Minna Hassinen

Zebrafish heart as a model for human cardiac electrophysiology

ABSTRACT The zebrafish (Danio rerio) has become a popular model for human cardiac diseases and pharmacology including cardiac arrhythmias and its electrophysiological basis. Notably, the phenotype of zebrafish cardiac action potential is similar to the human cardiac action potential in that both have a long plateau phase. Also the major inward and outward current systems are qualitatively similar in zebrafish and human hearts. However, there are also significant differences in ionic current composition between human and zebrafish hearts, and the molecular basis and pharmacological properties of human and zebrafish cardiac ionic currents differ in several ways. Cardiac ionic currents may be produced by non-orthologous genes in zebrafish and humans, and paralogous gene products of some ion channels are expressed in the zebrafish heart. More research on molecular basis of cardiac ion channels, and regulation and drug sensitivity of the cardiac ionic currents are needed to enable rational use of the zebrafish heart as an electrophysiological model for the human heart.

A novel inwardly rectifying K+ channel, Kir2.5, is upregulated under chronic cold stress in fish cardiac myocytes.

SUMMARY A new member of the inward-rectifier K+ channel subfamily Kir2 was isolated and characterised from the crucian carp (Carassius carassius) heart. When expressed in COS-1 cells this 422 amino acid protein produced an inward-rectifying channel with distinct single-channel conductance, mean open time and open probability. Phylogenetic sequence comparisons indicate that it is not homologous to any known vertebrate Kir channel, yet belongs to the Kir2 subfamily. This novel crucian carp channel increases the number of vertebrate Kir2 channels to five, and has therefore been designated as ccKir2.5 (cc for Carassius carassius). In addition to the ccKir2.5 channel, the ccKir2.2 and ccKir2.1 channels were expressed in the crucian carp heart, ccKir2.1 being present only in trace amounts (<0.8% of all Kir2 transcripts). Whole-cell patch clamp in COS-1 cells demonstrated that ccKir2.5 is a stronger rectifier than ccKir2.2 or ccKir2.1, and therefore passes weakly outward current. Single-channel conductance, mean open time and open probability of ccKir2.5 were, respectively, 1.6, 4.96 and 4.17 times as large as that of ccKir2.2. ccKir2.5 was abundantly expressed in atrium and ventricle of the heart and in skeletal muscle, but was a minor component of Kir2 in brain, liver, gill and kidney. Noticeably, ccKir2.5 was strongly responsive to chronic cold exposure. In fish reared at 4°C for 4 weeks, ccKir2.5 mRNA formed 59.1±2.1% and 65.6±3.2% of all ccKir2 transcripts in atrium and ventricle, respectively, while in fish maintained at 18°C the corresponding transcript levels were only 16.2±1.7% and 23.3±1.7%. The increased expression of ccKir2.5 at 4°C occurred at the expense of ccKir2.2, which was the main Kir2 isoform in 18°C acclimated fish. A cold-induced increase in the slope conductance of the ventricular IK1 from 707±49 to 1001±59 pS pF–1 (P<0.05) was thus associated with an isoform shift from ccKir2.2 towards ccKir2.5, suggesting that ccKir2.5 is a cold-adapted and ccKir2.2 a warm-adapted isoform of the inward-rectifying K+ channel.

Tetrodotoxin Sensitivity of the Vertebrate Cardiac Na+ Current

Evolutionary origin and physiological significance of the tetrodotoxin (TTX) resistance of the vertebrate cardiac Na+ current (INa) is still unresolved. To this end, TTX sensitivity of the cardiac INa was examined in cardiac myocytes of a cyclostome (lamprey), three teleost fishes (crucian carp, burbot and rainbow trout), a clawed frog, a snake (viper) and a bird (quail). In lamprey, teleost fishes, frog and bird the cardiac INa was highly TTX-sensitive with EC50-values between 1.4 and 6.6 nmol·L−1. In the snake heart, about 80% of the INa was TTX-resistant with EC50 value of 0.65 μmol·L−1, the rest being TTX-sensitive (EC50 = 0.5 nmol·L−1). Although TTX-resistance of the cardiac INa appears to be limited to mammals and reptiles, the presence of TTX-resistant isoform of Na+ channel in the lamprey heart suggest an early evolutionary origin of the TTX-resistance, perhaps in the common ancestor of all vertebrates.

