APs) is the result of interaction between inward and outward directed flow of ions across the plasma membrane. In studies on thermal tolerance of electrical excitability we have used fish ventricular myocytes as a model system, as they are well suited for patch-clamp experiments. Atrial myocytes are a less suitable model as they fail to maintain stable resting potential in the current-clamp mode of patch-clamp due to the tiny background inward rectifier current (IK1) and the absence of acetylcholine-dependent inward rectifier current (IKACh) in isolated cells (Vornanen et al., 2002; Molina et al., 2007). In a quiescent ventricular myocyte, there is a negative resting membrane potential (Vrest), which is maintained by K+ efflux via IK1. For initiation and propagation of cardiac AP, Vrest must be depolarized to the threshold potential (Vth) of an AP. This is accomplished by Na+ influx through the voltage-gated Na+ channels, which generate the sodium current (INa). AP is initiated only when the charge transfer of the inward INa (the source current) exceeds the charge transfer of the outward IK1 (the sink current or resting membrane leak). At high temperatures, electrical excitability of fish ventricular myocytes may fail due to mismatch between the source current (INa) and the sink current (IK1) (Vornanen et al., 2014). Acute warming reduces charge transfer via the INa, while K+ leak via IK1 increases: INa fails to depolarize Vrest to the threshold potential of AP. At the level of a working heart this appears as atrioventricular block and depression of ventricular beating rate (Haverinen and Vornanen, 2020), which may eventually result in a collapse of cardiac output and thermal death of the fish.

The present study aimed to test the source–sink mismatch hypothesis with respect to the source current, INa (Vornanen, 2020). Given the differences in temperature tolerances between rainbow trout (a cold-active temperate species) and zebrafish (a warm-dwelling subtropical species), it was hypothesized that thermal tolerance of the zebrafish INa is higher than that of the rainbow trout INa. This assumption was tested by comparing INa of zebrafish and rainbow trout ventricular myocytes at different temperatures. Another prediction of the source–sink hypothesis is that the channels that generate currents with slow gating kinetics can withstand high temperatures better than channels that produce currents with fast kinetics (Touska et al., 2018; Vornanen, 2020). This is based on the assumption that stiffer molecules have higher activation energy and slower kinetics, i.e. there is a trade-off between flexibility and thermal stability of the proteins (Somero, 1995; Zavodszky et al., 1998; Fields, 2001). Studies on mammalian Na+ channels have shown that the skeletal isoform, NaV1.4, has faster inactivation kinetics than the cardiac isoform, NaV1.5 (Wang et al., 1996). INa with slow inactivation kinetics is able to provide more depolarizing charge at high temperature than INa, which is rapidly inactivated. As zebrafish and trout ventricular INa are mainly generated by NaV1.5 and NaV1.4 channels, respectively (Haverinen et al., 2007; Haverinen et al., 2018), it was hypothesized that those channel isoforms are involved in adaptation of cardiac INa to high and low temperature, respectively. To this end, the inactivation kinetics and charge transfer of INa between rainbow trout and zebrafish ventricular myocytes were compared at different temperatures.

Finally, it was hypothesized that thermal stability and kinetics of fish INa depend in part on the cellular environment (e.g. biophysical properties of the lipid membrane and/or ancillary protein subunits) where they are expressed. To test this, the two main alpha subunits of zebrafish and rainbow trout Na+ channels were cloned and expressed in a mammalian cell line, the human embryonic kidney (HEK) cell. This allowed comparison of inactivation kinetics and thermal resistance of the Na+ channels in an identical cellular environment.

MATERIALS AND METHODS

Animals

The wild-type zebrafish, Danio rerio (F. Hamilton 1822) (ab strain, kindly donated by Dr Maxim Lovat, Lomonosov Moscow State University), were raised and maintained at the animal facilities of Lomonosov Moscow State University according to common practices (Westerfield, 2007). The rearing temperature of the fish was 28°C. Fish of either sex, about 1.5 years old, were used for electrophysiological experiments (N=8). Zebrafish were killed by immersion in an ice-water bath and cutting of the spine. Rainbow trout, Oncorhynchus mykiss (Walbaum, 1792), were obtained from a local fish farm (Kontiolahti, Finland). Fish weighing 118.7±20.6 g (N=11) of either sex, acclimated at 12°C for more than 3 weeks, were used in electrophysiological experiments and gene cloning. Trout were stunned by a quick blow to the head and killed by cutting of the spine immediately behind the head. The experiments conform to the ‘European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes’ (Council of Europe No. 123, Strasbourg 1985) and were authorized by the national animal experimental board in Finland (permission ESAVI/8877/2019).

