Animals

Hatchery-reared rainbow trout (Oncorhynchus mykiss; 73.43 ± 11.69 g, n = 18) were obtained from the local fish farm (Kontiolahti, Finland). In the animal facilities of the University of Eastern Finland, the trout were maintained in 500-L metal aquaria for a minimum of 3 wk before use in the experiments and fed aquarium fish food (Ewos) at least 5 times a week. Water temperature was regulated at 14 ± 0.5 °C (Computec Technologies), and oxygen saturation was maintained by aeration with compressed air. Groundwater (average pH 8.0, conductivity 13 µS/cm) was constantly flowing through the aquaria at a rate of 150 to 200 L/d (permission ESAVI/2832/04.10.07/2015).

Myocyte isolation

All experiments were conducted in vitro on enzymatically isolated ventricular myocytes. Fish were killed by a cranial concussion and pithing, and the heart was rapidly excised. Ventricular myocytes were isolated using retrograde perfusion of the heart and the standard concentrations of hydrolytic enzymes as reported for the method developed in our laboratory (Vornanen 1997). Cell isolation was conducted at room temperature (20–22 °C). Isolated myocytes were used in the experiments within 10 h from isolation.

Whole-cell patch clamp

Whole-cell current-clamp recordings were made by using an Axopatch 1D amplifier (Axon Instruments). Clampex 9.2 software was used for data acquisition, and off-line analysis of the recordings was done using the Clampfit 10.4 software package. During the experiments, myocytes were continuously superfused with external saline solution at a rate of 1.5 to 2 mL min^–1. The temperature of the external solution in the recording chamber was regulated at 14 °C by using a Peltier device (CL-100 from Warner Instruments or HCC-100A from Dagan) and continuously recorded on the same file with electrophysiological data. Patch pipettes were pulled (PP-83; Narishige) from borosilicate glass (King Precision) and had a resistance of 2.7 ± 0.06 MΩ when filled with the internal saline solution. After gaining a gigaohm seal, the membrane under the pipette tip was ruptured by a short-voltage pulse (zap) to get access to the cell, transients attributable to series resistance (7.3 ± 0.26 MΩ) and pipette capacitance were canceled, and the capacitive size of ventricular myocytes was determined.

For recording of APs and K⁺ currents, the external saline solution contained (mmol/L^–1) 150 NaCl, 5.4 KCl, 1.2 MgCl₂, 1.8 CaCl₂, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 10 glucose, with pH adjusted with NaOH to 7.6 at 20 °C (giving a pH of 7.68 at the experimental temperature). The composition of the pipette (internal) solution was as follows (mmol/L^–1): 140 KCl, 4 MgATP, 1 MgCl₂, 0.03 Tris-GTP, and 10 HEPES (pH adjusted with KOH to 7.2 at 20 °C). To elicit APs, ventricular myocytes were stimulated with current pulses of constant duration (4 ms) and with increasing amplitude. The initial stimulus strength was 200 pA, and it was raised with 20-pA increments until an all-or-none AP was elicited (Badr et al. 2018). The stimulation frequency was 1 Hz. The following AP parameters were analyzed off-line: resting membrane potential (V_rest, mV), threshold potential of AP (V_th, mV), threshold current (I_th, pA), critical depolarization (V_th – V_rest, mV), AP overshoot (mV), AP amplitude (AMP, 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; Figure 1). Measures V_th, I_th, and critical depolarization are for electrical excitability of ventricular myocytes, that is, the ease with which AP can be triggered by a depolarizing current.

Voltage dependency of the rapid component of the delayed rectifier K⁺ current (I_Kr) and the inward rectifier K⁺ current (I_K1) were measured using standard stimulation protocols (Vornanen et al. 2002a) from the holding potential of –80 mV. When recording I_K1, the external saline included 2 µM E-4031 (1-[2-(6-methyl-2-pyridyl)ethyl]-4-(4-methylsulfonyl-aminobenzoyl)piperidine), 0.5 µM tetrodotoxin (TTX; Tocris Cookson), and 10 µM nifedipine, to block I_Kr, I_Na, and I_CaL, respectively. Also, I_Kr was recorded in the presence of TTX (0.5 µM), nifedipine (10 µM), and 0.2 mM BaCl₂ (to block I_K1).

