Basic characteristics of the zebrafish heart and cardiac myocytes in comparison to those of the human heart.
There are several similarities and differences in electrical excitability between zebrafish and human ventricles. Major ion currents generating ventricular action potentials are largely the same in human and zebrafish hearts with some exceptions. A large T-type calcium current is unique to the zebrafish ventricle (absent in human ventricle), and two potassium currents (IKs and Ito) may be absent in zebrafish ventricular myocytes. However, there are substantial differences among alpha subunit isoforms of the ion channel families or subfamilies (e.g. zebrafish Kv11.2 vs. human Kv11.1; zebrafish Kir2.4 vs. human Kir2.1) between human and zebrafish hearts. Contraction of zebrafish ventricle is strongly dependent on extracellular calcium, while human ventricle relies heavily on calcium stores of the sarcoplasmic reticulum. These differences may affect the use of zebrafish as a model in drug screening and safety pharmacology.
- cardiac action potential
- ion currents
- ion channels
- drug screening
- e-c coupling
2. Cardiac action potential
Contraction of cardiac myocytes is triggered by electrical excitation of myocyte sarcolemma in the form of cardiac AP. Propagation of AP through the heart can be recorded as electrocardiogram (Figure 1A). Each functionally different cardiac tissue has a characteristic AP shape generated by the tissue-specific ion currents and ion channel compositions. The five different phases of the mammalian ventricular AP—with the exception of phase 1 fast repolarization—are readily discernible in the zebrafish ventricular AP (Figure 1B). Similar to the human ventricular AP (but unlike the murine AP), the zebrafish ventricular AP has a distinct plateau phase (phase 2) at positive voltages [8, 9, 20] (Figure 1B). Indeed, the only major difference between zebrafish and human ventricular APs is the absence of the fast phase 1 repolarization in the zebrafish AP . This may be due to the absence of the transient outward K+ current (Ito) in zebrafish ventricular myocytes .
Zebrafish are ectothermic vertebrates and therefore their AP characteristics may change depending on the rearing temperature of the fish, as has been reported for several teleost fish species [21, 22]. In the adult zebrafish, reared at 28°C, the duration of ventricular AP (APD50; at 28°C) is about 30% shorter than that of the human ventricular AP at 37°C (Table 1). At 36°C, the duration of zebrafish ventricular AP is only 80 ms, i.e. about 25% of the duration of human ventricular AP at 37°C. The shorter AP of the zebrafish heart may be associated with the higher heart rate of the fish, which at 28°C is about double (120–130 beats per minute) and at 37°C about quadruple (287 beats per minute) the human resting heart rate [19, 23] (Table 1). Temperature is an important environmental factor for an ectothermic vertebrate, which modifies cardiac gene expression and ion channel function [24, 25, 26]. Therefore, rearing and experimental temperatures should be carefully controlled and reported in zebrafish studies.
|Relative heart size (% of body mass)||0.1||||0.64|||
|Diastolic/systolic blood pressure (mm Hg)||0.42/2.51||||70/125|||
|Myocyte size (ventricle) (pF)||26–33||[8, 39]||117–227||[72, 73]|
|T-tubuli||No||[20, 48]||Yes||[67, 74]|
|Role of CICR1 in e-c coupling (%)||15||||~70||[75, 76]|
|Myofibril location||Subsarcolemmal||||Throughout the myocyte|
|Ventricular AP duration (ms)||~240 at 28°C|
~80 at 37°C
|||~330 at 37°C|||
|Resting heart rate (bpm)||120–130 at 28°C|
~287 at 37°C
|[8, 19, 23]||60–80 bpm at 37°C|||
3. Ion currents and ion channels
Density and kinetics of ion currents must be such that chamber-specific APs are generated and can be adjusted to heart rates according to the circulatory demands. This is reflected in the composition of ion channel assemblies and their abundances in different cardiac chambers and in a species-specific manner . This overview is limited to ventricular myocytes, since atrial ion currents/channels of the zebrafish heart are still relatively poorly known.