Thermal adaptation of the crucian carp (Carassius carassius) cardiac delayed rectifier current, IKs, by homomeric assembly of Kv7.1 subunits without MinK

Ectothermic vertebrates experience acute and chronic temperature changes which affect cardiac excitability and may threaten electrical stability of the heart. Nevertheless, ectothermic hearts function over wide range of temperatures without cardiac arrhythmias, probably due to special molecular adaptations. We examine function and molecular basis of the slow delayed rectifier K(+) current (I(Ks)) in cardiac myocytes of a eurythermic fish (Carassius carassius L.). I(Ks) is an important repolarizing current that prevents excessive prolongation of cardiac action potential, but it is extremely slowly activating when expressed in typical molecular composition of the endothermic animals. Comparison of the I(Ks) of the crucian carp atrial myocytes with the currents produced by homomeric K(v)7.1 and heteromeric K(v)7.1/MinK channels in Chinese hamster ovary cells indicates that activation kinetics and pharmacological properties of the I(Ks) are similar to those of the homomeric K(v)7.1 channels. Consistently with electrophysiological properties and homomeric K(v)7.1 channel composition, atrial transcript expression of the MinK subunit is only 1.6-1.9% of the expression level of the K(v)7.1 subunit. Since activation kinetics of the homomeric K(v)7.1 channels is much faster than activation of the heteromeric K(v)7.1/MinK channels, the homomeric K(v)7.1 composition of the crucian carp cardiac I(Ks) is thermally adaptive: the slow delayed rectifier channels can open despite low body temperatures and curtail the duration of cardiac action potential in ectothermic crucian carp. We suggest that the homomeric K(v)7.1 channel assembly is an evolutionary thermal adaptation of ectothermic hearts and the heteromeric K(v)7.1/MinK channels evolved later to adapt I(Ks) to high body temperature of endotherms.

Effects of seasonal acclimatization on action potentials and sarcolemmal K+ currents in roach (Rutilus rutilus) cardiac myocytes

Temperature sensitivity of electrical excitability is a potential limiting factor for high temperature tolerance of ectotherms. The present study examines whether heat resistance of electrical excitability of cardiac myocytes is modified by seasonal thermal acclimatization in roach (Rutilus rutilus), a eurythermal teleost species. To this end, temperature dependencies of ventricular action potentials (APs), and atrial and ventricular K+ currents were measured from winter-acclimatized (WiR) and summer-acclimatized (SuR) roach. Under patch-clamp recording conditions, ventricular APs could be triggered over a wide range of temperatures (4-43°C) with prominent changes in resting membrane potential (RMP), AP duration and amplitude. In general, APs of SuR were slightly more tolerant to high temperatures than those of WiR, e.g. the break point temperature (TBP) of RMP was 37.6±0.4°C in WiR and 41±1°C in SuR (p<0.05). Of the two major cardiac K+ currents, the inward rectifier K+ current (IK1) was particularly heat resistant in both SuR (TBP 39.4±0.4°C) and WiR (TBP 40.0±0.4°C) ventricular myocytes. The delayed rectifier K+ current (IKr) was not as heat resistant as IK1. Surprisingly, IKr of WiR tolerated heat better (TBP 31.9±0.8°C) than IKr of SuR (TBP 24.1±0.5°C) (p<0.05). IKr (Erg2) channel transcripts of both atrial and ventricular myocytes were up-regulated in WiR. IK1 (Kir2) channel transcripts were not affected by seasonal acclimatization, although ventricular IK1 current was up-regulated in summer. Collectively, these findings show that thermal tolerance limits of K+ currents in isolated myocytes between seasonally acclimatized roach are much less pronounced than the heat sensitivity of ECG variables in intact fish.