Cloning of SCN5LA and SCN4A Na+ channel genes of zebrafish and rainbow trout

Total cardiac RNA was extracted by TriReagent (Thermo Fisher Scientific, Vilnius, Lithuania) and the quality and quantity of RNA were determined by agarose gel electrophoresis and NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), respectively. RNA (2 µg) was treated with RNase free DNase (Thermo Fisher Scientific) and converted to cDNA using SuperScript IV reverse transcriptase (Invitrogen, Glasgow, UK) and oligo(dT) primers. cDNA (1 µl) was used as a template in 25 µl polymerase chain reaction (PCR) including a final concentration of 0.2 mmol l−1 dNTP mix, 0.2 mmol l−1 primers (synthesized by Invitrogen) as shown in Table 1, and 0.02 U µl−1 Phusion High Fidelity DNA polymerase (Thermo Fisher Scientific). Cycling conditions for all PCR reactions were as follows: initial denaturation at 98°C for 1 min, 35 cycles at 98°C for 10 s, at 60°C for 30 s and at 72°C for 100–260 s (40 s per kb; for the length of the products see Table 1), followed by final extension at 72°C for 5 min. The PCR products were separated on a 0.8% agarose gel, and the nucleotide chains were extracted from the gel using a GeneJET Gel Extraction Kit (Thermo Fisher Scientific). Overhang adenines were added to the 3′-ends of PCR products using Dynazyme II DNA polymerase (Thermo Fisher Scientific) and products were ligated into the pGEM-T Easy vector (Promega, Madison, WI, USA). The inserts were digested from the pGEM-T Easy vector and directionally cloned into the expression vector. If the coding sequence of the SCN gene was amplified by PCR in two parts, the pieces were ligated into the expression vector to form the entire coding sequence. Coding sequences of the SCN5LA genes of rainbow trout (om for O.mykiss) and zebrafish (dr for D.rerio) were cloned into the expression vector pcDNA3.1/Zeo(+) (Invitrogen). Several attempts to clone the full-length om/drSCN4A into the pcDNA3.1 failed. To overcome this problem, the high-copy number vector pcDNA3.1/Zeo(+) was converted to a low-copy number vector that was better tolerated by bacterial cells. To this end, the ColE1 origin was replaced by the pMB1 origin via digesting pcDNA3.1/Zeo(+) with BsmI and PvuI and replacing this fragment (bases 2768–4462) with the digested fragment from pBR322 (bases 1353–3733). The coding sequences of dr/omSCN4A were successfully cloned into this modified vector. Plasmids were isolated using PureLink HiPure Plasmid Midiprep Kit (Thermo Fisher Scientific) and sequenced by GATC Biotech AB (Köln, Germany). Nucleotide sequences were converted to protein sequences using EMBOSS Transeq software (https://www.ebi.ac.uk/Tools/st/emboss_transeq/), and the protein sequences of the species-specific isoforms were aligned with EMBOSS Needle (https://www.ebi.ac.uk/Tools/psa/emboss_needle/). The fish SCN4A and SCN5LA genes are orthologous to the mammalian SCN4A and SCN5A genes. For simplicity, the protein names (NaV1.4 and NaV1.5) of the orthologous genes are used throughout the text.

Table 1.

Primers used in cloning of the SCN genes of rainbow trout and zebrafish

Primers used in cloning of the SCN genes of rainbow trout and zebrafish
Primers used in cloning of the SCN genes of rainbow trout and zebrafish

Heterologous expression of SCN4A and SCN5LA genes

Due to the whole genome duplications of the teleost lineage, the number of gene paralogues expressed in fish striated muscles is high (Alderman et al., 2012; Glasauer and Neuhauss, 2014). All eight teleost Na+ channel genes are expressed in the heart of both zebrafish and rainbow trout (Haverinen et al., 2018; Hassinen et al., 2021). Because striated muscle isoforms, NaV1.4 and NaV1.5, make up the great majority (>99%) of all Na+ channel transcripts in zebrafish and trout hearts, genes for heterologous expression were selected from these paralogues. For both NaV1.5 and NaV1.4, there are three paralogues in the trout genome and two paralogues in the zebrafish genome. For NaV1.5 channels, drSCN5LAb and omSCN5LAba, the most abundant paralogues of ventricular myocytes, were expressed in HEK cells. For the expression of zebrafish NaV1.4 channels, drSCN4Ab, the most abundantly expressed SCNA paralogue of ventricular myocytes, was inserted in HEK cells. In the case of trout NaV1.4 channels, omSCN4Abb, the second most abundant SCN4A paralogue of ventricular myocytes (most abundant in atrial myocytes) was expressed in HEK cells. We failed to clone the most abundant paralogue of the ventricle, omSCN4Aba, and were therefore forced to use the major atrial isoform, omSCN4Abb. Human embryonic kidney (HEK293; ECACC) cells were grown at 37°C in DMEM (Biowest) supplemented with 10% fetal bovine serum (FBS, sterile-filtered and heat inactivated; Sigma-Aldrich) and 100 U ml−1 penicillin and streptomycin (Sigma-Aldrich) in a 5% CO2 environment. HEK cells were transiently co-transfected with pEGFP-N1 (Clontech), and either drSCN4Ab, drSCN5LAb, omSCN4Abb or omSCN5LAba construct using TurboFect transfection reagent (Thermo Fisher Scientific). Cells were transfected for 16–18 h at 37°C; plate medium was then refreshed, and the cells were further incubated at 28°C in a 5% CO2 environment for at least 24 h. Incubation at lower temperature increased the expression level of Na+ channels. Whole-cell patch-clamp experiments were conducted 48–72 h after transfection.

Patch-clamp measurements of Na+ current in fish cardiac myocytes and HEK cells

The procedure of isolating zebrafish cardiomyocytes was essentially similar to the original method of isolating crucian carp (Carassius carassius) cardiac cells (Vornanen, 1997), but scaled down to the size of small zebrafish hearts and using slightly higher enzyme concentrations (1 mg ml−1 collagenase Type IA and 0.67 mg ml−1 Trypsin IV; both from Sigma). A blunt-ended syringe needle (34 gauge, TE734025; Adhesive Dispensing Ltd, Milton Keynes, UK) cannula was inserted via the bulbus arteriosus into the ventricle and secured in place with a fine thread. The heart was perfused first with Ca2+-free solution for 5 min and then with the same solution but with added hydrolytic enzymes together with fatty acid-free serum albumin (1 mg ml−1; Sigma) for 25–30 min. Myocytes were stored at 5°C and used on the same day as they were isolated. The Ca2+-free solution contained (mmol l−1): 100 NaCl, 10 KCl, 1.2 KH2PO4, 4 MgSO4, 50 taurine, 20 glucose and 10 HEPES at pH 6.9 (adjusted with KOH at 20°C). From rainbow trout heart, myocytes were obtained using essentially the same procedure, but with lower enzyme concentrations (0.75 mg ml−1 collagenase Type IA and 0.5 mg ml−1 Trypsin IV) and shorter digestion time (10–12 min).

