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Birds acquired endothermy and a four-chambered heart independently from mammals in the course of evolution. Though avian embryos are widely used in experiments, little is known about the adult avian heart. Recent studies have shown that, despite a large evolutionary distance, the set of repolarizing potassium currents in avian myocardium resembles that in mammalian heart as well as that in humans. This allows for proposing birds as a potential model in experimental cardiology. The present study for the first time describes inward rectifier currents in working myocardium of quail. Using patch clamp method, we recorded main background inward rectifier current IK1 was recorded in isolated atrial and ventricular cardiomyocytes of quail. Both inward and outward components of IK1 in ventricular cells were larger than those in atrial cells, while there were no differences in voltage dependence of inward rectification. Acetylcholine and carbachol induced activation of acetylcholine-dependent inward rectifier current IKACh in atrial but not in ventricular myocytes. IKACh in atrial myocytes was sensitive to tertiapin. Constitutively active IKACh has not been detected. In multicellular preparations of the quail right atrium, carbachol induced hyperpolarization and shortening of action potentials, while no such effects were observed in preparations of the right ventricle. Activation of IKACh upon application of carbachol was dose-dependent with EC50 = 4.922 × 10–7 М. The described distribution of inward rectifier currents in avian myocardium is similar to that in mammalian species, which are widely used as model objects in experimental cardiology.

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).

Small G-proteins of Rho family modulate the activity of several classes of ion channels, including K+ channels Kv1.2, Kir2.1, and ERG; Ca2+ channels; and epithelial Na+ channels. The present study was aimed to check the RhoA potential regulatory effects on Na+ current (INa) transferred by Na+ channel cardiac isoform NaV1.5 in heterologous expression system and in native rat cardiomyocytes. Whole-cell patch-clamp experiments showed that coexpression of NaV1.5 with the wild-type RhoA in CHO-K1 cell line caused 2.7-fold decrease of INa density with minimal influence on steady-state activation and inactivation. This effect was reproduced by the coexpression with a constitutively active RhoA, but not with a dominant negative RhoA. In isolated ventricular rat cardiomyocytes, a 5-h incubation with the RhoA activator narciclasine (5 × 10−6 M) reduced the maximal INa density by 38.8%. The RhoA-selective inhibitor rhosin (10−5 M) increased the maximal INa density by 25.3%. Experiments with sharp microelectrode recordings in isolated right ventricular wall preparations showed that 5 × 10−6 M narciclasine induced a significant reduction of action potential upstroke velocity after 2 h of incubation. Thus, RhoA might be considered as a potential negative regulator of sodium channels cardiac isoform NaV1.5.

The effect of small G-proteins of the Rho family on sodium current conducted by cardiac isoform NaV1.5 of voltage-gated sodium channels was studied in heterologous expression system, CHO-K1 cell line transfected with a plasmid containing the NaV1.5 gene. The influence of cotransfection with genes of wild-type, constitutively-active, and dominant-negative small G-proteins RhoA, Rac1, and Cdc2 on the parameters of sodium current and its noninactivating component (INa,late) was estimated. Among three studied small G-proteins, only RhoA (wild-type and constitutively-active type) strongly affected sodium current reducing its peak amplitude, but not the value of INa,late. Cotransfection with wild-type Rac1 resulted in a minor decrease in sodium current. Thus, small G-protein RhoA has potential capability for suppression of sodium current, although physiological relevance of this property has to be verified.

