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Atrioventricular (AV) nodal tissue synchronizes activities of atria and ventricles of the vertebrate heart and is also a potential site of cardiac arrhythmia, e.g., under acute heat stress. Since ion channel composition and ion currents of the fish AV canal have not been previously studied, we measured major cation currents and transcript expression of ion channels in rainbow trout (Oncorhynchus mykiss) AV tissue. Both ion current densities and expression of ion channel transcripts indicate that the fish AV canal has a characteristic electrophysiological phenotype that differs from those of sinoatrial tissue, atrium and ventricle. Two types of cardiomyocytes were distinguished electrophysiologically in trout AV nodal tissue: the one (transitional cell) is functionally intermediate between working atrial/ventricular myocytes and the other (AV nodal cell) has a less negative resting membrane potential than atrial and ventricular myocytes and is a more similar to the sinoatrial nodal cells in ion channel composition. The AV nodal cells are characterized by a small or non-existent inward rectifier potassium current (IK1), low density of fast sodium current (INa) and relatively high expression of T-type calcium channels (CACNA3.1). Pacemaker channel (HCN4 and HCN2) transcripts were expressed in the AV nodal tissue butIfcurrent was not found in enzymatically isolated nodal myocytes. The electrophysiological properties of the rainbow trout nodal cells are appropriate for a slow rate of action potential conduction (smallINa) and a moderate propensity for pacemaking activity (absence ofIK1).

The spatial pattern of electrical activation is crucial for a full understanding of fish heart function. However, it remains unclear whether there is regional variation in action potential (AP) morphologies and underlying ion currents. Because the direction of depolarization and spatial differences in the durations of ventricular APs set limits to potential patterns of ventricular repolarization, we determined AP morphologies, underlying ion currents and ion channel expression in four different ventricular regions (spongy myocardium; and apex, base and middle of the compact myocardium), and correlated them with in vivo electrocardiograms (ECGs) in rainbow trout (Oncorhynchus mykiss). ECGs recorded from three leads indicated that the depolarization and repolarization of APs propagate from base to apex, and the main depolarization axis of the ventricle is between +90 and +120 deg. AP shape was uniform across the whole ventricle, and little regional differences were found in the density of repolarizing K+ currents or depolarizing Ca2+ and Na+ currents and the underlying transcripts of ion channels, providing compelling evidence for the suggested excitation pattern. The spatial uniformity of AP durations and base-to-apex propagation of activation with a relatively slow velocity of propagation indicates no special ventricular conduction pathway in the trout ventricle such as the His–Purkinje system of mammalian hearts. The sequence of repolarization is solely determined by activation time without being affected by regional differences in AP duration.

In cardiac myocytes, the slow component of the delayed rectifier K+ current (IKs) ensures repolarization of action potential during beta-adrenergic activation or when other repolarizing K+ currents fail. As a key factor of cardiac repolarization IKs should be present in model species used for cardiovascular drug screening, preferably with pharmacological characteristics similar to those of the human IKs. To this end, we investigated the effects of inhibitors and activators of the IKs on KCNQ1 and KCNQ1+KCNE1 channels of the zebrafish, an important model species, in Chinese hamster ovary cells. Inhibitors of IKs, chromanol 293B and HMR-1556, inhibited zebrafish IKs channels with approximately similar potency as that of mammalian IKs. Chromanol 293B concentration for half-maximal inhibition (IC50) of zebrafish IKs was at 13.1±5.8 and 13.4±2.8 μM for KCNQ1 and KCNQ1+KCNE1 channels, respectively. HMR-1556 was a more potent inhibitor of zebrafish IKs with IC50=0.1±0.1 μM and 1.5±0.8 μM for KCNQ1 and KCNQ1+KCNE1 channels, respectively. R-L3 and mefenamic acid, generally identified as IKs activators, both inhibited zebrafish IKs. R-L3 almost completely inhibited zebrafish IKs generated by KCNQ1 and KCNQ1+KCNE1 channels with similar affinity (IC50 1.1±0.4 and 1.0±0.4 μM, respectively). Mefenamic acid partially blocked zebrafish KCNQ1 (IC50=9.5±4.8 μM) and completely blocked KCNQ1+KCNE1 channels (IC50=3.3±1.8 μM). Although zebrafish IKs responds to IKs inhibitors in the same way as mammalian IKs, its response to activators is atypical, probably due to the differences in the binding domain of KCNE1 to KCNQ1. Therefore, care must be taken when translating the results from zebrafish to humans.