3.1. Sodium currents and channels
Sodium influx through the voltage-gated Na+ channels initiates the all-or-none action potential (AP) of atrial and ventricular myocytes, when the current flow from the upstream cell depolarizes the membrane of the downstream cell to the threshold level (about −55 mV). At the threshold voltage, the density of inward Na+ current (INa) exceeds the total density of the outward K+ currents (IK). The rapid opening of Na+ channels generates a fast upstroke (depolarization) of AP and an overshoot to the level of about +40 mV (phase 0) [19, 20] (Figure 1B). Then INa quickly inactivates due to the closing of the inactivation gate of the channel.
The density of INa is the main determinant for the rate of AP propagation over the heart. The rate of AP upstroke in zebrafish ventricular myocytes at 28°C is about 130 V s−1 (RMP ~ −84 mV) (Haverinen et al., submitted), which is less than half of the rate of AP upstroke in human ventricular myocytes at 37°C . These findings suggest that the density of ventricular INa is lower and the rate of AP propagation slower than in human ventricles at the species-specific temperatures (28°C vs. 37°C). However, a thorough analysis of the zebrafish INa is needed to reveal to what extent these differences are due to RMP (availability of Na+ channel for opening), Na+ channel density and kinetic properties of the cardiac Na+ channels.
The zebrafish heart expresses eight different Na+ channel alpha subunits. The main isoforms are Nav1.5Lb (83.1% of the transcripts) and Nav1.4b (16.2%), which are orthologues to the human cardiac Nav1.5 (71.1% in the right ventricle) and skeletal Nav1.4 channels, respectively  (Table 2). Nav2.1 is abundantly expressed in the human right ventricle (27.8%), but seems to be absent in zebrafish ventricular myocytes. Unlike the mammalian cardiac INa, which is tetrodotoxin-resistant (IC50 about 1 μM), the zebrafish INa is more than two orders of magnitude more sensitive to tetrodotoxin (IC50 about 6 nM) (Haverinen et al., submitted), similar to the INa of other fish species [29, 30]. Thus, there is a remarkable difference in tetrodotoxin-sensitivity and some minor differences in Na+ channel composition between zebrafish and human hearts.
|Ion current||Ion channels*||Ion channels**|
|Nav1.4b (16.2%)||Nav2.1 (27.8%)|
|Nav1.1b (0.48%)||Nav1.3 (7.1%)|
|Nav1.6b (0.12%)||Nav1.1 (2.7%)|
|Nav1.6a (0.03%)||Nav1.7 (1.4%)|
|Cav1.3a (0.07%)||Cav1.3 (0.039%)|
|IKs||Kv7.1 (14.6%)||Kv7.1 (15.7%)|
|IK1 (inward rectifying)|
|Kir2.2a (6.3%)||Kir2.2 (28.9%)|
|Kir2.3 (0.04%)||Kir2.3 (24.5%)|
3.2. Calcium currents and channels
The vertebrate heart usually has two main types of Ca2+ currents, a high-threshold or long-lasting L-type current (ICaL) and a low-threshold or transient T-type (ICaT) current. The former is activated at voltages more positive than −40 mV and with the peak amplitude at about +10 mV, while the latter is generated already at −60 mV and with the peak current amplitude at about −30 mV [31, 32].