MicroRNA is a small, single-stranded nucleotide sequence, which can regulate gene expression at the posttranscriptional level. To date, it is known that some microRNAs play an important role in physiology and pathophysiology of the cardio-vascular system. The effects of lipotransfection with microRNAs, such as miR-1-3p, miR-153-3p, or miR-133-3p, on the electrophysiological indices of the myocardium tissue were studied here for the first time. With the use of the standard microelectrode technique, action potentials (AP) in isolated perfused samples of the rat atrial tissue were recorded after their treatment with a transfection mixture of a liposome forming reagent and miR-1-3p, miR-153-3p, or miR-133-3p. It is demonstrated that the treatment of the myocardial tissue with a transfection agent results in the prolongation of the repolarization phase of AP. It was found that miR-1-3p and miR-153-3p did not affect the pattern of AP within 6 h after the treatment of tissue samples. However, miR133a-3p evoked a statistically significant increase in the AP duration under 90% repolarization. The duration was maximum 4 h after the transfection. Search and analysis of possible targets for miR133a-3p using bioinformatic methods were performed and it was assumed that this microRNA can interact with mRNA of some protein phosphatases. Suppression of protein phosphatases’ expression in cardiomyocytes may underlie the increased AP duration during the application of miR133a-3p found in the present study due to the effect on the proteins of calcium turnover.

A detailed description of the electrophysiological features of cardiomyocytes in the various contractile chambers of the vertebrate heart is essential to understand the evolution of cardiac electrical activity, yet very little is known about reptiles. The present study characterizes major ionic currents (I Na, I CaL, I Kr, I K1 and I KACh) and action potential (AP) configuration in cardiomyocytes from the ventricle, the right atrium and the sinus venosus (SV) of Burmese pythons (Python molurus) using sharp microelectrode and patch clamp recordings. Special attention was given to SV, since it consists of myocardial cells and appears to contribute to right atrial filling in snakes. We demonstrate that most of the SV in pythons has a stable resting potential of − 82.3 ± 2.6 mV (n = 9) and lacks pacemaker activity. AP duration at 50% repolarization was similar in cells from SV and atria (350.2 ± 8.7 and 330.4 ± 17.2 ms, respectively; n = 7), but shorter than ventricular APs (557.6 ± 19.2 ms, n = 5) at 30 °C. The densities of ionic currents, however, differed substantially between atrial and SV cells, where the latter had much lower densities of I Na, I CaL and I Kr than atrial and ventricular myocytes. I K1 in ventricle was ninefold greater than in atrial cells and 23-fold greater than in myocytes from SV. However, I KACh was absent in ventricular cells, while it was equally large in atrial and SV myocytes. Consistent with this observation, APs of atrium and SV, but not ventricle, were greatly shortened upon addition of acetylcholine (10 −6 M). Thus, snake SV, right atrium and ventricle have distinct patterns of ionic currents, but the resulting electrical activity is similar in atrium and SV.

Diadenosine polyphosphates (DAP) are now considered as a new class of endogenous regulatory cardiotropic compounds. In previous studies DAP were demonstrated to affect cardiac electrical activity and contractility in various animal species including rats. DAP decreased the action potential duration and reduced the contractility of the rat myocardium. At the same time, DAP did not affect repolarizing potassium currents (IK1, IKACh, Ito1, IKur), which normally participate in repolarization after the action potentials (AP), and had a little effect on L-type calcium current in isolated rat cardiomyocytes. However, in addition to these ionic currents, AP duration can be regulated via chloride currents. In this study the presence of a transient inward calcium-dependent chloride current Ito2 has been shown in rat ventricular myocardium and an influence of DAP on this current has been demonstrated for the first time. Ionic currents were recorded in isolated rat ventricular cardiomyocytes using whole-cell patch clamp method. Action potentials were recorded in isolated preparations of rat right ventricle with sharp glass microelectrodes. In the absence of Na+ and K+ and in the presence of potassium current blockers 4-aminopyridine (5 × 10–3 M) and tetraethylammonium (1.5 × 10–2 M) transient outward current was present in ventricular myocytes. This current was sensitive to non-selective chloride channel blocker 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS, 10–5 M), L-type calcium current blocker nifedipine (10–5 M), and a selective blocker of calcium-dependent chloride channels 6-(1,1-dimethyl ethyl ethyl)-2-[(2-furanyl carbonyl)amino]-4,5,6,7-tetrahydrobenzo[b]thiophen-3 carbonic acid (CaCCinh-A01, 10–5 M). In the presence of diadenosine tetraphosphate (Ap4A, 10–4 M) in the external solution the peak amplitude of the current increased by 44 ± 11%. Diadenosine pentaphosphate (Ap5A) and NAD+ failed to produce any significant effects on the current density. In isolated preparations of rat ventricular myocardium DIDS (10–5 M) and CaCCinh-A01 (10–5 M) blocked the Ap4A-induced acceleration of repolarization. Thus, the effects of Ap4A on cardiac electrical activity in rats are at least partially mediated by its influence on the amplitude of repolarizing chloride current Ito2.