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

Heat tolerance of heart rate in fish is suggested to be limited by impaired electrical excitation of the ventricle due to the antagonistic effects of high temperature on Na+(INa) and K+(IK1) ion currents (INais depressed at high temperatures while IK1is resistant to them). To examine the role of Na+channel proteins and the lipid matrix of the channels in heat tolerance of INa, we compared temperature-dependencies of zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss) ventricular INa, and INagenerated by the cloned zebrafish and rainbow trout NaV1.4 and NaV1.5 Na+channels in HEK cells. Whole-cell patch clamp recordings showed that zebrafish ventricular INahas better heat tolerance and slower inactivation kinetics than rainbow trout ventricular INa. In contrast, heat tolerance and inactivation kinetics of zebrafish and rainbow trout NaV1.4 channels are similar when expressed in the identical plasma membrane lipid matrix of HEK cells. The same applies to NaV1.5 channels. Thermal adaptation of the ventricular INais largely achieved by differential expression of Na+channel alpha subunits: zebrafish which tolerate well high temperatures mainly express the slower NaV1.5 isoform, while rainbow trout which prefer cold waters mainly express the faster NaV1.4 isoform. Differences in elasticity (stiffness) of the lipid bilayer may be also involved in thermal adaptation of INa. These findings suggest that both the protein component and its lipid bilayer matrix are involved in thermal adaptation of the voltage-gated Na+channels and therefore in heart rate regulation under thermal stress in fish.

Na+ channel α-subunit composition of the zebrafish heart and electrophysiological properties of Na+ current (INa) of zebrafish ventricular myocytes were examined. Eight Na+ channel α-subunits were expressed in both atrium and ventricle of the zebrafish heart. Nav1.5Lb, an orthologue to the human Nav1.5, was clearly the predominant isoform in both chambers representing 65.2 ± 4.1% and 83.1 ± 2.1% of all Na+ channel transcripts in atrium and ventricle, respectively. Nav1.4b, an orthologue to human Nav1.4, formed 34.1 ± 4.1 and 16.2 ± 2.0% of the Na+ channel transcripts in atrium and ventricle, respectively. The density of INa and the rate of action potential upstroke in zebrafish ventricular myocytes at 28 °C were similar to those of human ventricles at the comparable temperature. Na+ channel isoforms and the main electrophysiological characteristics of the INa are largely similar in zebrafish and human hearts indicating evolutionary conservation of Na+ channel composition and function. The zebrafish INa differs from the human cardiac INa in terms of higher tetrodotoxin sensitivity (IC50-value = 5.3 ± 0.1 nM) and slower inactivation kinetics. The zebrafish INa was inhibited with tricaine (MS-222) with an IC50-value of 1.2 ± 0.18 mM (336 mg l−1), suggesting some care in the use of MS-222 as an anesthetic.