Comparison of Gene Expression in the Gill of Salmon (Salmo salar) Smolts from Anadromous and Landlocked Populations

We examined whether gene expression in the young salmon (Salmo salar) gill differs in relation to the salinity of their migration habitat by comparing three salmon stocks: (1) fish that migrate from a river system to Lake Saimaa, (2) fish that migrate to the brackish waters of the Baltic Sea, and (3) fish that migrate to the full-strength salinity of the Arctic Ocean. Transcripts of the gill tissue were measured at three successive developmental stages (parr, smolt and postsmolt) using the cDNA microarray in fish reared under common conditions. The changes in gene expression were qualitatively and quantitatively similar in the three stocks irrespective of the salinity of the natural growing habitat. This suggests that the parr—smolt transformation in the gill tissue of the landlocked fresh-water salmon stock is similar to the seawater migrating salmon. The transformation of the gill to a hypoosmotic organ in the freshwater salmon has been retained in evolution, possibly due to its adaptive role as a signal for migration from a relatively poor-growth environment of the river to a more productive lake habitat.

Molecular basis and drug sensitivity of the delayed rectifier (IKr) in the fish heart.

Fishes are increasingly used as models for human cardiac diseases, creating a need for a better understanding of the molecular basis of fish cardiac ion currents. To this end we cloned KCNH6 channel of the crucian carp (Carassius carassius) that produces the rapid component of the delayed rectifier K(+) current (IKr), the main repolarising current of the fish heart. KCNH6 (ccErg2) was the main isoform of the Kv11 potassium channel family with relative transcript levels of 98.9% and 99.6% in crucian carp atrium and ventricle, respectively. KCNH2 (ccErg1), an orthologue to human cardiac Erg (Herg) channel, was only slightly expressed in the crucian carp heart. The native atrial IKr and the cloned ccErg2 were inhibited by similar concentrations of verapamil, terfenadine and KB-R7943 (P>0.05), while the atrial IKr was about an order of magnitude more sensitive to E-4031 than ccErg2 (P<0.05) suggesting that some accessory β-subunits may be involved. Sensitivity of the crucian carp atrial IKr to E-4031, terfenadine and KB-R7943 was similar to what has been reported for the Herg channel. In contrast, the sensitivity of the crucian carp IKr to verapamil was approximately 30 times higher than the previously reported values for the Herg current. In conclusion, the cardiac IKr is produced by non-orthologous gene products in fish (Erg2) and mammalian hearts (Erg1) and some marked differences exist in drug sensitivity between fish and mammalian Erg1/2 which need to be taken into account when using fish heart as a model for human heart.

Effects of prolonged anoxia on electrical activity of the heart in Crucian carp (Carassius carassius)

ABSTRACT The effects of sustained anoxia on cardiac electrical excitability were examined in the anoxia-tolerant crucian carp (Carassius carassius). The electrocardiogram (ECG) and expression of excitation–contraction coupling genes were studied in fish acclimatised to normoxia in summer (+18°C) or winter (+2°C), and in winter fish after 1, 3 and 6 weeks of anoxia. Anoxia induced a sustained bradycardia from a heart rate of 10.3±0.77 beats min−1 to 4.1±0.29 beats min−1 (P<0.05) after 5 weeks, and heart rate slowly recovered to control levels when oxygen was restored. Heart rate variability greatly increased under anoxia, and completely recovered under re-oxygenation. The RT interval increased from 2.8±0.34 s in normoxia to 5.8±0.44 s under anoxia (P<0.05), which reflects a doubling of the ventricular action potential (AP) duration. Acclimatisation to winter induced extensive changes in gene expression relative to summer-acclimatised fish, including depression in those genes coding for the sarcoplasmic reticulum calcium pump (Serca2a_q2) and ATP-sensitive K+ channels (Kir6.2) (P<0.05). Genes of delayed rectifier K+ (kcnh6) and Ca2+ channels (cacna1c) were up-regulated in winter fish (P<0.05). In contrast, the additional challenge of anoxia caused only minor changes in gene expression, e.g. depressed expression of Kir2.2b K+ channel gene (kcnj12b), whereas expression of Ca2+ (cacna1a, cacna1c and cacna1g) and Na+ channel genes (scn4a and scn5a) was not affected. These data suggest that low temperature pre-conditions the crucian carp heart for winter anoxia, whereas sustained anoxic bradycardia and prolongation of AP duration are directly induced by oxygen shortage without major changes in gene expression. Summary: Low temperature pre-conditions fish heart for prolonged anoxia by changes in activity of excitation–contraction coupling genes and thereby allows sustained bradycardia and prolongation of ventricular action potential when oxygen shortage sets in.