To provide the first description of the exact location of primary pacemaker of the squamate heart, we used sharp microelectrode impalements and optical mapping of isolated sinus venosus preparations from Burmese pythons. We located the dominant pacemaker site at the base of the right leaflet of the sinoatrial valve (SAV), but latent pacemakers were also identified in a circular region around the SAV. Acetylcholine (10−5 mol l−1) or noradrenaline (10−6 mol l−1) induced shifts of the leading pacemaker site to other points near the SAV. The ionic currents of most of the cardiomyocytes isolated enzymatically from the SAV region resembled those of typical working myocytes from the sinus venosus. However, seven cells lacked the background inward rectifier current (IK1) and had a time-dependent hyperpolarization-induced inward current identified as the ‘funny’ pacemaker current (If). Therefore, the region proximal to SAV demonstrates pacemaking activity and contains cells that resemble the electrophysiological properties of mammalian pacemaker myocytes.

The development of interatrial septum (IAS) is a complicated process, which continues during postnatal life. The hypertrophic signals in developing heart are mediated among others by α-adrenergic pathways. These facts suggest the presence of specific electrophysiological features in developing IAS. This study was aimed to investigate the electrical activity in the tissue preparations of IAS from rat heart in normal conditions and under stimulation of adrenoreceptors. Intracellular recording of electrical activity revealed less negative level of resting membrane potential in IAS if compared to myocardium of left atrium. In normal conditions, non-paced IAS preparations were quiescent, but noradrenaline (10

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.

We identified a single nucleotide variation (SNV) (c.1264A > G) in the KCNQ1 gene in a 5-year-old boy who presented with a prolonged QT interval. His elder brother and mother, but not sister and father, also had this mutation. This missense mutation leads to a p.Lys422Glu (K422E) substitution in the Kv7.1 protein that has never been mentioned before. We inserted this substitution in an expression plasmid containing Kv7.1 cDNA and studied the electrophysiological characteristics of the mutated channel expressed in CHO-K1, using the whole-cell configuration of the patch-clamp technique. Expression of the mutant Kv7.1 channel in both homo- and heterozygous conditions in the presence of auxiliary subunit KCNE1 results in a significant decrease in tail current densities compared to the expression of wild-type (WT) Kv7.1 and KCNE1. This study also indicates that K422E point mutation causes a dominant negative effect. The mutation was not associated with a trafficking defect; the mutant channel protein was confirmed to localize at the cell membrane. This mutation disrupts the poly-Lys strip in the proximal part of the highly conserved cytoplasmic A–B linker of Kv7.1 that was not shown before to be crucial for channel functioning. © 2022 by the authors. Russian Science Foundation, RSF: 22-14-00088; International Association for the Evaluation of Educational Achievement, IEA: 2773 This study was funded by Russian Science Foundation (22-14-00088 to O.S.S.). Authors thank Lisa Trifonova for proof-reading the manuscript and Lisha Mai for help with . G.L. would like to acknowledge support from the CNRS International Emerging Action (IEA) PRC RUSSIE 2019 (PRC no. 2773). O.S.S. and V.N. acknowledge the support from the Interdisciplinary Scientific and Educational School of Moscow Lomonosov University «Molecular Technologies of the Living Systems and Synthetic Biology». was created with the help of Biorender.com.