Zebrafish are increasingly used as a model for human cardiac electrophysiology, arrhythmias, and drug screening. However, K+ ion channels of the zebrafish heart, which determine the rate of repolarization and duration of cardiac action potential (AP) are still incompletely known and characterized. Here, we provide the first evidence for the presence of the slow component of the delayed rectifier K+channels in the zebrafish heart and characterize electrophysiological properties of the slow component of the delayed rectifier K+current, IKs. Zebrafish atrium and ventricle showed strong transcript expression of the kcnq1 gene, which encodes the Kv7.1 α-subunit of the slow delayed rectifier K+ channel. In contrast, the kcne1 gene, encoding the MinK β-subunit of the delayed rectifier, was expressed at 21 and 17 times lower level in ventricle and atrium, respectively, in comparison to the kcnq1. IKs was observed in 62% of ventricular myocytes with mean (± SEM) density of 1.23 ± 0.37 pA/pF at + 30 mV. Activation rate of IKs was 38% faster (τ50 = 1248 ± 215 ms) than kcnq1:kcne1 channels (1725 ± 792 ms) expressed in 3:1 ratio in Chinese hamster ovary cells. Microelectrode experiments demonstrated the functional relevance of IKs in the zebrafish heart, since 100 μM chromanol 293B produced a significant prolongation of AP in zebrafish ventricle. We conclude that AP repolarization in zebrafish ventricle is contributed by IKs, which is mainly generated by homotetrameric Kv7.1 channels not coupled to MinK ancillary β-subunits. This is a clear difference to the human heart, where MinK is an essential component of the slow delayed rectifier K+channel.

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., final draft, peerReviewed

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

Inward rectifier K+(Kir2) channels are critical for electrical excitability of cardiac myocytes. Here, we examine expression of Kir2 channels in the heart of three Gadiformes species, polar cod (Boreogadus saida) and navaga (Eleginus nawaga) of the Arctic Ocean and burbot (Lota lota) of the temperate lakes to find out the role of Kir2 channels in cardiac adaptation to cold. Five boreal freshwater species: brown trout (Salmo trutta fario), arctic char (Salvelinus alpinus), roach (Rutilus rutilus), perch (Perca fluviatilis) and pike (Esox lucius), and zebrafish (Danio rerio), were included for comparison. Transcript expression of genes encoding Kir2.1a, − 2.1b, − 2.2a, − 2.2b and − 2.4 was studied from atrium and ventricle of thermally acclimated or acclimatized fish by quantitative PCR. Kir2 composition in the polar cod was more diverse than in other species in that all Kir2 isoforms were relatively highly expressed. Kir2 composition of navaga and burbot differed from that of the polar cod as well as from those of other species. The relative expression of Kir2.2 transcripts, especially Kir2.2b, was higher in both atrium and ventricle of navaga and burbot (56–89% from the total Kir2 pool) than in other species (0.1–11%). Thermal acclimation induced only small changes in cardiac Kir2 transcript expression in Gadiformes species. However, Kir2.2b transcripts were upregulated in cold-acclimated navaga and burbot hearts. All in all, the cardiac Kir2 composition seems to be dependent on both phylogenetic position and thermal preference of the fish.

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.

Calcium channels are necessary for cardiac excitation–contraction (E–C) coupling, but Ca 2+ channel composition of fish hearts is still largely unknown. To this end, we determined transcript expression of Ca 2+ channels in the heart of zebrafish ( Danio rerio ), a popular model species. Altogether, 18 Ca 2+ channel α-subunit genes were expressed in both atrium and ventricle. Transcripts for 7 L-type (Ca v 1.1a, Ca v 1.1b, Ca v 1.2, Ca v 1.3a, Ca v 1.3b, Ca v 1.4a, Ca v 1.4b), 5 T-type (Ca v 3.1, Ca v 3.2a, Ca v 3.2b, Ca v 3.3a, Ca v 3.3b) and 6 P/Q-, N- and R-type (Ca v 2.1a, Ca v 2.1b, Ca v 2.2a, Ca v 2.2b, Ca v 2.3a, Ca v 2.3b) Ca 2+ 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 Ca 2+ channel transcripts. In the atrium, the relative expression of T-type and L-type Ca 2+ channel transcripts was 64.1% and 33.8%, respectively (others accounted for 2.1%). Thus, at the transcript level, T-type Ca 2+ channels are prevalent in zebrafish atrium and ventricle. At the functional level, peak densities of ventricular T-type ( I CaT ) and L-type ( I CaL ) Ca 2+ current were 6.3±0.8 and 7.7±0.8 pA pF −1 , respectively. I CaT mediated a sizeable sarcolemmal Ca 2+ influx into ventricular myocytes: the increment in total cellular Ca 2+ content via I CaT was 41.2±7.3 µmol l −1 , which was 31.7% of the combined Ca 2+ influx (129 µmol l −1 ) via I CaT and I CaL (88.5±20.5 µmol l −1 ). The diversity of expressed Ca 2+ channel genes in zebrafish heart is high, but dominated by the members of the T-type subfamily. The large ventricular I CaT is likely to play a significant role in E–C coupling.