The whole-cell voltage-clamp recording of INa was performed using an Axopatch 200B or an Axopatch 1-D amplifier (Molecular Devices, San Jose, CA, USA) as previously described in detail (Haverinen et al., 2018). Cardiac myocytes or coverslips containing cultured HEK cells were placed in a small chamber with a continuous flow of K+-based external saline solution containing (mmol l−1): 150 NaCl, 5.4 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 glucose and 10 HEPES, with pH adjusted to 7.7 at 20°C with NaOH. The temperature of the saline solution was set using a Peltier device (CL-100, Warner Instruments, Hamden, CT, USA; or TC-10, Dagan, Minneapolis, MN, USA) and monitored continuously with thermistors placed close to the cells. Patch pipettes with a resistance of 1.5–2.5 MΩ were drawn from borosilicate glass (Hilgenberg GmbH, Malsfeld, Germany) and filled with Cs+-based electrode solution containing (mmol l−1): 5 NaCl, 130 CsCl, 1 MgCl2, 5 EGTA, 5 Mg2ATP and 5 HEPES, with pH adjusted to 7.2 with CsOH. Current amplitudes were normalized to the capacitive cell size to obtain current density (pA pF−1).

During INa recording, myocytes were superfused with a Cs-based low-Na+ external saline solution, which contained (mmol l−1): 20 NaCl, 120 CsCl, 1 MgCl2, 0.5 CaCl2, 10 glucose and 10 HEPES at pH 7.7 (adjusted with CsOH at 20°C) (Haverinen and Vornanen, 2004). In experiments with cardiac myocytes, nifedipine (10 μmol l−1; Sigma) was included to block ICa. The low concentration of Na+ outside the cell reduced the driving force for Na+ influx and made it possible to achieve stable and complete voltage control of INa. Approximately 80% of the series resistance was compensated for when recording fast and large INa. INa was leakage-corrected using the P/N procedure of Clampex 9.2 software. For determination of the current–voltage (IV) relationship, INa was elicited from the holding potential of −120 mV with 60 ms depolarizing pulses (range −100 to +70 mV) at a frequency of 1 Hz (Fig. 1A). The voltage dependence of Na+ channel conductance was calculated from the IV recordings using the equation: GNa=INa/(VVrev), where GNa is the Na+ conductance of the membrane, INa is the peak current at a given membrane potential (V) and Vrev is the reversal potential of INa. The steady-state (SS) voltage dependence of activation was obtained by plotting the normalized conductance (G/Gmax) as a function of membrane potential and fitting it into the equation of Boltzmann distribution:
formula
(1)
where V is membrane potential, V0.5 the midpoint potential and S is the slope of the curve. SS inactivation was determined using a two-step protocol where a 300 ms conditioning pulse to potentials between −110 and −20 mV was followed by a 60 ms test pulse to −20 mV. The normalized test pulse currents (I/Imax) were plotted as a function of membrane potential and fitted to the Boltzmann function with a negative slope (–S).
Fig. 1.

Voltage and temperature dependence of current density and charge transfer of INa in zebrafish and rainbow trout ventricular myocytes. (A) The voltage protocol used to elicit INa. (B) A representative recording from a zebrafish ventricular myocyte indicating tracings of INa at different membrane voltages. (C–F) Mean results (±s.e.m.) for current–voltage relationship and charge transfer of INa at three different temperatures for zebrafish (ZF; N=47 myocytes from six fishes) (C,D) and rainbow trout (RT; N=33 myocytes from six fishes) (E,F) ventricle. (G) Current density of INa in heat ramp experiments with zebrafish (N=10 myocytes from two fishes) and rainbow trout (N=10 myocytes from four fishes) ventricle. Statistically significant differences (P<0.05) are shown as follows: *12 versus 28°C; 12 versus 20°C; §20 versus 28°C.

Fig. 1.

Voltage and temperature dependence of current density and charge transfer of INa in zebrafish and rainbow trout ventricular myocytes. (A) The voltage protocol used to elicit INa. (B) A representative recording from a zebrafish ventricular myocyte indicating tracings of INa at different membrane voltages. (C–F) Mean results (±s.e.m.) for current–voltage relationship and charge transfer of INa at three different temperatures for zebrafish (ZF; N=47 myocytes from six fishes) (C,D) and rainbow trout (RT; N=33 myocytes from six fishes) (E,F) ventricle. (G) Current density of INa in heat ramp experiments with zebrafish (N=10 myocytes from two fishes) and rainbow trout (N=10 myocytes from four fishes) ventricle. Statistically significant differences (P<0.05) are shown as follows: *12 versus 28°C; 12 versus 20°C; §20 versus 28°C.

The time constant of INa inactivation at different membrane potentials (–30 to 0 mV) was derived by fitting the decay phase of INa using the double exponential function of the Chebyshev transformation procedure of the Clampfit 10.3 software package. The amplitude of the fast component (τf) was over 90% of the current. The slow component could only be reliably determined at the voltages at which INa was close to its peak amplitude. Therefore, only the results of τf are reported. Thermal coefficient (Q10) values of INa inactivation rate were calculated using the equation: Q10=(R2/R1)^{10°C/(T2T1)}, where R1 and R2 are fast time constants of inactivation (τf) at temperatures T1 and T2.

All mentioned properties of INa were analysed in native trout or zebrafish myocytes and HEK cells expressing NaV1.4 or NaV1.5 channels at three different temperatures: 12, 20 and 28°C. However, additional experiments to estimate the dynamics of the temperature dependence of INa were done using the ‘heat ramp’ protocol. In these experiments INa was elicited by repetitive depolarizations to −20 mV and the temperature was steadily raised from 12 to 28°C (up to 39°C in the case of native zebrafish myocytes).

Statistics

Statistical analyses were performed using SPSS (IBM; version 25). One-way ANOVA (with Tukey's or Dunnett's T3 post hoc test) and unpaired t-test were used to compare normally distributed data with homogenous variances. If the assumptions of parametric tests were not met, non-parametric Kruskal–Wallis and post hoc Mann–Whitney U-tests were used. P<0.05 was considered to show a statistically significant difference between means.