Aim This study is aimed at investigation of electrophysiological effects of α1-adrenoreceptor (α1-AR) stimulation in the rat superior vena cava (SVC) myocardium, which is one of the sources of proarrhythmic activity. Methods α1-ARs agonists (phenylephrine-PHE or norepinephrine in presence of atenolol-NE + ATL) were applied to SVC and atrial tissue preparations or isolated cardiomyocytes, which were examined using optical mapping, glass microelectrodes or whole-cell patch clamp. α1-ARs distribution was evaluated using immunofluorescence. Kir2.X mRNA and protein level were estimated using RT-PCR and Western blotting. Results PHE or NE + ATL application caused a significant suppression of the conduction velocity (CV) of excitation and inexcitability in SVC, an increase in the duration of electrically evoked action potentials (APs), a decrease in the maximum upstroke velocity (dV/dtmax ) and depolarization of the resting membrane potential (RMP) in SVC to a greater extent than in atria. The effects induced by α1-ARs activation in SVC were attenuated by protein kinase C inhibition (PKC). The whole-cell patch clamp revealed PHE-induced suppression of outward component of IK1 inward rectifier current in isolated SVC, but not atrial myocytes. These effects can be mediated by α1A subtype of α-ARs found in abundance in rat SVC. The basal IK1 level in SVC was much lower than in atria as a result of the weaker expression of Kir2.2 channels. Conclusion Therefore, the reduced density of IK1 in rat SVC cardiomyocytes and sensitivity of this current to α1A-AR stimulation via PKC-dependent pathways might lead to proarrhythmic conduction in SVC myocardium by inducing RMP depolarization, AP prolongation, CV and dV/dtmax decrease.

Oil and gas exploration in the Arctic can result in the release of polycyclic aromatic hydrocarbons (PAHs) into relatively pristine environments. Following the recent spill of approximately 17 500 tonnes of diesel fuel in Norilsk, Russia, May 2020, our study focussed on the effects of phenanthrene, a low molecular weight PAH found in diesel and crude oil, on the isolated atrial and ventricular myocytes from the heart of the polar teleost, the Navaga cod (Eleginus nawaga). Acute exposure to phenanthrene in navaga cardiomyocytes caused significant action potential (AP) prolongation, confirming the proarrhythmic effects of this pollutant. We show AP prolongation was due to potent inhibition of the main repolarising current, IKr, with an IC50 value of ~2 µM. We also show a potent inhibitory effect (~55%) of 1 µM phenanthrene on the transient IKr currents that protects the heart from early-after-depolarizations and arrhythmias. These data, along with more minor effects on inward sodium (INa) (~17% inhibition at 10 µM) and calcium (ICa) (~17% inhibition at 30 µM) currents, and no effects on inward rectifier (IK1 and IKAch) currents, demonstrate the cardiotoxic effects exerted by phenanthrene on the atrium and ventricle of navaga cod. Moreover, we report the first data that we are aware of on the impact of phenanthrene on atrial myocyte function in any fish species. © 2021 The Authors This work is supported by British Heart Foundation Grant PG/17/77/33125 to HAS and SNK and by the Interdisciplinary Scientific and Educational School of Moscow University «Molecular Technologies of the Living Systems and Synthetic Biology» as part of the Scientific Project of the State Order of the Government of Russian Federation to Lomonosov Moscow State University No.121032300071-8 to DVA. We thank Prof Jules Hancox, University of Bristol, UK, and Dr Fabien Brette, Université de Bordeaux for insightful comments. We are grateful to White Sea Biological Station director, Professor Alexander Tzetlin for supporting the study. We thank Valo and Valentina Sivonen for collecting the fish.