Funny current (

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 signa...

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

SUMMARYA 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 (

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, published version, peerReviewed

Sodium current (I(Na)) of the mammalian heart is resistant to tetrodotoxin (TTX) due to low TTX affinity of the cardiac sodium channel (Na(v)) isoform Na(v)1.5. To test applicability of this finding to other vertebrates, TTX sensitivity of the fish cardiac I(Na) and its molecular identity were examined.Molecular cloning and whole-cell patch-clamp were used to examine alpha-subunit composition and TTX inhibition of the rainbow trout (Oncorhynchus mykiss) cardiac Na(v) respectively.I(Na) of the trout heart is about 1000 times more sensitive to TTX (IC50 = 1.8-2 nm) than the mammalian cardiac I(Na) and it is produced by three Na(v)alpha-subunits which are orthologs to mammalian skeletal muscle Na(v)1.4, cardiac Na(v)1.5 and peripheral nervous system Na(v)1.6 isoforms respectively. Oncorhynchus mykiss (om) omNa(v)1.4a is the predominant isoform of the trout heart accounting for over 80% of the Na(v) transcripts, while omNa(v)1.5a forms about 18% and omNa(v)1.6a only 0.1% of the transcripts. OmNa(v)1.4a and omNa(v)1.6a have aromatic amino acids, phenylalanine and tyrosine, respectively, in the critical position 401 of the TTX binding site of the domain I, which confers their high TTX sensitivity. More surprisingly, omNa(v)1.5a also has an aromatic tyrosine in this position, instead of the cysteine of the mammalian TTX-resistant Na(v)1.5.The ortholog of the mammalian skeletal muscle isoform, omNa(v)1.4a, is the predominant Na(v)alpha-subunit in the trout heart, and all trout cardiac isoforms have an aromatic residue in position 401 rendering the fish cardiac I(Na) highly sensitive to TTX.

Potassium currents are plastic entities that modify electrical activity of the heart in various physiological conditions including chronic thermal stress. We examined the molecular basis of the inward rectifier K+ current ( IK1) in rainbow trout acclimated to cold (4°C, CA) and warm (18°C, WA) temperature. Inward rectifier K+ channel (Kir)2.1 and Kir2.2 transcripts were expressed in atrium and ventricle of the trout heart, Kir2.1 being the major component in both cardiac chambers. The relative expression of Kir2.2 was, however, higher ( P < 0.05) in atrium than ventricle. The density of ventricular IK1 was ∼25% larger ( P < 0.05) in WA than CA trout. Furthermore, the IK1 of the WA trout was 10 times more sensitive to Ba2+ (IC50 0.18 ± 0.42 μM) than the IK1 of the CA trout (1.17 ± 0.44 μM) ( P < 0.05), and opening kinetics of single Kir2 channels was slower in WA than CA trout ( P < 0.05). When expressed in COS-1 cells, the homomeric Kir2.2 channels demonstrated higher Ba2+ sensitivity (2.88 ± 0.42 μM) than Kir2.1 channels (24.99 ± 7.40 μM) ( P < 0.05). In light of the different Ba2+ sensitivities of rainbow trout (om)Kir2.1 and omKir2.2 channels, it is concluded that warm acclimation increases either number or activity of the omKir2.2 channels in trout ventricular myocytes. The functional changes in IK1 are independent of omKir2 transcript levels, which remained unaltered by thermal acclimation. Collectively, these findings suggest that thermal acclimation modifies functional properties and subunit composition of the trout Kir2 channels, which may be needed for regulation of cardiac excitability at variable temperatures.