ICaL is the main ICa of atrial and ventricular myocytes. It has a significant physiological function in maintaining the long plateau (phase 2) of the cardiac AP and mediating Ca2+ influx into the myocyte (Figure 1B). ICaL contributes to the Ca2+ transient, which sets cardiac contraction in motion, either directly by increasing cytosolic Ca2+ concentration or triggering a further release of Ca2+ from the sarcoplasmic reticulum (SR) (for more details see excitation-contraction coupling). The mean density of ICaL in ventricular myocytes of the zebrafish heart at 28°C is 6–8 pA pF−1, which is about double the density of the human ventricular ICaL at 35°C (3–4 pA pF−1) [8, 20, 33, 34]. This difference may signify a larger role of sarcolemmal Ca2+ influx in e-c coupling of the zebrafish heart . In the human ventricle, the main L-type Ca2+ channel isoform is Cav1.2, which represents 98.4% of the total Ca2+ channel transcripts in the right ventricle  (Table 2). In the zebrafish ventricle, seven different L-type Ca2+ alpha subunits are expressed, among them three paralogue pairs . Similar to the human ventricle, Cav1.2 is the most abundant L-type Ca2+ channel isoform in zebrafish ventricle consisting of 38.3% of the all Ca2+ channels transcripts.
In vertebrate hearts ICaT is a sizeable current in nodal myocytes and it may be also present in atrial myocytes, but it is usually absent in ventricular myocytes. In this respect, the zebrafish is clearly different. A characteristic feature for zebrafish ventricular myocytes is a large ICaT with a current density almost equal to that of ICaL [8, 37]. T-type Ca2+ channels pass Ca2+ influx at more negative voltages than L-type Ca2+ channels. Therefore, they may contribute to upstroke and early plateau of the ventricular AP. Although ICaT inactivates faster than ICaL, it allows a significant sarcolemmal Ca2+ entry into zebrafish ventricular myocytes. During a 300 ms depolarizing pulse to −30 mV the Ca2+ influx through T-type Ca2+ channels is about 35% of the Ca2+ influx of L-type Ca2+ channels during 300 ms pulse to +10 mV . Therefore, ICaT may have a significant role in e-c coupling of zebrafish ventricular myocytes. T-type Ca2+ channels are abundantly expressed in the zebrafish ventricle constituting majority of the transcripts (about 55%) of the total Ca2+ channel population (Table 2). Altogether five alpha subunits of the T-type (Cav3) family are expressed in the zebrafish ventricle. Cav3.1 (alpha1G) is clearly the dominant isoform, not only among T-type Ca2+ channels, but also among all the cardiac Ca2+ channel types with the transcript abundance of 54.8%. The other T-type alpha subunits are expressed only in trace amounts. In human ventricular myocytes, the T-type Ca2+ channels are very weakly expressed. Cav3.1 and Cav3.2 alpha subunits constitute together less than 2% of the Ca2+ channels transcripts in the human right ventricle  (Table 2). Taken together the prominent expression of ICaT in zebrafish ventricular myocytes is one of the most striking differences in ion channel composition between zebrafish and human hearts. The exact role of ICaT in excitation and e-c coupling of zebrafish ventricular myocytes needs to be examined in detail.
Overall, the diversity of Ca2+ channels in zebrafish ventricle is larger than in the human ventricle. Most notably, T-type Ca2+ channels are more abundant than L-type Ca2+ channels.
3.3. Potassium currents and channels
Outward potassium currents (IK) are repolarizing, i.e. they maintain negative resting membrane potential (RMP) and limit the duration of cardiac AP.
3.3.1. Inward rectifier K+ currents and channels
The inward rectifier K+ current (IK1) maintains RMP of atrial and ventricular myocytes and provides K+ efflux for the final phase 3 repolarization of AP  (Figure 1B). This current is generated by the Kir2 subfamily channels in vertebrate hearts. Characteristic for the inward rectifier K+ channels of the Kir2 family is that they pass outward IK1 in the voltage range from about −80 to 0 mV with the peak current at −59 mV in zebrafish ventricular myocytes . At RMP, the net K+ flux at the RMP is almost zero, but the outward IK1 activates instantaneously on depolarization of sarcolemma and generates a fast outward surge of IK1 at the rising phase of AP. At the plateau voltage, K+ efflux is small due to the voltage-dependent block of the channel pore by intracellular polyamines and Mg2+ ions. When membrane potential starts to repolarize (due to the activation of IKr and IKs and inactivation of ICa), the polyamine block of Kir2 channels relaxes and K+ efflux through Kir2 channel accelerates repolarization at phase 3. Different members of the Kir2 family differ in the ease with which they allow K+ efflux through the sarcolemma. Large difference in Ba2+-sensitivity between Kir2 isoforms suggests that their interaction with potential medicinal drug molecules might also differ .