RESULTS

Temperature dependence of INa density and charge transfer in zebrafish and rainbow trout ventricular myocytes

Temperature dependency of ventricular INa was markedly different between zebrafish and rainbow trout myocytes, as shown by the current–voltage (IV) relationships (Fig. 1). In zebrafish myocytes, an acute rise of temperature from 12 to 20°C resulted in a significant increase in INa density at voltages between −50 and −30 mV (Fig. 1C). A similar difference was detected in INa density between 12 and 28°C (P<0.05). However, the current densities at 20 and 28°C were almost identical, suggesting plateauing of INa in this temperature range (Fig. 1C). In rainbow trout myocytes, warming from 12 to 20°C increased INa density at voltages from −30 to −10 mV, but further warming to 28°C caused a strong depression of INa (Fig. 1E). Although the density of the zebrafish INa was higher at 20 and 28°C than at 12°C, the charge transfer (integral of INa) was significantly smaller than at 12°C (Fig. 1D). In trout ventricular myocytes, the integral of INa decreased with increasing experimental temperature (Fig. 1F). Notably, charge transfer/INa density ratio, i.e. depolarizing power, was 4.5–22.2 times higher (at −20 mV) for zebrafish than rainbow trout INa at all experimental temperatures (0.6, 0.7 and 0.9 V A−1 for rainbow trout and 2.7, 4.5 and 20.0 V A−1 for zebrafish at 28, 20 and 12°C, respectively).

Temperature tolerance of peak density and charge transfer of INa (at −20 mV) were further studied using acute heat ramps. To this end, the cells were warmed from 12°C to their upper thermal tolerance limit while INa was elicited by depolarization from −100 to −20 mV for every second (Fig. 1G). Consistent with the I–V data, there was a dramatic interspecies difference in temperature dependence of the peak INa (Fig. 1G). Initially, when the temperature was raised above 12°C, the density of INa increased in both species. However, the breakpoint temperature (TBP, the temperature above which peak INa started to decrease steadily) was much lower for rainbow trout INa (18.3±0.6°C, N=13) than for zebrafish INa (26.6±0.5°C, N=16) (P<0.05).

Taken together, these findings indicate that the ventricular INa of rainbow trout heart is much less tolerant of high temperatures than the zebrafish ventricular INa.

Inactivation kinetics of INa in zebrafish and rainbow trout ventricular myocytes

Temperature dependence of INa inactivation kinetics was measured in the voltage range −30 to 0 mV at 12, 20 and 28°C (Fig. 2). Kinetics of zebrafish or rainbow trout INa accelerated with increasing temperature and membrane depolarization. There was a striking difference in the rate of INa inactivation between trout and zebrafish ventricular myocytes (Fig. 2). At 12, 20 and 28°C, the time constant (τf) of INa inactivation (at 0 mV) was about 10.0, 6.3 and 4.0 times faster in rainbow trout ventricular myocytes in comparison with zebrafish ventricular myocytes (P<0.05).

Fig. 2.

Temperature dependence of inactivation kinetics of INa in zebrafish and rainbow trout ventricular myocytes.INa was elicited from a holding potential of −120 mV to −30 to 0 mV for 30 ms. (A,C,E) Representative tracings of INa at −20 mV at temperatures of 12°C (A), 20°C (C) and 28°C (E). (B,D,F) Means (±s.e.m.) of inactivation time constant (τ) at 12°C (B), 20°C (D) and 28°C (F). The results are from 31 and 30 myocytes from six zebrafish (ZF) and four rainbow trout (RT), respectively. Error bars of RT data are smaller than the size of the symbols. Statistically significant differences (P<0.05) between ZF and RT are shown by dissimilar letters (a and b).

Fig. 2.

Temperature dependence of inactivation kinetics of INa in zebrafish and rainbow trout ventricular myocytes.INa was elicited from a holding potential of −120 mV to −30 to 0 mV for 30 ms. (A,C,E) Representative tracings of INa at −20 mV at temperatures of 12°C (A), 20°C (C) and 28°C (E). (B,D,F) Means (±s.e.m.) of inactivation time constant (τ) at 12°C (B), 20°C (D) and 28°C (F). The results are from 31 and 30 myocytes from six zebrafish (ZF) and four rainbow trout (RT), respectively. Error bars of RT data are smaller than the size of the symbols. Statistically significant differences (P<0.05) between ZF and RT are shown by dissimilar letters (a and b).

Steady-state activation and inactivation of INa in zebrafish and rainbow trout ventricular myocytes

Voltage dependence of steady-state (SS) activation and inactivation of INa was studied at 12, 20 and 28°C (Fig. 3). In zebrafish ventricular myocytes, warming from 12 to 20°C and further to 28°C decreased the slope factor of the SS inactivation curve (P<0.05), while V0.5 remained unaffected (P>0.05; Fig. 3C; Table 2). Voltage dependence of SS activation of INa in zebrafish myocytes was more temperature sensitive, as warming to either 20 or 28°C shifted V0.5 to the left by almost 10 mV and decreased the slope factor (P<0.05) (Table 2; Fig. 3C). In trout ventricular myocytes, warming from 12 to 20°C and further to 28°C failed to shift V0.5 or change slope factor of both curves (Table 2; Fig. 3D).

Fig. 3.

Voltage and temperature dependence of steady-state activation and inactivation of INa in ventricular myocytes of zebrafish and rainbow trout. (A) The two-step voltage protocol used to elicit INa. (B) A representative recording from a zebrafish ventricular myocyte indicating tracings of INa at −20 mV following 1 s depolarizations from −120 mV to −110 to +30 mV. (C) Steady-state (SS) activation and inactivation of zebrafish (ZF) ventricular INa (N=87 myocytes from seven fishes). (D) Steady-state activation and inactivation of rainbow trout (RT) ventricular INa (N=58 myocytes from six fishes).