Large-scale vector manufacturing for phase III and beyond has proven to be challenging. Upscaling the process with suspension cells is increasingly feasible, but many viral production applications are still applicable only in adherent settings. Scaling up the adherent system has proven to be troublesome. The iCELLis(®) disposable fixed-bed bioreactors offer a possible option for viral vector manufacturing in large quantities in an adherent environment. In this study, we have optimized adenovirus serotype 5 manufacturing using iCELLis Nano with a cultivation area up to 4 m(2). HEK293 cell cultivation, infection, and harvest of the virus (by lysing the cells inside the bioreactor) proved possible, reaching total yield of up to 1.6×10(14) viral particles (vp)/batch. The iCELLis 500 is designed to satisfy demand for large-scale requirements. Inoculating a large quantity of cell mass into the iCELLis 500 was achieved by first expanding the cell mass in suspension. Upscaling the process into an iCELLis 500/100 m(2) cultivation area cassette was practical and produced up to 6.1×10(15) vp. Flask productivity per cm(2) in iCELLis Nano and iCELLis 500 was in the same range. As a conclusion, we showed for the first time that iCELLis 500 equipment has provided an effective way to manufacture large batches of adenoviral vectors.

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

Cold-acclimated (CA) phenotype of trout heart was induced by 4-wk acclimation at 4°C and was characterized by 32.7% increase in relative heart mass and 49.8% increase in ventricular myocyte size compared with warm-acclimated (WA; 18°C) fish ( P < 0.001). Effect of temperature acclimation on transcriptome of the rainbow trout heart was examined using species-specific microarray chips containing 1,380 genes. After 4 wk of temperature acclimation, 8.8% (122) of the genes were differently expressed in CA and WA hearts, and most of them (82%) were upregulated in the cold ( P < 0.01). Transcripts of genes engaged in protein synthesis and intermediary metabolism were most strongly upregulated, whereas genes contributing to the connective tissue matrix were clearly repressed. Extensive upregulation of the genes coding for ribosomal proteins and translation elongation and initiation factors suggest that the protein synthesis machinery of the trout heart is enhanced in the cold and is an essential part of the compensatory mechanism causing and maintaining the hypertrophy of cardiac myocytes. The prominent depression of collagen genes may be indicative of a reduced contribution of extracellular matrix to the remodeling of the CA fish heart. Temperature-related changes in transcripts of metabolic enzymes suggest that at mRNA level, glycolytic energy production from carbohydrates is compensated in the heart of CA rainbow trout, while metabolic compensation is absent in mitochondria. In addition, the analysis revealed three candidate genes: muscle LIM protein, atrial natriuretic peptide B, and myosin light chain 2, which might be central for induction and maintenance of the hypertrophic phenotype of the CA trout heart. These findings indicate that extensive modification of gene expression is needed to maintain the temperature-specific phenotype of the fish heart.

The prevention of flower formation is important for avoiding the spread of transgenes from genetically modified plants into wild populations. Moreover, the resources not expended for the generation of flowers and fruits might be allocated to increased vegetative growth. We have been developing methods for preventing flower formation in silver birch (Betula pendula), a tree species of considerable economical importance in the boreal region. Here we study the suitability of the promoter of BpFRUITFULL-LIKE1 (BpFULL1, formerly BpMADS5) for tissue-specific ablation of inflorescences in Arabidopsis, tobacco and birch. With all these species, the development of inflorescences was successfully prevented. The results show that the BpFULL1::BARNASE construct has potential biotechnological applications in different plant species.

