Expression of calcium channel transcripts in the zebrafish heart: dominance of T-type channels

Voltage-gated Ca²⁺ channels enable Ca²⁺ influx into cells and mediate a wide variety of physiological responses including muscle contraction, hormone secretion, neuronal transmission, gene expression and cell division (McDonald et al., 1994; Hofmann et al., 2014). In the sarcolemma of cardiac myocytes, two major types of Ca²⁺ channel are usually present: L-type (long-lasting, high-threshold) Ca²⁺ channels of the subfamily Ca_v1 and T-type (transient, low-threshold) Ca²⁺ channels of the subfamily Ca_v3 (Ertel et al., 2000; Catterall et al., 2005).

In the mammalian heart, L-type Ca²⁺ channels are abundantly expressed in atrial and ventricular myocytes, sinoatrial pacemaker cells and atrioventricular nodal cells (Hagiwara et al., 1988; Zamponi et al., 2015; Mesirca et al., 2015). L-type Ca²⁺ current (I_CaL) is a critical component in excitation–contraction (E–C) coupling of atrial and ventricular myocytes by inducing Ca²⁺ release (Ca²⁺-induced Ca²⁺ release, CICR) from the sarcoplasmic reticulum (SR) (Fabiato, 1983; Bers, 2002). In sinoatrial myocytes, I_CaL generates the upstroke of pacemaker action potential (AP) (Irisawa et al., 1993). In cardiac myocytes of fetal and neonatal mammals and ectothermic vertebrates, I_CaL directly contributes to the cytosolic Ca²⁺ transient, as CICR is less powerful in these cells (Fabiato and Fabiato, 1978; Morad et al., 1981; Vornanen, 1996; Vornanen et al., 2002).

The distribution of T-type Ca²⁺ channels and current (I_CaT) is more limited in the adult mammalian heart. T-type Ca²⁺ channels are strongly expressed in sinoatrial and atrioventricular nodes, weakly expressed in atrial myocytes and are usually not present in healthy ventricular myocytes (Perez-Reyes, 2003; Ono and Iijima, 2010). It should be noted, however, that a small I_CaT is present in guinea-pig and dog ventricular myocytes (Nilius et al., 1985; Mitra and Morad, 1986; Balke et al., 1992; Wang and Cohen, 2003). I_CaT has a significant role in cardiac pacemaking by contributing to the diastolic depolarization of pacemaker AP, while its significance in E–C coupling of atrial and ventricular myocytes is incompletely resolved (Zhou and January, 1998; Kitchens et al., 2003; Jaleel et al., 2008; Ono and Iijima, 2010; Mesirca et al., 2015). I_CaT is upregulated in diseased mammalian heart, including genetic hypertension, cardiac hypertrophy and atherosclerosis (Takebayashi et al., 2006; Chiang et al., 2009).

I_CaL has been recorded in atrial and ventricular myocytes of several fish species including the zebrafish (Danio rerio) (Maylie and Morad, 1995; Vornanen, 1997, 1998; Hove-Madsen and Tort, 1998; Vornanen et al., 2002; Shiels et al., 2006; Brette et al., 2008; Zhang et al., 2011; Galli et al., 2011; Haworth et al., 2014; Haverinen et al., 2014). I_CaT has been shown to be present in atrial and ventricular myocytes of the dogfish (Squalus acanthias) and zebrafish hearts, and in atrial myocytes of the Siberian sturgeon (Acipenser baerii) heart (Maylie and Morad, 1995; Warren et al., 2001; Nemtsas et al., 2010; Haworth et al., 2014). In contrast to the rich data on the mammalian cardiac Ca²⁺ channels, very little is known about Ca²⁺ channels of the fish heart, even though sarcolemmal Ca²⁺ channels play a central role in cardiac E–C coupling of fish (Vornanen et al., 2002; Zhang et al., 2011). There is also little knowledge about the molecular and genetic background of cardiac Ca²⁺ channels in zebrafish (Rottbauer et al., 2001), which is somewhat surprising considering that this species is a popular model for cardiovascular drug screening and human cardiac diseases (Bakkers, 2011; MacRae and Peterson, 2015). To be a useful model for the human heart, it is crucial to know the molecular basis of Ca²⁺ currents in the zebrafish heart, as Ca²⁺ channels are important therapeutic targets for the treatment of cardiovascular diseases (Belardetti and Zamponi, 2012). To this end, we measured Ca²⁺ channel transcript expression in the zebrafish heart. In addition, Ca²⁺ influx via I_CaT and I_CaL into zebrafish ventricular myocytes was determined using the whole-cell patch-clamp method. Because I_CaL is generally regarded as the most important cardiac Ca²⁺ current, our working hypothesis was that different L-type Ca²⁺ channels form the dominant Ca²⁺ channel subfamily in the zebrafish atrium and ventricle.