Small functional If current in sinoatrial pacemaker cells of the brown trout (Salmo trutta fario) heart despite strong expression of HCN channel transcripts

Funny current (If), formed by hyperpolarization-activated cyclic nucleotide-gated channels (HCN channels), is supposed to be crucial for the membrane clock regulating the cardiac pacemaker mechanism. We examined the presence and activity of HCN channels in the brown trout (Salmo trutta fario) sinoatrial (SA) pacemaker cells and their putative role in heart rate (fH) regulation. Six HCN transcripts (HCN1, HCN2a, HCN2ba, HCN2bb, HCN3, and HCN4) were expressed in the brown trout heart. The total HCN transcript abundance was 4.0 and 4.9 times higher in SA pacemaker tissue than in atrium and ventricle, respectively. In the SA pacemaker, HCN3 and HCN4 were the main isoforms representing 35.8 ± 2.7 and 25.0 ± 1.5%, respectively, of the total HCN transcripts. Only a small If with a mean current density of -1.2 ± 0.37 pA/pF at -140 mV was found in 4 pacemaker cells out of 16 spontaneously beating cells examined, despite the optimization of recording conditions for If activity. If was not found in any of the 24 atrial myocytes and 21 ventricular myocytes examined. HCN4 coexpressed with the MinK-related peptide 1 (MiRP1) β-subunit in CHO cells generated large If currents. In contrast, HCN3 (+MiRP1) failed to produce If in the same expression system. Cs+ (2 mM), which blocked 84 ± 12% of the native If, reversibly reduced fH 19.2 ± 3.6% of the excised multicellular pacemaker tissue from 53 ± 5 to 44 ± 5 beats/min (P < 0.05). However, this effect was probably due to the reduction of IKr, which was also inhibited (63.5 ± 4.6%) by Cs+ These results strongly suggest that fH regulation in the brown trout heart is largely independent on If.

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

ABSTRACT Calcium channels are necessary for cardiac excitation–contraction (E–C) coupling, but Ca2+ channel composition of fish hearts is still largely unknown. To this end, we determined transcript expression of Ca2+ channels in the heart of zebrafish (Danio rerio), a popular model species. Altogether, 18 Ca2+ channel α-subunit genes were expressed in both atrium and ventricle. Transcripts for 7 L-type (Cav1.1a, Cav1.1b, Cav1.2, Cav1.3a, Cav1.3b, Cav1.4a, Cav1.4b), 5 T-type (Cav3.1, Cav3.2a, Cav3.2b, Cav3.3a, Cav3.3b) and 6 P/Q-, N- and R-type (Cav2.1a, Cav2.1b, Cav2.2a, Cav2.2b, Cav2.3a, Cav2.3b) Ca2+ channels were expressed. In the ventricle, T-type channels formed 54.9%, L-type channels 41.1% and P/Q-, N- and R-type channels 4.0% of the Ca2+ channel transcripts. In the atrium, the relative expression of T-type and L-type Ca2+ channel transcripts was 64.1% and 33.8%, respectively (others accounted for 2.1%). Thus, at the transcript level, T-type Ca2+ channels are prevalent in zebrafish atrium and ventricle. At the functional level, peak densities of ventricular T-type (ICaT) and L-type (ICaL) Ca2+ current were 6.3±0.8 and 7.7±0.8 pA pF−1, respectively. ICaT mediated a sizeable sarcolemmal Ca2+ influx into ventricular myocytes: the increment in total cellular Ca2+ content via ICaT was 41.2±7.3 µmol l−1, which was 31.7% of the combined Ca2+ influx (129 µmol l−1) via ICaT and ICaL (88.5±20.5 µmol l−1). The diversity of expressed Ca2+ channel genes in zebrafish heart is high, but dominated by the members of the T-type subfamily. The large ventricular ICaT is likely to play a significant role in E–C coupling. Summary: Zebrafish heart expresses a diversity of Ca2+ channel genes dominated by the T-type (Cav3.1) subfamily; the associated current (ICaT) is likely to play a significant role in excitation–contraction coupling.