The development of flowers is regulated by a complex network of transcriptional activators and repressors, many of which belong to the MADS box gene family. In this study, we describe two MADS box genes of silver birch (Betula pendula Roth), BpMADS1 and BpMADS6, which are similar to SEPALLATA3 and AGAMOUS in Arabidopsis thaliana, respectively. In situ hybridization showed that BpMADS1 was expressed in the inflorescence meristem at a very early stage, but not later. Both genes were expressed in developing carpels, ovules and stamens but not in tepals or scales. Ectopic expression of BpMADS1 in Arabidopsis resulted in a reduced number of floral organs or whole whorls and in petaloid or carpelloid sepals, a phenotype reminiscent of that of fil mutants. 35S::BpMADS6 caused very early flowering in Arabidopsis. In tobacco, both 35S::BpMADS1 and 35S::BpMADS6 accelerated flowering and, in addition, 35S::BpMADS6 caused changes in sepals and petals. In some transgenic birch plants, 35S::BpMADS1 antisense resulted in the development of both male and female organs in the axil of a single bract and in a change of some inflorescences into vegetative shoots. In two plants, either 35S::BpMADS6 sense or antisense constructs resulted in an increase in the number of tepals and in complete lack of stamens in some male inflorescences. These results suggest that BpMADS1 participates both in inflorescence and in flower formation and BpMADS6 participates in flower formation and that they are functional homologues to SEPALLATA3 and AGAMOUS, respectively.

Electrophysiological properties and molecular background of the zebrafish (Danio rerio) cardiac inward rectifier current (IK1) were examined. Ventricular myocytes of zebrafish have a robust (-6.7 ± 1.2 pA pF(-1) at -120 mV) strongly rectifying and Ba(2+)-sensitive (IC50 = 3.8 μM) IK1. Transcripts of six Kir2 channels (drKir2.1a, drKir2.1b, drKir2.2a, drKir2.2b, drKir2.3, and drKir2.4) were expressed in the zebrafish heart. drKir2.4 and drKir2.2a were the dominant isoforms in both the ventricle (92.9 ± 1.5 and 6.3 ± 1.5%) and the atrium (28.9 ± 2.9 and 64.7 ± 3.0%). The remaining four channels comprised together less than 1 and 7 % of the total transcripts in ventricle and atrium, respectively. The four main gene products (drKir2.1a, drKir2.2a, drKir2.2b, drKir2.4) were cloned, sequenced, and expressed in HEK cells for electrophysiological characterization. drKir2.1a was the most weakly rectifying (passed more outward current) and drKir2.2b the most strongly rectifying (passed less outward current) channel, whilst drKir2.2a and drKir2.4 were intermediate between the two. In regard to sensitivity to Ba(2+) block, drKir2.4 was the most sensitive (IC50 = 1.8 μM) and drKir2.1a the least sensitive channel (IC50 = 132 μM). These findings indicate that the Kir2 isoform composition of the zebrafish heart markedly differs from that of mammalian hearts. Furthermore orthologous Kir2 channels (Kir2.1 and Kir2.4) of zebrafish and mammals show striking differences in Ba(2+)-sensitivity. Structural and functional differences needs to be taken into account when zebrafish is used as a model for human cardiac electrophysiology, cardiac diseases, and in screening cardioactive substances.