The fast Na⁺ current (I_Na) was measured under a reduced Na⁺ gradient (20 mM [Na⁺]_o, 5 mM [Na⁺]_i) across the sarcolemma to obtain good control of the membrane voltage. The composition of the external saline was (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). Nifedipine (10 µmol L^–1) was included in the external solution to block I_CaL. The pipette solution consisted of (in mmol L^–1) 5 NaCl, 130 CsCl, 1 MgCl₂, 5 egtazic acid (EGTA), 5 Mg₂ATP, and 5 HEPES (pH adjusted to 7.2 with CsOH at 20 °C). Using established stimulus protocols, I_Na was elicited from a holding potential of –120 mV (Haverinen and Vornanen 2006; Haverinen et al. 2018b).

The composition of the external saline solution for recording I_CaL was as follows (mmol/L^–1): 150 NaCl, 5.4 CsCl, 1.8 CaCl₂, 1.2 MgCl₂, 10 HEPES, and10 glucose (pH adjusted to 7.6 at 20 °C with CsOH). We included TTX (0.5 μM) in this saline to block Na⁺ current (I_Na; Vornanen 1998). Because Cs⁺ may flow through the Erg K⁺ channels, 2 μM E-4031 was included in the external solution to prevent contamination by I_Kr. The pipette solution contained (mmol L⁻¹) 130 CsCl, 15 tetraethylammonium chloride, 5 MgATP, 1 MgCl₂, 5 oxaloacetate, 10 HEPES, and 5 EGTA (pH adjusted to 7.2 at 20 °C with CsOH; all chemicals from Sigma). We elicited I_CaL from a holding potential of –80 to +10 mV at a frequency of 0.2 Hz. Recording of I_CaL is complicated by time-dependent rundown (decline) of the current. To minimize the effect of rundown on results, time-dependent changes in I_CaL were monitored after getting access to the whole configuration. For the same reason, the analysis of PAH effects was limited to the 2 highest concentrations. Only those cells where I_CaL stabilized within approximately 5 min from the start of recording were accepted for analysis.

PAHs

The stocks of phenanthrene (Sigma-Aldrich) and retene (MP Biomedicals) were made in dimethyl sulfoxide (DMSO) at 20 mM. Test solutions at concentrations of 0.3, 1.0, 10, and 30 µM for phenanthrene and 0.1, 1.0, and 10 µM for retene were made daily in external saline solutions. Effects of the highest DMSO concentration in the experimental solutions on AP parameters and ion currents were tested in separate experiments. No statistically significant effects were noticed.

Statistical analyses

After checking the normality of distribution and equality of variances, one-way analysis of variance (with Tukey's or Dunnett's T3 post hoc test) or nonparametric test (with Friedman's test) were used for evaluating the effect of different PAH concentrations on AP parameters and maximum ion currents. All statistical tests were performed using SPSS (IBM; Ver 21.0) software. Data are presented as mean ± standard error of the mean (SEM), and p < 0.05 was considered statistically different.

Phenanthrene and retene differentially modify the AP in rainbow trout ventricular myocytes

Phenanthrene had no effect on the duration of AP at the level of 50% repolarization (APD50; Figure 2A and E) but shortened it at the zero voltage level (APD0) at 30 µM (Figure 2A). Phenanthrene increased the maximum upstroke velocity (+dV/dt) at 1 and 10 µM and accelerated the maximum rate of AP repolarization (–dV/dt) at 10 and 30 µM (Figure 2C). Retene had more pronounced effects on APs than phenanthrene. The duration of the AP (APD50 and APD0) was strongly shortened at 1 and 10 µM concentrations (Figure 3A and E). Retene augmented the AP amplitude at 10 µM and increased the overshoot at 1 and 10 µM (Figure 3B). The maximum rate of AP upstroke became faster at all concentrations of retene, but the effect on –dV/dt was significant only at 10 µM (Figure 3C). Excitability of ventricular myocytes was decreased at the highest (10 µM) retene concentration as the critical depolarization needed to elicit AP was approximately 18% higher than in the control (Figure 3B).