Fig. 3.

Voltage and temperature dependence of steady-state activation and inactivation of INa in ventricular myocytes of zebrafish and rainbow trout. (A) The two-step voltage protocol used to elicit INa. (B) A representative recording from a zebrafish ventricular myocyte indicating tracings of INa at −20 mV following 1 s depolarizations from −120 mV to −110 to +30 mV. (C) Steady-state (SS) activation and inactivation of zebrafish (ZF) ventricular INa (N=87 myocytes from seven fishes). (D) Steady-state activation and inactivation of rainbow trout (RT) ventricular INa (N=58 myocytes from six fishes).

Table 2.

Midpoint membrane potential and slope factor of steady-state inactivation and activationfor INa of zebrafish and rainbow trout ventricular myocytes and INa generated by their NaV1.4 and NaV1.5 channels in HEK cells

Midpoint membrane potential and slope factor of steady-state inactivation and activationfor INa of zebrafish and rainbow trout ventricular myocytes and INa generated by their NaV1.4 and NaV1.5 channels in HEK cells
Midpoint membrane potential and slope factor of steady-state inactivation and activationfor INa of zebrafish and rainbow trout ventricular myocytes and INa generated by their NaV1.4 and NaV1.5 channels in HEK cells

Temperature dependence of INa density and charge transfer generated by NaV1.4 or NaV1.5 channels in HEK cells

Zebrafish and rainbow trout NaV1.4 and NaV1.5 Na+ channels were expressed in the same cellular environment for a direct comparison between temperature dependencies of the orthologous gene products (Fig. 4). Protein sequences of these Na+ channels suggest a relatively high degree of structural similarity between the species. Trout and zebrafish NaV1.4 shared 64.5% identity and 76.0% similarity, whereas identity and similarity for NaV1.5 were 79.9 and 87.1%, respectively (Figs S1 and S2).

Fig. 4.

Voltage and temperature dependence of current density and charge transfer of INa generated by zebrafish and rainbow trout NaV1.4 and NaV1.5 channels in HEK cells. (A–D) Mean results (±s.e.m.) for current–voltage relationship (A,C) and charge transfer (B,D) of INa at three different temperatures for zebrafish (ZF; N=49 cells) (A,B) and rainbow trout (RT; N=15 cells) (C,D) NaV1.4 channels. (E–H) Mean results (±s.e.m.) for current–voltage relationship (E,G) and charge transfer (F,H) of INa at three different temperatures for ZF (N=30 cells) (E,F) and RT (N=27 cells) (G,H) NaV1.5 channels. (I,J) Current density of INa in heat ramp experiments with ZF and RT NaV1.4 (I) and NaV1.5 (J) channels. Arrows (blue and red) mark the breakpoint temperature of each curve. Number of tested cells are shown in the figure. Statistically significant differences (P<0.05) are shown as follows: *12 versus 28°C; 12 versus 20°C; §20 versus 28°C.

Fig. 4.

Voltage and temperature dependence of current density and charge transfer of INa generated by zebrafish and rainbow trout NaV1.4 and NaV1.5 channels in HEK cells. (A–D) Mean results (±s.e.m.) for current–voltage relationship (A,C) and charge transfer (B,D) of INa at three different temperatures for zebrafish (ZF; N=49 cells) (A,B) and rainbow trout (RT; N=15 cells) (C,D) NaV1.4 channels. (E–H) Mean results (±s.e.m.) for current–voltage relationship (E,G) and charge transfer (F,H) of INa at three different temperatures for ZF (N=30 cells) (E,F) and RT (N=27 cells) (G,H) NaV1.5 channels. (I,J) Current density of INa in heat ramp experiments with ZF and RT NaV1.4 (I) and NaV1.5 (J) channels. Arrows (blue and red) mark the breakpoint temperature of each curve. Number of tested cells are shown in the figure. Statistically significant differences (P<0.05) are shown as follows: *12 versus 28°C; 12 versus 20°C; §20 versus 28°C.

INa generated by the zebrafish NaV1.4 channels responded to acute temperature increases in a similar manner to native ventricular INa: the current density increased, and the charge transfer decreased with increasing temperature (Fig. 4A,B). INa generated by the trout NaV1.4 channels did not show any statistically significant differences between the three temperatures for either current density or charge transfer (Fig. 4C,D). (The expression level of trout NaV1.4 channels in HEK cells was much lower than zebrafish NaV1.4 channels, which may have reduced the resolution of the analysis.) The INa generated by trout NaV1.5 channels was, however, much more heat tolerant than the native trout ventricular INa: current density and charge transfer strongly increased with increasing temperature in HEK cells (Fig. 4G,H), while in ventricular myocytes both variables were strongly reduced at 28°C (Fig. 1E,F).

INa density of the zebrafish NaV1.5 channels in HEK cells increased with increasing temperature (P<0.05) (Fig. 4E). Differently from ventricular INa, the heterologously expressed NaV1.5 channels positively responded to warming (at −50 mV) up to 28°C, i.e. INa indicated a better heat tolerance in HEK cell membranes than in ventricular myocytes. No statistically significant differences were found in charge transfer by the heterologously expressed NaV1.5 channels (Fig. 4F). INa density of the rainbow trout NaV1.5 channels in HEK cells increased with warming from 12 to 28°C (Fig. 4G). This is a striking deviation from the thermal response of the ventricular INa, which was strongly depressed at 28°C. Charge transfer by rainbow trout NaV1.5 channels was significantly increased by acute warming in HEK cells, in contrast to the observations of ventricular INa (Fig. 4H).