The wild-type Turku zebrafish line (kindly donated by Prof. Pertti Panula, University of Helsinki) was raised and maintained at the animal facilities of UEF (University of Eastern Finland, Joensuu) according to the established principles (Westerfield, 2007). The rearing temperature of the fish was 28°C. 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/2832/04.10.07/2015).

Fish were killed by immersing them in ice water and the heart was excised. Total RNA was isolated from three atrial and ventricular samples (see ‘Patch-clamp analysis of I_CaT and I_CaL’, below) (each containing cardiac tissue of eight adult zebrafish, 1.5 years old) using TriReagent (Thermo Scientific, Waltham, MA, USA). RNA was DNase treated with RNase-free DNaseI (Thermo Scientific) and reverse-transcribed with random hexamer and oligo(dT) primers and Maxima RNase H-reverse transcriptase (Thermo Scientific) following the manufacturer's protocols. In order to design gene-specific primers for qPCR, a and b isoforms of each cacna (α1) gene were aligned and primers were designed to non-homologous regions. Moreover, all 18 α1 genes were aligned using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and the specificity of primers was checked. Finally, PrimerBlast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) was used to validate the targets of each primer pair. qPCR was performed using Maxima SYBR Green qPCR Master Mix (Thermo Scientific) in an Aria MX Real-Time PCR machine (Agilent Technologies, Santa Clara, CA, USA). Accession numbers of the genes and primers used in this study are listed in Table 1. The thermal conditions were as follows: enzyme activation at 95°C for 10 min followed by 40 cycles at 95°C for 10 s, 58°C for 20 s and 72°C for 30 s, and final extension at 72°C for 3 min. After the qPCR reaction, the specificity of amplification was checked by melting curve analysis (from 65 to 95°C) and by running the qPCR products on an agarose gel. Data were normalized to the geometric mean of dnaja2 (DnaJ homologue subfamily A member 2) and actb1 (β-actin) mRNA expression levels.

Electrophysiological experiments were conducted on enzymatically isolated ventricular myocytes of adult zebrafish (1.5 years old, 0.68±0.03 g). Fish were stunned with a quick blow to the head, the spine was cut and the heart was excised. The yield of atrial myocytes was low and therefore extensive characterization of atrial electrophysiology was not possible. The myocyte isolation procedure was essentially similar to our original isolation method for fish hearts (Vornanen, 1997), but scaled down to the size of small zebrafish hearts. For retrograde perfusion of the heart, a small cannula (34 gauge, TE734025, Adhesive Dispensing Ltd, Milton Keynes, Bucks, UK) was inserted via the bulbus arteriosus into the ventricle and the bulbus was secured with a fine thread around the cannula. The heart was perfused first with Ca²⁺-free solution for 5 min and then with the same solution but with added hydrolytic enzymes [Collagenase Sigma IA, Trypsin type VI (Serva, Heidelberg, Germany) together with fatty acid-free serum albumen] for 15–20 min. Myocytes were stored at 5°C and used on the same day they were isolated. The composition of the Ca²⁺-free solution was (in mmol l⁻¹): 100 NaCl, 10 KCl, 1.2 KH₂PO₄, 4 MgSO₄, 50 taurine, 20 glucose and 10 Hepes, with pH adjusted to 6.9 with KOH at 20°C.