Mir-133a-3p is the most abundant myocardial microRNA. The impact of mir-133a-3p on cardiac electrophysiology is poorly explored. In this study, we investigated the effects of mir-133a-3p on the main ionic currents critical for action potential (AP) generation and electrical activity of the heart. We used conventional ECG, sharp microelectrodes and patch-clamp to clarify a role of mir-133a-3p in normal cardiac electrophysiology in rats after in vivo and in vitro transfection. Mir-133a-3p caused no changes to pacemaker APs and automaticity in the sinoatrial node. No significant changes in heart rate (HR) were observed in vivo; however, miR transfection facilitated HR increase in response to β-adrenergic stimulation. Mir-133a-3p induced repolarization abnormalities in the atrial working myocardium and the L-type calcium current (ICa,L) was significantly increased. The main repolarization currents, including the transient outward (Ito), ultra-rapid (IK,ur), and inward rectifier (IK1) remained unaffected in atrial cardiomyocytes. Mir-133a-3p affected both ICa,L and Ito in ventricular cardiomyocytes. Systemic administration of mir-133a-3p induced QT-interval prolongation. Bioinformatic analysis revealed protein phosphatase 2 (PPP2CA/B) and Kcnd3 (encoding Kv4.3 channels generating Ito) as the main miR-133a-3p targets in the heart. No changes in mRNA expression of Cacna1c (encoding Cav1.2 channels generating ICa,L) and Kcnd3 were seen in mir-133a-3p treated rats. However, the expression of Ppp2cA, encoding PPP2CA, and Kcnip2 encoding KChIP2, a Kv4.3 regulatory protein, were significantly decreased. The accumulation of mir-133a-3p in cardiac myocytes causes chamber-specific electrophysiological changes. The suppression of PPP2CA, involved in adrenergic signal transduction, and Kchip2 may indirectly mediate mir-133a-3p-induced augmentation of ICa,L and attenuation of Ito.

Mir-133a-3p is the most abundant myocardial microRNA. The impact of mir-133a-3p on cardiac electrophysiology is poorly explored. In this study, we investigated the effects of mir-133a-3p on the main ionic currents critical for action potential (AP) generation and electrical activity of the heart. We used conventional ECG, sharp microelectrodes and patch-clamp to clarify a role of mir-133a-3p in normal cardiac electrophysiology in rats after in vivo and in vitro transfection. Mir-133a-3p caused no changes to pacemaker APs and automaticity in the sinoatrial node. No significant changes in heart rate (HR) were observed in vivo; however, miR transfection facilitated HR increase in response to β-adrenergic stimulation. Mir-133a-3p induced repolarization abnormalities in the atrial working myocardium and the L-type calcium current (I

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.

Birds occupy a unique position in the evolution of cardiac design. Their hearts are capable of cardiac performance on par with, or exceeding that of mammals, and yet the structure of their cardiomyocytes resembles those of reptiles. It has been suggested that birds use intracellular Ca2+ stored within the sarcoplasmic reticulum (SR) to power contractile function, but neither SR Ca2+ content nor the cross-talk between channels underlying Ca2+-induced Ca2+ release (CICR) have been studied in adult birds. Here we used voltage clamp to investigate the Ca2+ storage and refilling capacities of the SR and the degree of trans-sarcolemmal and intracellular Ca2+ channel interplay in freshly isolated atrial and ventricular myocytes from the heart of the Japanese quail (Coturnix japonica). A trans-sarcolemmal Ca2+ current (ICa) was detectable in both quail atrial and ventricular myocytes, and was mediated only by L-type Ca2+ channels. The peak density of ICa was larger in ventricular cells than in atrial cells, and exceeded that reported for mammalian myocardium recorded under similar conditions. Steadystate SR Ca2+ content of quail myocardium was also larger than that reported for mammals, and reached 750.6±128.2 μmol lâ'1 in atrial cells and 423.3±47.2 μmol lâ'1 in ventricular cells at 24°C. We observed SR Ca2+-dependent inactivation of ICa in ventricular myocytes, indicating cross-talk between sarcolemmal Ca2+ channels and ryanodine receptors in the SR. However, this phenomenon was not observed in atrial myocytes. Taken together, these findings help to explain the high-efficiency avian myocyte excitation-contraction coupling with regard to their reptilian-like cellular ultrastructure. © 2020 Company of Biologists Ltd. All rights reserved. The study was supported by the Russian Foundation for Basic Research (19-34-90142 to D.V.A.).