Inactivation kinetics of INa generated by NaV1.4 and NaV1.5 channels in HEK cells

The inactivation rate of INa increased with increasing temperature and with membrane depolarization. There were marked gene-specific differences in the rate of INa inactivation (Fig. 5). The rate of inactivation of INa produced by NaV1.5 channels was much slower than that produced by NaV1.4 channels for both zebrafish and trout genes (P<0.05). In contrast to gene-specific differences, interspecies differences in inactivation rate of INa were non-existent or small. The time constant of INa inactivation of NaV1.5 channels was almost identical for zebrafish and trout variants of the channel. In contrast, the INa generated by NaV1.4 channels was faster for zebrafish than the trout channel variant at more negative voltages (at −30 mV at 20°C, and at −30 and −20 mV at 28°C) (Fig. 5D,F). Temperature dependence (Q10 values) of INa inactivation varied between 1.8 and 6.0 with no differences between channel isoforms. However, the Q10 value of INa inactivation rate was higher in zebrafish ventricular myocytes than in trout ventricular myocytes at voltages of −10 and 0 mV (P<0.05) (Table S1).

Fig. 5.

Temperature dependence of inactivation kinetics of INa for zebrafish NaV1.4 and NaV1.5 channels in HEK cells.INa was elicited from the holding potential of −120 mV to −30 to 10 mV for 30 ms. (A–F) Means (±s.e.m.) of inactivation time constant (τf) for NaV1.5 (A,C,E) and NaV1.4 (B,D,F) at 12, 20 and 28°C as indicated. The results are from 39 and 42 cells from zebrafish (ZF) and rainbow trout (RT) channels, respectively. Representative tracings of INa at −30 mV are shown in the inset to each panel. Statistically significant differences (P<0.05) between ZF and RT are shown by dissimilar letters (a and b).

Fig. 5.

Temperature dependence of inactivation kinetics of INa for zebrafish NaV1.4 and NaV1.5 channels in HEK cells.INa was elicited from the holding potential of −120 mV to −30 to 10 mV for 30 ms. (A–F) Means (±s.e.m.) of inactivation time constant (τf) for NaV1.5 (A,C,E) and NaV1.4 (B,D,F) at 12, 20 and 28°C as indicated. The results are from 39 and 42 cells from zebrafish (ZF) and rainbow trout (RT) channels, respectively. Representative tracings of INa at −30 mV are shown in the inset to each panel. Statistically significant differences (P<0.05) between ZF and RT are shown by dissimilar letters (a and b).

Interestingly, the rate of INa inactivation of heterologously expressed zebrafish NaV1.4 and NaV1.5 Na+ channels was much faster than the inactivation kinetics of the endogenous ventricular INa. Opposite to the findings in zebrafish, the INa inactivation rate of trout NaV1.5 channels was much slower than that of trout ventricular myocytes. In contrast, the inactivation rates of INa for heterologously expressed trout NaV1.4 channels and trout ventricular myocytes were similar.

Steady-state activation and inactivation of INa generated by NaV1.4 and NaV1.5 channels in HEK cells

Acute increases in temperature had only weak effects on the voltage dependence of SS activation and inactivation of INa generated by the heterologously expressed Na+ channels (Table 2; Fig. 6). The only change for the zebrafish INa produced by NaV1.5 channels was the reduced slope of SS activation at 20 and 28°C relative to that at 12°C (P<0.05). Similar changes were observed for the rainbow trout INa generated by NaV1.5 channels (P<0.05) (Fig. 6). In addition, the rainbow trout NaV1.5 channels had a positive shift in the voltage dependence and a decrease in the slope factor of the INa SS inactivation (P<0.05) (Table 2; Fig. 6D). Elevated temperatures also reduced the slope factor of SS activation of the trout NaV1.4 channels (P<0.05) (Table 2; Fig. 6B).

Fig. 6.

Voltage and temperature dependence of steady-state activation and inactivation of INa for NaV1.4 and NaV1.5 channels in HEK cells. The voltage protocol was the same as in Fig. 2. (A,B) Steady-state activation and inactivation of zebrafish (ZF; N=49 cells) (A) and rainbow trout (RT; N=33 cells) (B) NaV1.4 channels. (C,D) Steady-state (SS) activation and inactivation of zebrafish (N=49 cells) (C) and rainbow trout (N=50 cells) (D) NaV1.5 channels.

Fig. 6.

Voltage and temperature dependence of steady-state activation and inactivation of INa for NaV1.4 and NaV1.5 channels in HEK cells. The voltage protocol was the same as in Fig. 2. (A,B) Steady-state activation and inactivation of zebrafish (ZF; N=49 cells) (A) and rainbow trout (RT; N=33 cells) (B) NaV1.4 channels. (C,D) Steady-state (SS) activation and inactivation of zebrafish (N=49 cells) (C) and rainbow trout (N=50 cells) (D) NaV1.5 channels.

DISCUSSION

The present results can be summarized in three major findings. (1) The properties of the endogenous INa of zebrafish and rainbow trout ventricular myocytes differ markedly in terms of heat tolerance and inactivation kinetics, with the zebrafish INa being more heat tolerant and more slowly inactivating. (2) The major Na+ channel isoforms of zebrafish and rainbow trout ventricles, NaV1.5 and NaV1.4, respectively, show only minor interspecies differences, when expressed in HEK cells, i.e. the orthologous Na+ channel alpha subunits are functionally (heat tolerance, inactivation kinetics) similar in the same membrane matrix. (3) When expressed in HEK cells, INa generated by NaV1.4 and NaV1.5 isoforms of both species show large channel-specific differences in inactivation kinetics, with NaV1.4 being fast and NaV1.5 slow. Taken together, the species-specific properties of ventricular INa seem to be determined partly by the expressed Na+ channel alpha subunit – NaV1.5 in zebrafish and NaV1.4 in rainbow trout – and partly by the biophysical properties of the lipid matrix/ancillary subunits of the channel assembly. Notably, the better heat tolerance of INa seems to be related to the slower inactivation kinetics of Na+ channels.