An Axopatch 1D amplifier (Axon Instruments, Saratoga, CA, USA) with a CV-4 1/100 head-stage was used for whole-cell patch-clamp experiments. The external solution used for I_CaT and I_CaL recordings contained (in mmol l⁻¹): 20 CsCl, 120 tetraethylammonium chloride, 1 MgCl₂, 2 CaCl₂, 10 glucose and 10 Hepes (pH adjusted to 7.7 with CsOH). The solution was Na⁺ free and included 0.5 µmol l⁻¹ tetrodotoxin (Tocris Cookson, Bristol, UK) to prevent Na⁺ current (I_Na). As Cs⁺ can flow through Erg K⁺ channels, 2 μmol l⁻¹ E-4031 (Tocris Cookson) was included in the external saline solution to prevent the rapid component of the delayed rectifier K⁺ current (I_Kr). Temperature was regulated at 28°C by using a Peltier device (HCC-100A, Dagan, MN, USA). The pipette solution contained (in 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, St Louis, MO, USA), giving a mean (±s.e.m.) resistance of 2.5±0.03 MΩ.

After gaining a giga-ohm seal and access to the cell, current transients due to series resistance (6.17±0.22 MΩ) and pipette capacitance (8.20±0.10 pF) were cancelled out, and the capacitive size of ventricular myocytes (27.77±1.11 F, n=62) was determined. The total I_Ca, including both I_CaT and I_CaL, was elicited by 300 ms square wave pulses from the holding potential (V_h) of −90 mV with 10 mV increments to cover a voltage range from −80 to +50 mV. I_CaL was elicited from a V_h of −50 mV to voltages ranging from −40 to +50 mV. I_CaT was obtained as the difference current from these two protocols.

Charge transfer through Ca²⁺ channels was determined by integrating the inactivating portion of the Ca²⁺ current for 300 ms voltage pulses from −90 mV to −30 mV for I_CaT and from −50 mV to 0 mV for I_CaL. The change in total intracellular cellular Ca²⁺ concentration ([Ca²⁺]_i) due to Ca²⁺ influx via T-type and L-type Ca²⁺ channels was calculated from the measured cell capacitance and the experimentally determined surface-to-volume ratio of the cells (1.15): as described earlier in detail (Vornanen, 1997). [Ca²⁺]_i was expressed for the non-mitochondrial cell volume assuming a non-mitochondrial volume fraction of 0.55.

Statistics were performed using SPSS version 21.0. Statistically significant differences between sarcolemmal Ca²⁺ influxes by I_CaT versus I_CaL were detected through Mann–Whitney U non-parametric test for two independent samples, after checking the normality of distribution and carrying out necessary transformation of the data. Differences between mean transcript expression values of the five Ca²⁺ channel types and concentration-dependent I_CaT inactivation by Ni²⁺ were compared using one-way ANOVA followed by Tukey's post hoc test. Paired comparisons between transcripts of Ca²⁺ channel genes and T- and L-type Ca²⁺ current were done by two-tailed Student's t-test. The significance threshold was P<0.05.

We performed qPCR to investigate the mRNA expression of T-type (Ca_v3.1, Ca_v3.2a, Ca_v3.2b, Ca_v3.3a and Ca_v3.3b), L-type (Ca_v1.1a, Ca_v1.1b, Ca_v1.2, Ca_v1.3a, Ca_v1.3b, Ca_v1.4a and Ca_v1.4b), P/Q-type (Ca_v2.1a and Ca_v2.1b), N-type (Ca_v2.2a and Ca_v2.2b) and R-type (Ca_v2.3a and Ca_v2.3b) Ca²⁺ channel α-subunit genes in the atrium and ventricle of 1.5 year old zebrafish. Interestingly, in the atrium, transcript expression of T-type Ca²⁺ channels (64.1±3.7% of all Ca_v transcripts) was significantly higher than that of L-type Ca²⁺ channels (33.8±3.4%; P<0.05) (Fig. 1). In the ventricle, T-type channel transcripts (54.9±4.4%) seemed to be more numerous than L-type channel transcripts (41.1±4.4%), but the difference was not statistically significant (P>0.05). Transcripts of P/Q-type channels constituted 1.7±0.3% and 4.0±0.5% of all Ca²⁺ channel transcripts in the atrium and ventricle, respectively (Fig. 1). N- and R-type Ca²⁺ channel transcripts were also present, but the expression levels were low in both cardiac chambers (less than 0.5% of all Ca_v transcripts).