The main Kir2 channel isoform of the zebrafish ventricle is Kir2.4, as in the most teleost fishes studied thus far . In the zebrafish atrium Kir2.2a channels are abundantly expressed in addition to Kir2.4 channels. There are striking differences in isoform composition between zebrafish and human ventricles. The main isoform in the human ventricles is Kir2.1 (46.5% of the transcripts in the right ventricle), which appears only in trace amounts (0.8%) in the zebrafish ventricle. Kir2.3 is abundantly expressed in human ventricle (24.5%), but almost totally absent in the zebrafish ventricle (Table 2). Since the Kir2 isoforms differ in their rectification properties those differences in isoform composition are likely to have functional consequences for repolarization of AP.
3.3.2. Voltage-gated K+ currents and channels
Voltage-gated K+ currents provide sarcolemmal K+ efflux for repolarization of AP. Several different voltage-gated K+ currents are expressed in human ventricle including fast and slow components of the delayed rectifier current (IKr and IKs, respectively) and transient outward current (Ito) [27, 40, 41]. Transcripts for the ultra-rapid component of the delayed rectifier channel (Kv1.5) are expressed in the human ventricle, but the current seems to be specific for atrial myocytes and not expressed in the ventricles . Much less is known about the voltage-gated K+ currents of the zebrafish heart. Similar to the human ventricle, IKr seems to be the main repolarizing current also in zebrafish ventricle [8, 37, 43]. Until now, IKs has not been recorded in zebrafish cardiac myocytes, even though transcripts of the Kv7.1 (KCNQ1) channel are expressed in the zebrafish ventricle (Hassinen et al., unpublished) (Table 2). Neither has been Ito found in zebrafish cardiac myocytes . These findings suggest that repolarization of zebrafish ventricular myocytes might be more strongly dependent on single voltage-gated K+ current, IKr, than human ventricular myocytes. However, a closer examination of the K+ ion composition of zebrafish ventricular myocytes is needed to verify/falsify these assumptions.
IKr is the dominant repolarizing current in human and zebrafish ventricular myocytes. Notably the current is generated by different isoforms in human and zebrafish heart. In the human cardiac myocytes, IKr flows through the erg1 (Herg or KCNH2 or Kv11.1) channels (Table 2). In zebrafish myocytes IKr is generated almost exclusively by KCNH6 (Kv11.2a,b or zebrafish erg) channels [19, 44], although it is often referred to as an orthologue to human KCNH2 channels [9, 11, 45]. Indeed, the expression of Kv11.1a,b transcripts in the zebrafish ventricle is only 0.2%, while Kv11.2a,b channel transcripts constitute 84.9% of the total voltage-gated K+ channel alpha subunit population. Transcripts of the Kv7.1 channels form about 15% of the total transcripts of the voltage-gated K+ channels in both human and zebrafish ventricles (Table 2). Kv1.5 channels are expressed in the human ventricle, but no reports exist about any zebrafish orthologues.
4. Excitation-contraction coupling
There are prominent differences in size, shape and fine structure between zebrafish and human ventricular myocytes (Table 1). Zebrafish ventricular myocytes are 5–10 times smaller (26.1–33.3 pF) than human ventricular myocytes (117–227 pF). Zebrafish ventricular myocytes are almost as long as human ventricular myocytes, but are much thinner [20, 46]. In human ventricular myocytes, myofibrils are evenly distributed throughout the myocyte, while in zebrafish ventricular myocytes myofibrils locate immediately under the sarcolemma . Due to the small diameter of ventricular myocytes and cortical location of myofibrils T-tubuli are unnecessary for cellular signaling and probably therefore completely absent in zebrafish myocytes [20, 48].