The endogenous INa of zebrafish and rainbow trout ventricular myocytes have very different inactivation kinetics: the INa of zebrafish ventricular myocytes inactivates much more slowly than INa of rainbow trout ventricular myocytes. At 12°C, the inactivation time constant of the zebrafish INa was almost an order of magnitude bigger than that of the rainbow trout (5.0±0.7 versus 0.5±0.03 ms at −20 mV). This difference largely, but not completely, disappeared when the rate of INa inactivation was measured at the acclimation temperatures of the fish (0.8±0.09 ms for zebrafish at 28°C versus 0.5±0.03 ms for trout at 12°C). As thermal acclimation does not have any effect on the inactivation kinetics of the rainbow trout ventricular INa (Haverinen and Vornanen, 2004), the differences in the inactivation rate between the two species can be regarded as adaptations of INa to the respective habitat temperatures of the species. In general, electrical excitation in nerves and muscle tissues of ectothermic animals is adapted to work best at lower temperatures in comparison with the tissues of the endothermic animals. For instance, at the typical mammalian body temperatures (36–38°C), the AP conduction of ectothermic nerve fibres suffers from heat block (Hodgkin and Katz, 1949; Volgushev et al., 2000). The gating kinetics of the plasma membrane ion channels are probably responsible for the thermal adaptation of AP conduction, allowing APs to propagate at the proper rate and frequency at the typical habitat temperatures of the species. Because zebrafish live at warmer habitats than rainbow trout, the slow inactivation kinetics of its ventricular INa may provide better excitability at higher temperatures than the fast inactivating INa of the rainbow trout. However, when comparing the intrinsic heart rates of the two species at their acclimation temperatures (130 beats min–1 for zebrafish versus 60 beats min–1 for rainbow trout) (Aho and Vornanen, 2001; Vornanen and Hassinen, 2016), the rate of INa inactivation appears to be slow. Apparently, the rate of INa recovery from inactivation does not limit heart rate in zebrafish, although this was not experimentally confirmed.

The slow rate of INa inactivation in zebrafish ventricular myocytes may protect against heat-induced impairment of electrical excitability (Park et al., 2016; Touska et al., 2018). This is consistent with our hypothesis that Na+ channels with slow gating kinetics better maintain electrical excitability at high temperatures (Vornanen, 2020). Indeed, findings from the pain receptors of human skin strongly suggest that the slow gating kinetics of Na+ channels are needed for heat resistance of INa (Touska et al., 2018). At mammalian body temperatures, most Na+ channel isoforms operate close to their optimum and a slight increase in temperature above the typical body temperature limits the density and charge transfer of INa (Touska et al., 2018). However, INa of the mammalian pain receptors works well at temperatures much above body temperature (43–50°C), largely owing to the thermal properties of the NaV1.9 Na+ channel isoform. Inactivation kinetics of this channel are more than an order of magnitude slower than those of NaV1.1–NaV1.8 isoforms (Balbi et al., 2017; Touska et al., 2018). Due to the slow inactivation of NaV1.9, it provides enough charge for depolarization of the plasma membrane at high temperatures. Analogously, the slow inactivation rate of the zebrafish cardiac INa provides more inward charge for the same peak amplitude of INa or the same number of active Na+ channels than the fast inactivating trout cardiac INa (Fig. 4). Indeed, the charge transfer/peak current ratio of the zebrafish INa is much higher at 28°C than that of the rainbow trout INa at 12°C.

The species-specific difference in the inactivation kinetics of the ventricular INa is partly explained by the difference in the alpha subunit composition of the Na+ channels. In zebrafish ventricular myocytes, the main alpha subunit is NaV1.5, which at the transcript level represents 83.1% of all ventricular Na+ channels. Transcripts of the NaV1.4 comprise only 16.2% of the zebrafish ventricular Na+ channels (Haverinen et al., 2018). In the ventricle of the rainbow trout, the situation is opposite: NaV1.4 channels form 80% of all Na+ channel transcripts, while NaV1.5 channels represent only 20% of the total channel population (Haverinen et al., 2007). Thus, in zebrafish ventricle Na+ channels are mainly of the slow isoform, while in the ventricle of the rainbow trout they are mainly the fast isoform. Based on their tissue distribution in mammals, NaV1.5 and NaV1.4 are often called ‘cardiac’ and ‘skeletal’ isoforms, respectively (Zimmer et al., 2015). The mammalian NaV1.4 is kinetically faster than NaV1.5 and therefore functionally better at eliciting fast twitches at high frequencies in skeletal muscle fibres (Wang et al., 1996; Sheets and Hanck, 1999). Although the classification of NaV1.5 and NaV1.4 to cardiac and skeletal isoforms, respectively, is not valid for fish, the kinetic similarities between the orthologous mammalian and piscine Na+ channels persist. The fast inactivation kinetics of the rainbow trout NaV1.4 can be regarded adaptive for heart function of this cold-dwelling fish.

The dominance of NaV1.5 alpha subunits in the zebrafish heart only partially explains the slow inactivation kinetics of the ventricular INa. Unlike the rainbow trout, where the inactivation rate of NaV1.4 channels in HEK cells relatively closely matches the inactivation rate of the endogenous ventricular INa, in zebrafish there is a large difference in the inactivation kinetics of the heterologously expressed NaV1.5 and the endogenous ventricular INa. As NaV1.4 and NaV1.5 Na+ channel alpha subunits of zebrafish and rainbow trout share the inactivation kinetics in the heterologous environment of HEK cells, the slow inactivation rate of the zebrafish ventricular INa is probably partly due to the general biophysical properties of the bulk lipid bilayer of ventricular myocytes in the membrane domain where they are located (Lundbaek et al., 2004). Phospholipid bilayer composition of the plasma membrane affects function of the integral membrane proteins mainly in a non-specific manner by its biophysical properties (Lundbaek et al., 2004). Elasticity (stiffness) of the lipid bilayer, determined by the lipid composition, regulates the inactivation of the voltage-gated Na+ channels. Decrease in membrane stiffness induced by amphiphiles like Triton-X or β-octyl-glucoside accelerate the rate of INa inactivation, while increase in membrane stiffness due to elevated cholesterol content decreases the rate of INa inactivation (Lundbaek et al., 2004). Therefore, it is possible that the lipid composition of the sarcolemma in zebrafish ventricular myocytes differs from that of HEK cells and rainbow trout myocytes, and results in increased stiffness, which slows the rate of INa inactivation.