At transcript level, the main T-type Ca²⁺ channel isoform in the zebrafish heart was α1G (encoding Ca_v3.1), representing 99.7±0.05% and 98.9±0.3% of T-type transcripts in the ventricle and atrium, respectively (Fig. 2). The dominant L-type Ca²⁺ channel isoform was Ca_v1.2 (encoded by the α1C gene), comprising 93.0±1.2% and 95.9±0.7% of all Ca_v1 transcripts in the ventricle and atrium, respectively (Fig. 2). Transcript expression of α1G was significantly (P<0.05) higher – in both the ventricle (54.8±4.4% of all Ca_v transcripts) and the atrium (63.4±3.9%) – than that of the dominant L-type Ca²⁺ channel, α1C (38.3±4.6% in the ventricle and 32.4±3.4% in the atrium) (Table 2). P/Q-type Ca²⁺ channel isoform Ca_v2.1b (encoded by α1Ab) showed the third highest expression level, representing 3.8±0.5% and 1.6±0.3% of all Ca_v transcripts in the ventricle and atrium, respectively (Table 2). Collectively, these data show that α1G mRNA expression is dominant in both cardiac chambers of the adult zebrafish heart.

I_CaL was determined as the current elicited from a V_h of −50 mV. I_CaT was obtained by subtracting I_CaL from the current elicited from a V_h of −90 mV, where both channels are available for opening. Ventricular myocytes of the zebrafish heart had large I_CaT and I_CaL (Fig. 3). I_CaT had a peak current density of 6.3±0.8 pA pF⁻¹ at −30 mV, whereas I_CaL peaked at 0 mV with a density of 7.7±0.8 pA pF⁻¹ (P>0.05) (Fig. 3B). Densities of I_CaT and I_CaL varied markedly between individual ventricular myocytes (Fig. 3C). Typical densities for I_CaL were 4–6 pA pF⁻¹. The frequency distribution of I_CaT density was flatter than that of I_CaL without any typical density value.

T-type Ca²⁺ channels are blocked by Ni²⁺. As Ca_v3.1 (α1G), Ca_v3.2 (α1H) and Ca_v3.3 (α1I) isoforms are differently sensitive to Ni²⁺ (Lee et al., 1999), inhibition of I_CaT by Ni²⁺ can be used to trace isoform composition of cardiac myocytes. To this end, Ni²⁺ sensitivity of I_CaT was examined by cumulative additions of NiCl₂ (0.3–1000 µmol l⁻¹) to the external saline solution. I_CaT was inhibited by Ni²⁺ in a concentration-dependent manner (Fig. 4A). Half-maximal inhibition (IC₅₀) occurred at 92.1±0.1 µmol l⁻¹ Ni²⁺ (Fig. 4B). Ni²⁺ also slowed recovery from inactivation of I_CaT (Fig. 4B, inset).

Sarcolemmal Ca²⁺ influx was obtained from the integrals of I_CaT and I_CaL at −50 and 0 mV, respectively (Fig. 5A). In ventricular myocytes, cytosolic Ca²⁺ increments via I_CaT and I_CaL were 41.2±7.3 and 88.5±20.5 µmol l⁻¹ per non-mitochondrial cell volume, respectively (Fig. 5B). This means that I_CaT was responsible for 31.7% of the total Ca²⁺ influx via sarcolemmal Ca²⁺ channels.

Ten Ca²⁺ channel α-subunit genes, divided into three subfamilies (Ca_v1–3), exist in vertebrate genomes (Catterall et al., 2005). Because of the whole-genome duplication in the early teleosts, fish genomes include a number of gene paralogues. Therefore, a higher diversity of Ca²⁺ channel genes is expected to exist in fish genomes (Jegla et al., 2009). Indeed, 21 α-subunit genes were found in the genome of fugu (Fugu rubripes) (Wong et al., 2006). In the current study, we provide a complete survey of Ca²⁺ channel α-subunit expression in the zebrafish heart. Transcript abundance of 18 Ca²⁺ channel α-subunit genes was quantified in the atrium and ventricle of the zebrafish heart. Several studies have shown that expression of ion channel proteins in the heart is predominantly determined at the level of transcription (Rosati and McKinnon, 2004; Marionneau et al., 2005; Gaborit et al., 2007; Chandler et al., 2009; Abd Allah et al., 2012). Therefore, we believe that the relative abundance of gene transcripts is a good surrogate for Ca²⁺ channel composition and expression in atrial and ventricular muscle of the zebrafish heart. Overall, the present findings show that the diversity of Ca²⁺ channels expressed in the zebrafish heart is higher than that in mammalian hearts and T-type channels are more abundant than L-type Ca²⁺ channels.