The marked differences in myocyte size and structure appear as significant differences in the excitation-contraction (e-c) coupling between zebrafish and human (mammalian) cardiac myocytes (Figure 2). The contraction of human ventricular myocyte mainly relies on intracellular Ca2+ stores of the sarcoplasmic reticulum (SR) in generating cytosolic Ca2+ transients. A small Ca2+ influx through L-type Ca2+ channels triggers a large Ca2+ release via ryanodine-sensitive Ca2+ release channels of the SR so that about 77% of the cytosolic Ca2+ transient originates from the SR [49, 50]. In ventricular myocytes of the zebrafish heart Ca2+ release from the SR makes only about 15% of the total Ca2+ transient . Voltage-dependence of cell shortening and Ca2+ transients also suggest that sarcolemma Ca2+ influx is the main source of cytosolic Ca2+ in zebrafish cardiomyocytes . In human cardiac myocytes, voltage dependence of the Ca2+ transients is bell-shaped reflecting the voltage dependence of the trigger for Ca2+ release from the SR, ICaL . In zebrafish myocytes both ICaL and Na+-Ca2+-exchange directly contribute to cytosolic Ca2+ transient resulting in monophasic voltage dependence of cell shortening and Ca2+ transients . Sarcolemmal Ca2+ influx via ICaL and ICaT during a single twitch is almost 130 μM L−1 from which about 32% occurs through T-type Ca2+ channels . These differences in Ca2+ handling are associated with 71% lower expression of ryanodine receptors in the zebrafish ventricle in comparison to mammalian (rabbit) ventricle, whereas little differences exist in the SR Ca2+ content . Ca2+ sensitivity of ryanodine receptors of the fish heart is often low in comparison to that of mammalian cardiac ryanodine receptors , which might also contribute to the small SR Ca2+ release in zebrafish cardiomyocytes . Overall, the main differences in e-c coupling between zebrafish and human ventricular myocytes are the smaller role of intracellular Ca2+ stores of the SR, the presence of large ICaT and the absence of T-tubuli in the zebrafish myocytes.
5. Implications for the use of zebrafish in drug screening
The use of animal models for studies of human cardiac electrophysiology is based on the similarity of animal and human hearts concerning ion current densities, ion channel compositions and mechanisms of ion channel regulation by rate changes and autonomic nervous system agonists [54, 55]. However, electrophysiological properties of cardiac myocytes are species-specific and significantly different even between mammalian species (e.g. human vs. dog, rabbit and guinea pig) [54, 55]. Therefore, it is necessary to consider, whether the noticed differences in electrophysiology between zebrafish and human ventricular myocyte might affect the status of zebrafish as a model for drug screening and safety pharmacology. In this respect, quantitative differences in repolarizing currents between model and human hearts are regarded significant [54, 55]. Since ion channel isoforms often differ concerning activation and inactivation kinetics, voltage-dependence and drug affinity [39, 43, 56, 57, 58], isoform-composition of the expressed ion channels is also expected to be important for drug screening.