Small G-proteins of Rho family modulate the activity of several classes of ion channels, including K

The purpose of this study was to examine the effects of interferon-γ (IFN-γ) on calcium movement in rat ventricular myocytes. L-type Ca2+ currents (ICa,L) were recorded with the whole-cell configuration of the patch-clamp techniques. IFN-γ induces current density reduction at the test potential of 0 mV by 47.6 ± 7.4%. Heparin, a selective inhibitor of inositol-1,4,5-triphosphate (IP3)–induced Ca2+ release, applied via a patch pipette, induced an ICa,L amplitude decrease of about 46 ± 5.6%. The addition of IFN-γ to heparin-treated cells has no effect on ICa,L. Ryanodine induced an ICa,L current amplitude decrease of 35.1 ± 6.2%. The addition of IFN-γ to ryanodine-treated cells caused an additional ICa,L inhibiting of 17.6 ± 4.8%. Both cyclopiazonic acid (CPA), a specific SERCA inhibitor, and a combination of CPA and ryanodine caused a significant reduction of the ICa,L amplitudes. Subsequent addition of IFN-γ inhibited ICa,L for an additional 16.3 ± 4.4%. The employment of chelerythrine in this study prevented IFN-γ-induced L-type Ca2+ channel inhibition in only 10 min from the start of perfusion. Proposed mechanisms of regulation involved IFN-γ-induced IP3-sensitive Ca2+ release probably by a Ca2+-dependent translocation of PKC from the cytoplasm to the cell membrane as the obligatory first step of the IFN-γ-induced PKC-dependent L-type Ca2+ channel inhibition.

Birds occupy a unique position in the evolution of cardiac design. Their hearts are capable of cardiac performance on par with, or exceeding that of mammals, and yet the structure of their cardiomyocytes resembles those of reptiles. It has been suggested that birds use intracellular Ca

Diadenosine tetraphosphate (Ap4A) belongs to a group of endogenous purine compounds that have recently been considered as new neurotransmitters or cotransmitters in the autonomic nervous system. It has been shown that Ap4A affects electrophysiology of a pacemaker and working myocardium and modulates adrenergic control of the heart in adult mammals. Nevertheless, the physiological role of Ap4A in the regulation of bioelectric properties in the pulmonary vein (PV) myocardium has not yet been investigated. It is well known that myocardial tissue in the wall of the PV acts as source of the ectopic proarrhythmic activity that underlies supraventricular arrhythmias like atrial fibrillation. The aim of the present study was to elucidate the effects Ap4A on bioelectrical properties and proarrhythmic ectopy in PVs in adult rats and at early postnatal ontogenesis. Thus, the resting potentials and the electrically evoked and spontaneous action potentials were recorded with the use of the standard microelectrode technique in multicellular isolated PV specimens from male Wistar rats at postnatal days 1-, 7-, 14-, and 21- and also from 60-day-old animals, which were considered as mature. The application of Ap4A caused significant reduction of action potential duration in PV specimens from rats of all ages. Ap4A also caused significant resting membrane potential hyperpolar-ization in quiescent PVs specimens from 14-, 21-, and 60-day-old rats. In addition, Ap4A caused complete and significant suppression of ectopic automaticity caused by preliminary noradrenaline administration in PV from 21- and 60-day-old rats, but Ap4A was unable to alter spontaneous intrinsic activity in PV from neo-nate (1-day-old) rats. The Ap4A-caused attenuation of noradrenaline-induced ectopy in PV was accompanied by substantial resting membrane potential hyperpolarization in all cases. Our results allow suggesting that the release of Ap4A as a cotransmitter from autonomic nerve endings can reduce proarrhythmic ectopy caused by sympathetic stimulation of the PV myocardium in vivo.