To our knowledge, this is the first quantitative analysis of Ca²⁺ channel composition and expression in the fish heart. The main finding of the present study is that, at the transcript level, T-type Ca²⁺ channels with a relative abundance of 55–64% form the biggest Ca²⁺ channel subfamily in the zebrafish heart. This is in striking contrast to the human heart, where L-type Ca²⁺ channels make up 96–98% of the Ca²⁺ channel transcripts in both atria and ventricles, while T-type Ca²⁺ channels represent only 2–4% of the transcripts (Gaborit et al., 2007). The predominance of T-type Ca²⁺ channels is probably not common among fish hearts in the light of the Ca²⁺ current recordings. I_CaL is the main Ca²⁺ current in atrial and ventricular myocytes of many fish species including crucian carp (Carassius carassius), rainbow trout (Oncorhynchus mykiss), burbot (Lota lota) and bluefin tuna (Thunnus orientalis) (Vornanen, 1997, 1998; Hove-Madsen and Tort, 1998; Shiels et al., 2006; Shiels et al., 2015). It remains to be shown whether the strong expression of T-type Ca²⁺ channels in ventricles is typical for Danio species in general (a phylogenetic trait) or is an adaptation of tropical fishes to high environmental temperatures.

In zebrafish heart, five isoforms of T-type Ca²⁺ channels are expressed, while in the human heart only two gene products (α1H, α1G) are present (Gaborit et al., 2007). Different from mammalian genomes, in the zebrafish genome two paralogues exist for α1H and α1I, and both gene products are expressed to some extent in the heart. Interestingly, the main T-type Ca²⁺ channel isoform of the zebrafish ventricle is Ca_v3.1 (α1G). In the right ventricle and Purkinje fibres of the human heart, T-type Ca²⁺ channel transcripts are weakly expressed, and Ca_v3.2 (α1H) is the predominant isoform in both tissues (Gaborit et al., 2007). In the human right atrium, Ca_v3.1 (α1G) is the main T-type Ca²⁺ channel isoform, comprising 73.6% of T-type Ca²⁺ channel transcripts and 3.0% of all Ca²⁺ channel transcripts (Gaborit et al., 2007).

The density of I_CaT is significantly higher in the ventricles of fetal and neonatal mammals than in the ventricles of adult mammals (Perez-Reyes, 2003; Ono and Iijima, 2010). The developmental decrease in I_CaT density of the rat ventricle is associated with a significant shift in isoform composition from predominantly α1G channels to α1H channels (Ferron et al., 2002). mRNA expression of T-type Ca²⁺ channel α1 subunits (α1G, α1H) in the diseased (cardiac hypertrophy and failure) mammalian heart is significantly higher than that in healthy controls (Perez-Reyes, 2003; Ono and Iijima, 2010) (see ‘Pathophysiology of T-type Ca²⁺ channels’, below).

Taken together, the diversity of isoform composition of T-type Ca²⁺ channels is wider in zebrafish ventricle than in human (mammalian) ventricles and the major isoform is Ca_v3.1 (encoded by α1G) instead of the human Ca_v3.2 (encoded by α1H). With respect to expression of T-type Ca²⁺ channels, the zebrafish ventricle more closely resembles perinatal and diseased mammalian ventricles than healthy adult mammalian ventricles. Overall, isoform composition of T-type Ca²⁺ channels in zebrafish ventricle is more diverse and relative transcript abundance is markedly higher than in adult human ventricles.