To date many early phase gene therapy trials have been successful. However, phase III and commercial phase have brought further challenges in producing viral vectors in sufficient large quantities. Upscaling the process on suspension cells is feasible, but many viral production applications are still applicable only in adherent settings. Scaling up the adherent system has proven to be troublesome and costly. The PALL iCELLisTM disposable fixed-bed bioreactors offer an efficient option for viral vector manufacturing in large quantities in an adherent environment. In iCELLisTM Nano, the cultivation area 0.53-4 m2 for smaller batches, is ideal for process development purposes. In iCELLIs500 the cultivation area varies between 66 and 500 m2 and is ideal to satisfy demand for phase3/commercial requirements.We have optimized adenovirus type 5 manufacturing using iCELLisTM Nano. HEK293 cell cultivation, infection and harvest of the virus by lysing the cells inside the bioreactor were efficient, reaching total yield of 3.4 × 10^14 vp/batch. When upscaling the process into 100 m2 cultivation area with iCELLis500, 1.2 × 10^16 vp/batch were produced.iCELLisTM technology is applicable also to other vector types which require for example plasmid transfection. Virus can also be harvested by perfusion from the medium. Lentiviral vector production in 293T cells was tested in iCELLisTM Nano and we achieved high plasmid transfection efficiency, leading to the comparable titers and productivity as in flasks. To conclude, iCELLisTM equipment has provided us an efficient way to manufacture large batches of different kinds of gene therapy products suitable for large preclinical animal models and up to phase III trial and beyond.

Freshwater fishes of north-temperate latitudes adjust electrical excitability of the heart to seasonal temperature changes by changing expression levels of ion channel isoforms. However, little is known about thermal responses of action potential (AP) in the hearts of marine polar fishes. To this end, we examined cardiac AP in the atrial myocardium of the Arctic navaga cod (Eleginus navaga) from the White Sea (Russia) acclimatized to winter (March) and summer (September) seasons. Acute increases in temperature from 4 to 10 °C were associated with increases in heart rate, maximum velocity of AP upstroke and negative resting membrane potential, while duration of AP was shortened in both winter-acclimatized and summer-acclimatized navaga hearts. In winter, there was a compensatory shortening (41.1%) of atrial AP duration and this was associated with a strong increase in transcript expression of Erg K(+) channels, known to produce the rapid component of the delayed rectifier K(+) current, I(Kr). Smaller increases were found in the expression of Kir2.1 channels that produce the inward rectifier K(+) current, I(K1). These findings indicate that the heart of navaga cod has a good acclimatory capacity in electrical excitation of cardiac myocytes, which enables cardiac function in the cold-eurythermal waters of the subarctic White Sea.

rAd-IFN is a recombinant adenoviral gene therapy vector encoding IFNα2b gene for the treatment of refractory non-muscle invasive bladder cancer. The vector transduces bladder wall cells where IFNα2b gene is expressed leading to death of cancer cells. The advanced testing strategy to determine the pharmacological activity of rAd-IFN drug product involves three key assays: 1. Infectious titer of the virus, quantitative assay 2. Expression of the transgene (IFNα2b), semiquantitative assay 3. Potency (IFNα2b mediated cell killing), quantitative assay The infectivity and transgene expression assays have been performed for batch release and stability monitoring of activity during Phase 2 and will remain unchanged in principle for Phase 3 and commercial use. In the infectivity assay the cells supporting adenovirus replication are infected with three concentrations of adenovirus and left to produce the virus for two days. The percentage of infected cells is then determined with a flow cytometer utilizing a fluorescently conjugated antibody against an adenoviral structural protein. Samples are analysed in parallel with a reference standard and infectivity is given as relative Infectious Units / ml. In expression assay, the IFNα expression capability of the virus preparation is determined by infecting IFN insensitive cells with the rAd-IFN virus and the concentration of produced IFNα is measured with a commercial IFNα ELISA (enzyme-linked immunosorbent assay) from cell culture supernatants For Phase 3 a new potency assay is developed and added to release and stability testing in order to provide evidence that batches of rAd-IFN are able to produce active IFNα2b which has a relevant pharmacological effect. In this assay cells are transduced using multiple dilutions of reference standard and test samples leading to expression of IFNα2b and subsequent cell death. Cell killing efficiency is determined using colorimetric method measuring dehydrogenase activity of the living cells. Relative potency of test sample is determined against reference standard response curve after testing parallelism by equivalence test. All activity assays will be fully validated according to ICH Q2 (R1) prior to release testing of Phase 3 clinical study material (Accuracy, Precision, Specificity, Linearity and Range, System Suitability and Robustness). The three validated assays will provide enhanced quantitative measure of biologic function of the rAd-IFN vector and thus demonstrate the quality and efficacy of drug product batches.