The orderly contraction of the vertebrate heart is determined by generation and propagation of cardiac action potentials (APs). APs are generated by the integrated activity of time- and voltage-dependent ionic channels which carry inward Na

Catecholamines mediate the 'fight or flight' response in a wide variety of vertebrates. The endogenous catecholamine adrenaline increases heart rate and contractile strength to raise cardiac output. The increase in contractile force is driven in large part by an increase in myocyte Ca

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.

Extracellular diadenosine polyphosphates (ApnA) are recently considered as an endogenous signaling compounds with transmitter-like activity which present in numerous tissues, including heart. It has been demonstrated previously that extracellular ApnA cause alteration of the heart functioning via purine receptors in different mammalian species. Nevertheless, principal intracellular pathways which underlie ApnA action in the heart remain unknown. In the present study the role of the P2Y-associated intracellular regulatory pathway in the mediation of diadenosine tetraphosphate (Ap4A) effects in the rat heart has been investigated for the first time. Extracellular Ap4A caused significant decreasing of the ventricular inotropy. Ap4A evoked reduction of the left ventricle contractility in the isolated Langendorff-perfused rat hearts, decreasing of the Ca2+ transients in the enzymatically isolated ventricular cardiomyocytes and induced shortening of action potentials in the ventricle multicellular preparations. The inhibitory effects of Ap4A in the rat heart were significantly attenuated by protein kinase C (PKC) inhibitor chelerythrine but these effects were not affected by NO-synthase inhibitor L-NAME and guanylyl cyclase (sGC) inhibitor ODQ. In addition, substantial attenuation of Ap4A-caused negative inotropy in the left ventricle was produced by nonselective phsophodiesterase (PDE) inhibitor IBMX, while PDE type 2 inhibitor EHNA was ineffective. In conclusion, our results allow suggesting that Ap4A-induced inhibitory effects in the rat heart are mediated by PKC, but not by NO/sGC/PKG-related signaling pathway. In addition, PDE stimulation may contribute to Ap4A-caused inhibition of the rat heart contractility.

A new class III antiarrhythmic drug niferidil (RG-2) has been introduced as a highly effective therapy for cases of persistent atrial fibrillation, but ionic mechanisms of its action are poorly understood. In the present study, the effects of niferidil on action potential (AP) waveform and potassium currents responsible for AP repolarization were investigated in guinea pig atrial myocardium. APs were recorded with sharp glass microelectrodes in multicellular atrial preparations. Whole-cell patch-clamp technique was used to measure K+ currents in isolated myocytes. In multicellular atrial preparations, 10−8 M niferidil effectively prolonged APs by 15.2 ± 2.8% at 90% repolarization level. However, even the highest tested concentrations, 10−6 M and 10−5 M failed to prolong APs more than 32.5% of control duration. The estimated concentration of niferedil for half-maximal AP prolongation was 1.13 × 10−8 M. Among the potassium currents responsible for AP repolarization phase, I K1 was found to be almost insensitive to niferidil. However, another inward rectifier, I KACh, was effectively suppressed by micromolar concentrations of niferidil with IC50 = 9.2 × 10−6 M. I KATP was much less sensitive to the drug with IC50 = 2.26 × 10−4 M. The slow component of delayed rectifier, I Ks, also demonstrated low sensitivity to niferidil—the highest used concentration, 10−4 M, decreased peak I Ks density to 46.2 ± 5.5% of control. Unlike I Ks, the rapid component of delayed rectifier, I Kr, appeared to be extremely sensitive to niferidil. The IC50 was 1.26 × 10−9 M. I Kr measured in ventricular myocytes was found to be less sensitive to niferidil with IC50 = 3.82 × 10−8 M. Niferidil prolongs APs in guinea pig atrial myocardium via inhibition of I Kr.