Instead of the four mammalian L-type Ca²⁺ channel genes, α1C, α1D, α1F and α1S, seven isoforms are known for the zebrafish, because α1D, α1F and α1S genes are duplicated. All seven L-type Ca²⁺ channel genes are expressed to some extent in the zebrafish heart. Similar to the human heart (Gaborit et al., 2007), α1C (encoding Ca_v1.2) is the most expressed L-type Ca²⁺ channel isoform gene in zebrafish atrium and ventricle, and it is almost equally expressed in the two cardiac chambers. Another L-type Ca²⁺ channel expressed in the human heart is Ca_v1.3 (α1D), but the expression level is quite low, representing 0.04% and 1.13% of all Ca_v1 transcripts in ventricular and atrial myocardia, respectively (Gaborit et al., 2007) (Table 2). In the zebrafish heart, both Ca_v1.3 (α1D) isoforms are very weakly expressed, whereas Ca_v1.1 (α1Sa) shows the second highest expression level among the L-type channels in both atrium and ventricle (Fig. 2). Collectively, the relative expression of L-type Ca²⁺ channel transcripts is much lower (41.1% versus 98.4%), and L-type Ca²⁺ channel composition is more diverse in zebrafish than in human heart.

Ventricular myocytes of the zebrafish heart have a large I_CaT. The peak current density of I_CaT is 83.1% of the respective value of I_CaL. The slightly smaller density of I_CaT in comparison to I_CaL is probably due to the lower single channel conductance of the T-type Ca²⁺ channels (Nilius et al., 1985). It should also be noted that the variability of current densities between cells is large for both I_CaT and I_CaL. Our findings are similar to those of Nemtsas et al. (2010), although our values for I_CaT are slightly higher and those for I_CaL slightly smaller than theirs. Notably, I_CaT has not been found in human atrial and ventricular myocytes, which accords with the minimal expression of the T-type Ca²⁺ channels in the human heart (Beuckelmann et al., 1991; Ouadid et al., 1991; Leuranguer et al., 2001). In contrast, a large cardiac I_CaT has previously been documented for a few endothermic and ectothermic vertebrates. A relatively large I_CaT exists in ventricular myocytes of finch (5.9 pA pF⁻¹; 56% of I_CaL; Bogdanov et al., 1995) and shark (S. acanthias; −9.8 pA pF⁻¹; 92.4% of I_CaL; Maylie and Morad, 1995), and atrial myocytes of the Siberian sturgeon (A. baerii; 3.52 pA pF⁻¹; 242% of I_CaL) (Haworth et al., 2014). The peak density of I_CaT in zebrafish ventricular myocytes is 2–6 times higher than the reported I_CaT density in atrial and nodal tissues of other vertebrate species (for references, see Maylie and Morad, 1995). The presence of a large I_CaT in zebrafish ventricular myocytes is one of the most prominent differences in ion current composition between zebrafish and human hearts. I_CaT density is significantly higher in the neonatal than in the adult mammalian heart. For example, in the rat ventricle, the density of I_CaT decreases from about 3 pA pF⁻¹ at the fetal stage F16 to practical absence of the current in the adult ventricle (Ferron et al., 2002).

Ni²⁺ blocks different T-type Ca²⁺ channels with different affinities, and Ni²⁺ sensitivity of the channels is dependent on the cellular environment (Lee et al., 1999). When Ca²⁺ channels were expressed in Xenopus oocytes, IC₅₀ values were 167, 5.7 and 87 µmol l⁻¹ Ni²⁺ for α1G (rat), α1H (human) and α1I (rat), respectively. In HEK-293 cells, the respective IC₅₀ values were markedly higher (250, 12 and 216 µmol l⁻¹), even though the relative sensitivity order remained the same. Furthermore, the Ni²⁺ block of α1G and α1I channels (but not of α1H) is voltage dependent, e.g. IC₅₀ value of α1G is 200 µmol l⁻¹ at 0 mV and only 70 µmol l⁻¹ at −40 mV (Lee et al., 1999). In the present study, Ni²⁺ inhibition of I_CaT was determined at −30 mV. The IC₅₀ value of the zebrafish ventricular I_CaT was 92.1 µmol l⁻¹, which is consistent with the T-type Ca²⁺ channel composition dominated by α1G (70 µmol l⁻¹ at −40 mV) (Lee et al., 1999). Others have reported a slightly higher IC₅₀ value (124 µmol l⁻¹) for the zebrafish ventricular I_CaT at −40 mV (Nemtsas et al., 2010). The high Ni²⁺ sensitivity of α1H channels is related to a unique histidine residue at position 191 in the S3–S4 loop of domain I (Kang et al., 2007). Sequence analysis of zebrafish T-type Ca²⁺ channel genes indicates that channels encoded by α1H and α1G, but not α1I, share histidine-191 with the mammalian α1H.