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 ( IKs) in cardiac myocytes of a eurythermic fish ( Carassius carassius L.). IKs 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 IKs of the crucian carp atrial myocytes with the currents produced by homomeric Kv7.1 and heteromeric Kv7.1/MinK channels in Chinese hamster ovary cells indicates that activation kinetics and pharmacological properties of the IKs are similar to those of the homomeric Kv7.1 channels. Consistently with electrophysiological properties and homomeric Kv7.1 channel composition, atrial transcript expression of the MinK subunit is only 1.6–1.9% of the expression level of the Kv7.1 subunit. Since activation kinetics of the homomeric Kv7.1 channels is much faster than activation of the heteromeric Kv7.1/MinK channels, the homomeric Kv7.1 composition of the crucian carp cardiac IKs 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 Kv7.1 channel assembly is an evolutionary thermal adaptation of ectothermic hearts and the heteromeric Kv7.1/MinK channels evolved later to adapt IKs to high body temperature of endotherms.

In ectotherms, compensatory changes in ion channel number and activity are needed to maintain proper cardiac function at variable temperatures. The rapid component of the delayed rectifier K+current ( IKr) is important for repolarization of cardiac action potential and, therefore, crucial for regulation of cellular excitability and heart rate. To examine temperature plasticity of cardiac IKr, we cloned the ether-à- go- go-related gene (ERG) channel and measured its electrophysiological properties in thermally acclimated rainbow trout ( Oncorhynchus mykiss; omERG). The present findings demonstrate a complete thermal compensation in the whole cell conductance of the atrial IKrin rainbow trout acclimated to 4°C (cold acclimation) and 18°C (warm acclimation). In situ hybridization indicates that transcripts of the omERG channel are present throughout the muscular tissue of the heart, and quantitative PCR shows increased expression of the omERG in cold-acclimated trout compared with warm-acclimated trout. In both acclimation groups, omERG expression is higher in atrium than ventricle. In addition, the omERG has some functional features that support IKractivity at low temperatures. Voltage dependence of steady-state activation is completely resistant to temperature changes, and steady-state inactivation and activation kinetics are little affected by temperatures below 11°C. Collectively, these findings suggest that high density of cardiac IKris achieved by cold-induced increase in the number of functional omERG channels and inherent insensitivity of the omERG to temperature below 11°C. These adaptations are probably important in maintaining high heart rates and proper excitability and contractility of trout cardiac myocytes in the cold.

Acceleration of flowering would be beneficial for breeding trees with a long juvenile phase; conversely, inhibition of flowering would prevent the spread of transgenes from the genetically modified trees. We have previously isolated and characterized several MADS genes from silver birch (Betula pendula Roth). In this study, we investigated the more detailed function of one of them, BpMADS4, a member of the APETALA1/FRUITFULL group of MADS genes. The expression of BpMADS4 starts at very early stage of the male and female inflorescence development and the activity is high in the apex of the developing inflorescence. Later, some expression is detected in the bracts and in the flower initials. Ectopic expression of BpMADS4 accelerates flowering dramatically in normally flowering clones and also in the early-flowering birch clone, in which the earliest line flowered about 11 days after rooting, when the saplings were only 3 cm high. The birches transformed with the BpMADS4 antisense construct showed remarkable delay in flowering and the number of flowering individuals was reduced. Two of the transformed lines did not show any signs of flower development during our 2-year study, whereas all the control plants formed inflorescences within 107 days. Our results show that BpMADS4 has a critical role in the initiation of birch inflorescence development and that BpMADS4 seems to be involved in the transition from vegetative to reproductive development. Therefore, BpMADS4 provides a promising tool for the genetic enhancement of forest trees.