Phenanthrene and retene modulate cardiac I_Na, I_CaL, and I_Kr currents but have no effect on I_K1

Phenanthrene and retene affected all studied ventricular ion currents except I_K1, but retene caused the effects at lower concentrations than phenanthrene. Under exposure of 10 µM phenanthrene or 1 µM retene, the peak density of I_Na was increased by 12 and 17%, respectively (Figure 4A and B). The effects of the highest concentrations (30 µM phenanthrene, 10 µM retene) were slightly less and statistically nonsignificant (Figure 4B).

After getting electrical access to the cell, there was a clear increase in the amplitude of I_CaL attributable to the buffering of intracellular free Ca²⁺ by EGTA of the pipette solution (removal of Ca²⁺-dependent inactivation of I_CaL). Then, the current stabilized and enabled the recording of drug effects on I_CaL (Figure 5A and B). Phenanthrene reduced I_CaL, but the effect was statistically significant only at the highest concentration tested, 30 µM (Figure 5C). Retene diminished I_CaL at 1 and 10 µM (Figure 5C).

Whereas phenanthrene attenuated I_Kr at 10 and 30 µM, retene was effective even at the lowest test concentration (0.1 µM; Figure 6). Both phenanthrene and retene decreased the I_Kr tail currents at all voltages, where the tail current was activated (Figure 6C and D). The maximum inhibition of I_Kr tail at +40 mV was 79.3 and 59.2% for phenanthrene and retene, respectively. During the depolarizing prepulse, phenanthrene and retene inhibited I_Kr (I_Kractiv) in the voltage range between 0 and +20 mV but did not have any effect at +40 and +60 mV (Figure 6E and F). This suggests that there is a phenanthrene- and retene-resistant current underlying I_Kr, probably the slow component of the delayed rectifier K⁺ current, I_Ks. Neither of the PAHs had an effect on the background inward rectifier, I_K1 (Supplemental Data, Figure S1A and B).

Effects on AP

Both 3-ring PAHs affected the ventricular AP of the rainbow trout heart, but retene was a much stronger AP modifier than phenanthrene. Phenanthrene did not change V_rest or AMP, consistent with the findings from bluefin tuna cardiomyocytes (Brette et al. 2017); V_rest is maintained by the I_K1, which remained untouched by phenanthrene. Phenanthrene had only minor effects on APD; APD was slightly reduced at the zero-voltage level but remained unchanged at the 50% repolarization level. In this respect, rainbow trout clearly differs from bluefin tuna, where phenanthrene lengthened ventricular APD (Brette et al. 2017). The APD is regulated by a delicate balance between influx of Ca²⁺ via I_CaL and efflux of K⁺ via I_Kr, I_Ks, and I_K1 (Grant 2009). Because the resistance of the sarcolemma at the AP plateau is high (Ca²⁺ and K⁺ fluxes are small), small changes in the amplitude and activation/inactivation rate of Ca²⁺ and K⁺ currents will affect APD (Zaza 2010). Shortening of AP at the zero-voltage level suggests that in the early plateau I_CaL is reduced more than I_Kr by phenanthrene. The results of the present study show a small but clear increase in +dv/dt in rainbow trout with 10 and 30 µM phenanthrene. Phenanthrene also steepened the rate of repolarization (–dv/dt). These are novel actions of PAHs on the AP of the fish heart. Collectively, the present findings show that phenanthrene has partly different effects on cardiac APs in rainbow trout and bluefin tuna.

In contrast to phenanthrene, retene strongly shortened APD, and the effect occurred at the lower drug concentrations (1 and 10 µM). The strong shortening of APD suggests that the relative effect of retene on I_CaL is stronger than its effect on I_Kr, the main repolarizing current. The effects of retene differ from those of phenanthrene in that AMP and overshoot were enhanced by retene but not by phenanthrene. This difference may be associated with slightly stronger stimulation of I_Na by retene. Notably, the highest retene concentration (10 µM) attenuated excitability of ventricular myocytes as critical depolarization was increased. In vivo, the decrease in excitability could cause interruptions in AP propagation between ventricular myocytes (Vornanen 2016).