In canine Purkinje myocytes, guinea-pig ventricular myocytes and mouse ventricular myocytes overexpressing α1G T-type Ca²⁺ channels, Ca²⁺ admitted through T-type Ca²⁺ channels is capable of activating contraction via CICR from the SR (Sipido et al., 1998; Zhou and January, 1998; Jaleel et al., 2008). However, contractions initiated by Ca²⁺ entry through T-type Ca²⁺ channels are characterized by a long delay to the onset of shortening, slow rates of shortening and relaxation, low peak shortening, and long time-to-peak shortening (Zhou and January, 1998; Sipido et al., 1998; Jaleel et al., 2008). These findings show that Ca²⁺ entry through I_CaT is less effective than that through I_CaL in triggering CICR. T-type Ca²⁺ channels are primarily located in the peripheral sarcolemma and therefore are more distant from the Ca²⁺ release channels of the SR than L-type Ca²⁺ channels of the T-tubule membrane (Jaleel et al., 2008).

The contraction of zebrafish ventricle is strongly dependent on sarcolemmal Ca²⁺ influx, while CICR from the SR makes only a small contribution (about 15%) to the Ca²⁺ transient (Zhang et al., 2011; Bovo et al., 2013). In keeping with this, the combined sarcolemmal Ca²⁺ influx via I_CaL and I_CaT in zebrafish ventricular myocytes (129 µmol l⁻¹ at 28°C) is markedly large, bigger than that in other fish species like rainbow trout (32.1–45.8 µmol l⁻¹ at 21°C) and crucian carp (14.7–42.9 µmol l⁻¹ at 19–23°C) (Vornanen, 1997, 1998; Hove-Madsen and Tort, 1998). Moreover, in zebrafish ventricular myocytes, a significant portion of this Ca²⁺ influx is mediated by I_CaT. Indeed, T-type Ca²⁺ channels could be important for E–C coupling of cardiac myocytes in zebrafish and other tropical ectotherms, which rely strongly on sarcolemmal Ca²⁺ influx for cardiac contraction. Because of its low voltage threshold (about −70 mV), I_CaT starts to contribute to the intracellular Ca²⁺ transient earlier in the AP than I_CaL, which has a more depolarized threshold (about −40 mV). This is expected to reduce the delay between membrane depolarization and the onset of the intracellular Ca²⁺ transient, and make the Ca²⁺ transient fast rising. Fast Ca²⁺ transients might be adaptive for the tropical Danio species, which have high heart rates at temperatures close to their upper thermal tolerance limit (Sidhu et al., 2014). For example, in D. rerio at 36°C, the heart beats about 300 times per minute and ventricular AP lasts (AP duration at 50% repolarization) only 66 ms (Vornanen and Hassinen, 2016). At high temperatures, I_CaT and fast Ca²⁺ transients may be needed to cope with the requirements of short cycle lengths and AP durations. The role of I_CaT in cardiac E–C coupling of zebrafish and other tropical fish species will be an interesting future topic.

T-type Ca²⁺ channels and I_CaT are present in ventricular myocytes of perinatal mammalian (e.g. rat and mouse) heart, but during early postnatal development they gradually disappear (Ferron et al., 2002; Yasui et al., 2005). T-type Ca²⁺ channels and I_CaT reappear in ventricular myocytes of hypertrophied mammalian heart under different pathological stresses (Nuss and Houser, 1993; Martinez et al., 1999; Takebayashi et al., 2006), suggesting that they are somehow involved in the pathogenesis of hypertrophy. Indeed, studies on transgenic mouse models suggest that Ca_v3.2 (α1H) channels might be pro-hypertrophic and Ca_v3.1 (α1G) channels anti-hypertrophic (Chiang et al., 2009; Nakayama et al., 2009). It should be noted, however, that in ventricular myocytes of perinatal mammals and adult zebrafish, I_CaT is a normal physiological component of the sarcolemmal ion current complex. In the ventricle of zebrafish, I_CaT is largely generated by Ca_v3.1 (α1G) channels, while in the ventricles of embryonic and neonatal mouse, Ca_v3.2 (α1H) channels predominate (Ferron et al., 2002; Yasui et al., 2005).