The human erg (KCNH2) channel is known for its propensity of being blocked by wide variety of small molecules, which may lead to AP prolongation and lethal cardiac arrhythmias. Therefore, preclinical drug screening procedures aim to assess drug-induced inhibition of IKr and prolongation of AP and QTc interval of electrocardiogram in order to remove proarrhythmic molecules from the drug development programs. However, drugs which inhibit IKr do not always produce QT prolongation due to simultaneous inhibition of the depolarizing INa and ICa currents, or since other ion channels provide a repolarization reserve to compensate for IKr inhibition. Proarrhythmic effects appear only when the drug changes the balance between inward and outward currents. To improve preclinical drug screening, the consortium of international stakeholders has recently launched an initiative called Comprehensive in-vitro Proarrhythmia Assay (CiPA). One of the central tenets of this initiative is that drug molecules are tested
Potential confounding factors in the applicability of zebrafish as a model is the presence of large ICaT and the putative absence of IKs and Ito. Since different channel isoforms of the same subfamily may have different electrophysiological properties and drug affinities, the erg (human KCNH2 vs. zebrafish KCNH6) and Kir2 (human Kir2.1 vs. zebrafish Kir2.4) channel isoform compositions may be also important. Zebrafish might be a useful high-throughput drug screening platform with the advantages of both phenotype screening, if it fulfils the qualifications of the CiPA procedure. Therefore, the calibration routine of the CiPA initiative should be conducted on zebrafish cardiac myocytes . This routine involves key cardiac ion channels (IKr, ICaL, INa, Ito, IKs and IK1), which should be examined under standardized voltage-clamp conditions for inhibition potency (IC50) of 12 selected drugs of the “minimally acceptable” dataset . These compounds are categorized into high, intermediate and low risk of torsades de pointes arrhythmia according to their currently known properties. The list of target channels should also include ICaT in the zebrafish.
One more factor that may be important in regard to proarrhythmia potency of zebrafish heart is the e-c coupling of ventricular myocytes. Factors recognized as significant causes of cardiac arrhythmias in mammals include APs that are too long or too short. If APs are abnormally long, early afterdepolarizations during the AP plateau (Phase 2 or 3) may be provoked by reactivation of Ca2+ or Na+ currents in the voltage “window,” where all Ca2+ and Na+ channels have not yet been inactivated and can be reactivated. In addition, early afterdepolarizations are promoted by spontaneous Ca2+ release from the SR that activates inward current via the reverse mode operation of Na+-Ca2+ exchange . APs that are too short can predispose the heart to delayed afterdepolarizations, which occur in early diastole (Phase 4), when spontaneous Ca2+ releases from the SR activate the inward Na+-Ca2+-exchange current. Afterdepolarizations may depolarize membrane potential to the AP threshold and induce extra systoles (triggered activity). Generation of delayed afterdepolarizations requires spontaneous Ca2+ release from the SR, which subsequently activates Ca2+ efflux via Na+-Ca2+ exchange and membrane depolarization to the AP threshold . In zebrafish heart, SR makes only a minor contribution to cardiac e-c coupling, possibly due to the low Ca2+ sensitivity of the ryanodine receptors. The relative independence of fish heart contraction from Ca2+-induced Ca2+ release is expected to make the zebrafish heart relatively resistant against early and delayed afterdepolarizations and therefore less suitable as an arrhythmia model .
The similarities between human and zebrafish cardiac electrophysiology are often emphasized, while the differences are overlooked or neglected. However, it may be that those physiological functions and electrophysiological properties that are unique to the zebrafish heart are the most useful features for cardiac research. Perhaps the most spectacular example is the exceptional regeneration power of the zebrafish heart, which may reveal to us the molecular underpinnings needed to heal the damaged human heart . Similarly, the exceptionally strong expression of the ICaT in the zebrafish ventricle may provide a test system to examine the role of T-type Ca2+ channels in e-c coupling and its significance as a drug target. In the human heart, ICaT is re-expressed and T-tubuli are lost, when the heart is subjected to pathological stressors that induce hypertrophy and failure [65, 66, 67]. Thus, zebrafish heart could be a “natural” model for testing proarrhythmic propensity of the drugs in the diseased heart. Structural (small cell size, absence of T-tubuli) and functional characteristics (minor role of SR in e-c coupling, presence of ICaT) of the zebrafish ventricle are more like those of fetal or neonatal mammalian heart than those of the adult human heart [68, 69]. Therefore, the zebrafish heart might be a better model for fetal/neonatal and diseased human heart than for the adult human heart in drug screening.
This study was supported by a grant from Jane and Aatos Erkko Foundation to MV (project N0. 64579).
Conflict of interest
The authors declare that they have no conflict of interest.
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