Effects on cardiac ion currents

The Na⁺ current (I_Na) is active during the upstroke of the AP, causing depolarization of the sarcolemma by fast and large influx of Na⁺. Both phenanthrene and retene increased the peak I_Na density in the ventricular myocytes of rainbow trout. Retene was more potent than phenanthrene at enhancing I_Na, which is in line with its larger effect on +dv/dt and overshoot of the AP. At the level of intact tissue the larger I_Na means a faster propagation of AP in the ventricular wall. To our knowledge, there are no earlier data on the effects of PAHs on fish cardiac I_Na. However, in bluefin tuna ventricular myocytes, phenanthrene did not affect the upstroke velocity of the AP, thus suggesting species-specific differences in PAH modulation of I_Na.

Currents I_CaL and I_Kr are the main determinants of the long AP plateau. They are counteracting currents because I_CaL is depolarizing and I_Kr repolarizing. The net outcome of the inhibition of these currents can be seen as changes in APD. Notably, both PAHs caused shortening of APD, but retene was much more powerful than phenanthrene. Strong shortening of APD by retene indicates that the net charge influx via I_CaL is inhibited more than the K⁺ efflux via I_Kr. The final phase 3 repolarization is accelerated by the background inward rectifier I_K1. The increase in the rate of –dV/dt by PAHs is probably attributable to the resistance of I_K1 to retene and phenanthrene whereby the uninhibited I_K1 overwhelms the reduced I_CaL.

The lowering of Ca²⁺ influx in phenanthrene-treated rainbow trout cardiac myocytes is in line with previous research showing that phenanthrene decreased Ca²⁺ transients in ventricular myocytes of scombrid fishes, even though scombrids may be more dependent on intracellular Ca²⁺ stores for contractile activation (Brette et al. 2017). The magnitude of I_CaL inhibition by phenanthrene seems to differ between tuna and rainbow trout: whereas 5 µM phenanthrene decreased I_CaL by approximately 30% and 25 µM phenanthrene by approximately 75% in bluefin tuna, in rainbow trout 10 and 30 µM phenanthrene attenuated I_CaL only by approximately 20 and approximately 40%, respectively (Brette et al. 2017). On the other hand, in tuna the reduction of I_CaL should be partly compensated by the prolonged AP plateau. Brette et al. (2017) did not report whether the rundown of I_CaL was taken into account, which might have also affected their results. In rainbow trout, retene was more potent than phenanthrene at attenuating I_CaL because the same reduction of I_CaL by approximately 20 and approximately 40% was achieved with lower concentrations of retene of 1 and 10 µM, respectively.

The strong shortening of APD and inhibition of I_CaL by retene are expected to have a strong reducing effect on the intracellular free Ca²⁺ concentration. In trout ventricular myocytes, the activation of contraction is largely dependent on the sarcolemmal Ca²⁺ influx during the AP plateau because approximately two-thirds of the activator Ca²⁺ is estimated to come from the extracellular space (Vornanen et al. 2002b). Inhibition of I_CaL and shortening of the plateau means that Ca²⁺ influx is smaller and there is less time for Ca²⁺ entry. In the intact ventricle, this should appear as reduced force of contraction. Indeed, exposure to PAHs or oil reduces atrial and ventricular contraction and diminishes cardiac stroke volume in larval fish (Incardona et al. 2013; Jung et al. 2013; Edmunds et al. 2015; Esbaugh et al. 2016; Sørhus et al. 2016; Khursigara et al. 2017; Perrichon et al. 2018). In rainbow trout yolk sac larvae, retene causes pericardial and yolk sac edemas (Billiard et al. 1999; Scott et al. 2011; Vehniäinen et al. 2016), phenomena often seen with PAH and oil exposures and proposed to be caused by reduced cardiac output (Incardona and Scholz 2016). Taken together, inhibition of I_CaL and shortening of the AP plateau would compromise contractility and cardiac output of the heart with the outcome of reduced physical performance level and fitness of the fish. These effects would be particularly strong under the intoxication by retene.