Hypertrophic heart is prone to cardiac arrhythmias, but the role of the re-expressed T-type Ca²⁺ channels in arrhythmogenesis of mammalian heart is not yet resolved (Kinoshita et al., 2009). Steady-state activation and inactivation curves of mammalian I_CaT overlap, which shows that a small portion of the channels do not inactivate (Vassort et al., 2006). The non-inactivating T-type Ca²⁺ channels could be involved in arrhythmogenesis by inducing early and delayed after-depolarizations and associated arrhythmias (Kinoshita et al., 2009). The role of I_CaT is, however, difficult to discern as many other ion currents are changed in parallel with I_CaT (Kinoshita et al., 2009). It should be noted, however, that delayed after-depolarizations are induced by spontaneous Ca²⁺ release from the SR and SR leak may also be involved in triggering early after-depolarizations (Choi et al., 2002). In fish ventricular myocytes, Ca²⁺ release channels of the SR have low affinity to Ca²⁺ and therefore spontaneous Ca²⁺ releases are rare (Shiels and White, 2005; Vornanen, 2006; Bovo et al., 2013). The minor role of CICR in fish cardiac E–C coupling is suggested to make fish hearts less susceptible to early and delayed after-depolarizations and therefore triggered arrhythmias (Vornanen, 2017).

Zebrafish ventricular myocytes have a large I_CaT, which contributes a distinct depolarizing current and relatively large sarcolemmal Ca²⁺ influx. This is a prominent difference to human ventricles, where I_CaL is apparently the sole Ca²⁺ current type (Beuckelmann et al., 1991; Leuranguer et al., 2001). These differences are due to the dominance of T-type Ca²⁺ channels in the zebrafish heart (present study) and L-type Ca²⁺ channels in the human heart (Gaborit et al., 2007). As the zebrafish is a popular model for drug screening and human cardiac toxicology (Barros et al., 2008; Chakraborty et al., 2009; Peterson and MacRae, 2012), these species-specific differences in electrical excitation and E–C coupling warrant some care when adapting results from zebrafish studies to human heart. Because of the significant differences in Ca²⁺ and K⁺ currents/channels between zebrafish and human ventricles (Hassinen et al., 2015; Vornanen and Hassinen, 2016), the zebrafish is probably not an optimal model for screening of cardiovascular drugs. Interspecies differences in cardiac ion currents are so marked (even among mammals) that non-human cells and tissues are considered unsatisfying for preclinical drug screening and safety pharmacology. According to the novel CiPA (Comprehensive in vitro Proarrhythmia Assay) initiative of preclinical drug screening, only human cardiac ion channels, human stem cell-induced cardiomyocytes and in silico models of human cardiac AP are considered acceptable for drug screening and safety pharmacology (Gintant et al., 2016). CIPA is based on analysis of drug effects on multiple (7) human cardiac ion channels (Colatsky et al., 2016; Vicente et al., 2016), and I_CaT is not among those channels. If zebrafish are used for preclinical drug screening and safety pharmacology, the potential impact of the large I_CaT on drug responses and arrhythmia propensity should be carefully examined.

Although zebrafish may not be an optimal general model for human cardiac safety pharmacology and screening of cardiac drugs, they may be useful in solving more specific problems of ion channel function in cardiac excitation and E–C coupling. Owing to the large ventricular I_CaT, zebrafish could be a useful model species when the role of I_CaT in cardiac E–C coupling and as a putative target for cardiovascular drugs is examined. With their large I_CaT, lower dependence on CICR from the SR and absence of T-tubules, zebrafish ventricular myocytes more closely resemble perinatal ventricular myocytes than adult ventricular myocytes of the human heart. Considering that there is a paucity of information regarding the cardiac safety pharmacology of human neonates and premature infants (Pesco-Koplowitz et al., 2018), zebrafish could be a useful model for this population.

Prof. Pertti Panula (University of Helsinki) is acknowledged for donating the zebrafish for the establishment of our own zebrafish population in Joensuu. Anita Kervinen kindly prepared the solutions for electrophysiological experiments.