Although we do not have a complete answer about the slow inactivation rate of the zebrafish ventricular INa, some possible explanations (in addition to membrane stiffness) can be mentioned to guide future studies. Na+ channels are heteromultimers of large pore-forming alpha subunits and small accessory beta subunits. Beta subunits are needed for proper transportation of the alpha subunits into the plasma membrane and they may also affect kinetics of the INa. However, beta subunits are unlikely to cause the slow inactivation of the zebrafish INa as they tend to enhance the rate of Na+ channel inactivation and recovery from inactivation (Chen and Cannon, 1995; Goldin, 2003). A more likely contributing factor is the fibroblast growth factor orthologous factor 2 (FGF2), which binds to the inactivation domain of the C-terminus in the Na+ channel alpha subunit and strongly slows the rate of inactivation (Liu et al., 2003; Li et al., 2020). Interestingly, FGF2 knock-out mice are highly sensitive to temperature change and show more cardiac conduction defects when their core body temperature is elevated (Park et al., 2016). Future studies should examine the role of FGFs in temperature dependence of electrical excitability of the fish heart.

Acute heat challenge experiments indicated that the zebrafish ventricular INa is much more resistant to high temperatures than the rainbow trout INa. Thus, the heat tolerance of INa seems to positively correlate with the upper thermal tolerance of the fish and its heart rate, although in both species the optimum temperature of INa is lower than the critical thermal maximum temperature of the fish and the TBP of the intrinsic heart rate (Beitinger, 2000; Aho and Vornanen, 2001; Cortemeglia and Beitinger, 2005; López-Olmeda and Sánchez-Vázquez, 2011; Vornanen and Hassinen, 2016). It is clear that INa of the trout ventricle would be almost non-functional at the acclimation temperature of the zebrafish, and it is likely that the kinetics of the zebrafish INa would be too slow for the trout heart at freezing temperatures.

Warming-induced decrease of heart rate is shown to be caused by atrioventricular block, probably due to the reduced excitability of the ventricle (Haverinen and Vornanen, 2020). Therefore, the thermal properties of INa are likely to affect the species-specific TBP of heart rate. Warming-induced decrease of INa and simultaneous increase in membrane K+ leak via IK1 results in source–sink mismatch, which may prevent AP generation (Vornanen, 2016; Haverinen and Vornanen, 2020; Vornanen, 2020). At low temperatures there is some excess of INa relative to the membrane leak via IK1 (INa/IK1≥1.0) called safety factor. When temperature rises the charge transfer of INa starts to decline (at the species-specific TBP) due to increased rate of inactivation: the safety factor is lost (INa/IK1<1.0) and excitation fails. In this respect, the difference in heat tolerance between the ventricular INa of rainbow trout and the INa generated by the heterologously expressed NaV1.4 and NaV1.5 channels of the trout may be important. Expression of trout INa in the HEK cell membrane makes it much more heat tolerant than it is in the native membrane surroundings. Notably, when zebrafish and trout NaV1.4 and NaV1.5 were expressed in HEK cells, inactivation rate and heat tolerance of INa were similar for the orthologous channels. It seems that the biophysical properties of the mammalian cell membrane shift the thermal tolerance window of the trout Na+ channels to higher temperatures which would, however, be suboptimal at the habitat temperature of the cold-dwelling fish.

Summary and perspectives

Heat tolerance and inactivation kinetics of INa differ strongly between zebrafish and rainbow trout ventricular myocytes. In contrast, heat tolerance and inactivation kinetics of NaV1.4 and NaV1.5 channels of zebrafish and rainbow trout are similar when expressed in the same cellular environment of HEK cells. Species-specific thermal adaptation of the ventricular INa is largely achieved by expressing a specific alpha isoform subunit of Na+ channel: the slowly inactivating NaV1.5 in zebrafish that tolerate higher temperatures, and the fast inactivating NaV1.4 in rainbow trout that favour cold waters. Differences in elasticity (stiffness) of the lipid bilayer and/or accessory protein components may also be involved in the thermal adaptation of INa. These findings are consistent with the hypothesis that slow Na+ channel kinetics are associated with increased heat tolerance of cardiac excitation. Future studies should examine the extent to which these components are flexible under temperature acclimation and therefore able to accommodate the electrical excitability of the heart for seasonal temperature changes and peak summer temperatures. As electrical excitability is regulated in basically the same way in all excitable cells, studying the biophysical properties of neuronal and muscular INa could provide clues to its role in thermal homeostasis and death of ectotherms.

Acknowledgements

We would like to thank Kontiolahti fish farm for providing the trout. Anita Kervinen is acknowledged for taking care of the fish at the university and preparing solutions for experiments.

Footnotes

Author contributions

Conceptualization: M.V.; Methodology: J.H., I.D., M.H.; Investigation: J.H., I.D., D.V.A., M.H.; Writing - original draft: J.H., D.V.A., M.H.; Writing - review & editing: M.V.; Visualization: J.H., I.D.; Project administration: M.V.; Funding acquisition: M.V.

Funding

The present study was funded by the Academy of Finland (Research Council for Biosciences and Environment; project number 15051) to M.V. and by the Interdisciplinary Scientific and Educational School of Moscow University ‘Molecular Technologies of the Living Systems and Synthetic Biology’ to D.V.A.

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Competing interests

The authors declare no competing or financial interests.

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