The I_Kr channels are notorious for their susceptibility to inhibition by low concentrations of various small-molecule compounds (Sanguinetti and Tristani-Firouzi 2006). The wide pore cavity of the channel allows access of small molecules to the pore (Vandenberg et al. 2001). Therefore, it is no surprise that also PAHs can block these channels in fish cardiac myocytes. In rainbow trout ventricular myocytes, 10 and 30 µM phenanthrene reduced I_Kr by 43 and 75%, respectively. This is slightly less than the inhibition in bluefin tuna, where 5 and 25 µM phenanthrene decreased I_Kr by 60 and more than 85%, respectively (Brette et al. 2017). In rainbow trout, the effect of retene on I_Kr was more pronounced because 10 µM retene caused a 60% reduction in I_Kr (Supplemental Data, Figure S2).

In mammalian heart, blockade of I_Kr by many drugs is shown to be proarrhythmic and able to induce chaotic ventricular tachycardia, torsades de pointes (Vandenberg et al. 2001). However, if both I_Kr and I_CaL are inhibited simultaneously and at similar drug concentrations, the effect is antiarrhythmic, even when drugs prolong, shorten, or triangulate ventricular APs (Kramer et al. 2013; Obejero-Paz et al. 2015). A typical example is verapamil, a useful human cardiovascular medicine, which inhibits human I_Kr and I_CaL at similar concentrations (Shetuan et al. 1999; Kang et al. 2012). Because PAHs inhibit both I_Kr and I_CaL at similar micromolar concentrations, they should not be proarrhythmic in fish ventricle. However, I_Kr and I_CaL are essential components of the cardiac pacemaker, which determines the rate and rhythm of the heartbeat (Schram et al. 2002). Half-maximal inhibition of I_Kr by E-4031 is known to reduce the beating rate of rainbow trout sinoatrial preparations, and therefore inhibition of I_Kr might explain the PAH-induced bradycardia of larval fish (Haverinen and Vornanen 2007). Inhibition of I_CaL is likely to affect impulse generation and conduction of the nodal tissues (sinoatrial pacemaker and atrioventricular canal) because I_CaL is the main determinant for the rate of AP upstroke and impulse conduction (I_Na is absent or small in nodal cells; Schram et al. 2002). Inhibition of I_CaL might therefore appear as atrioventricular block and ventricular bradycardia, phenomena seen in larval fish exposed to PAHs or oil (Incardona et al. 2004, 2005, 2009, 2011; Zeltser et al. 2004; Perrichon et al. 2016, 2018). However, care must be taken when applying results from mature fish to embryos or larval fish.

Because retene is quite hydrophobic (logK_OW ~6), the actual concentrations in the test chamber most probably were lower than nominal. It must also be borne in mind that retene is quickly metabolized by cytochrome P450 (CYP1A) in fish, and this may lower the concentration of parent retene that reaches cardiac myocytes in vivo (Hawkins et al. 2002). In nature, however, fish are exposed to PAH mixtures that frequently contain CYP1A inhibitors, which in turn decrease the metabolism of PAHs and thus increase the concentration of parent compounds (Hawkins et al. 2002).

Retene is an AhR agonist, and it disturbs cardiovascular development in fish via activating AhR and altering transcription (Scott et al. 2011; Vehniäinen et al. 2016). The present study shows that in addition to this transcriptional route, retene has a direct effect on cardiac function via modulating voltage-gated ion channel activity. Because normal cardiac function is important for cardiovascular development (Glickman and Yelon 2002; Incardona et al. 2015), as well as the development of other tissues and organs (Incardona et al. 2004), retene may cause developmental defects also independently of the AhR. The ability to modulate the activity of cardiac ion channels also means that in addition to early-life stages, retene may be cardiotoxic to juveniles and adults.