The Alaska blackfish (Dallia pectoralis) remains active at cold temperatures when experiencing aquatic hypoxia without air access. To discern the cardiophysiological adjustments that permit this behaviour, we quantified the effect of acclimation from 15°C to 5°C in normoxia (15N and 5N fish), as well as chronic hypoxic submergence (6-8 weeks; ∼6.3-8.4 kPa; no air access) at 5°C (5H fish), on in vivo and spontaneous heart rate (fH), electrocardiogram, ventricular action potential (AP) shape and duration (APD), the background inward rectifier (IK1) and rapid delayed rectifier (IKr) K+ currents and ventricular gene expression of proteins involved in excitation-contraction coupling. In vivo fH was ∼50% slower in 5N than in 15N fish, but 5H fish did not display hypoxic bradycardia. Atypically, cold acclimation in normoxia did not induce shortening of APD or alter resting membrane potential. Rather, QT interval and APD were ∼2.6-fold longer in 5N than in 15N fish because outward IK1 and IKr were not upregulated in 5N fish. By contrast, chronic hypoxic submergence elicited a shortening of QT interval and APD, driven by an upregulation of IKr. The altered electrophysiology of 5H fish was accompanied by increased gene expression of kcnh6 (3.5-fold; Kv11.2 of IKr), kcnj12 (7.4-fold; Kir2.2 of IK1) and kcnj14 (2.9-fold; Kir2.4 of IK1). 5H fish also exhibited a unique gene expression pattern that suggests modification of ventricular Ca2+ cycling. Overall, the findings reveal that Alaska blackfish exposed to chronic hypoxic submergence prioritize the continuation of cardiac performance to support an active lifestyle over reducing cardiac ATP demand. © 2020. Published by The Company of Biologists Ltd This research was funded by the National Science Foundation, Division of Integrative Organismal Systems (1557818) and UAA Innovate Award (J.A.W.S.); the Russian Science Foundation (19-15-00163) (D.V.A.); Alaska INBRE (IDeA Network of Biomedical Research Excellence from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103395; the content is solely the responsibility of the authors and does not necessarily reflect the official views of the NIH) and LGL Limited Environmental Research Associates graduate research awards (K.L.K.). Deposited in PMC for release after 12 months.

Recently, we found cardioprotective effects of ischemic preconditioning (IPC), and from a blocker of KCNQ voltage-gated K+ channels (KV7), XE991 (10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone), in isolated rat hearts. The purpose of the present study was to investigate the cardiovascular effects of IPC and XE991 and whether they are cardioprotective in intact rats. In conscious rats, we measured the effect of the KV7 channel blocker XE991 on heart rate and blood pressure by use of telemetry. In anesthetized rats, cardiac ischemia was induced by occluding the left coronary artery, and the animals received IPC (2 × 5 min of occlusion), XE991, or a combination. After a 2 h reperfusion period, the hearts were excised, and the area at risk and infarct size were determined. In both anesthetized and conscious rats, XE991 increased blood pressure, and the highest dose (7.5 mg/kg) of XE991 also increased heart rate, and 44% of conscious rats died. XE991 induced marked changes in the electrocardiogram (e.g., increased PR interval and prolonged QTC interval) without changing cardiac action potentials. The infarct size to area at risk ratio was reduced from 53 ± 2% (n = 8) in the vehicle compared to 36 ± 3% in the IPC group (P < 0.05, n = 9). XE991 (0.75 mg/kg) treatment alone or on top of IPC failed to reduce myocardial infarct size. Similar to the effect in isolated hearts, locally applied IPC was cardioprotective in intact animals exposed to ischemia-reperfusion. Systemic administration of XE991 failed to protect the heart against ischemia-reperfusion injury suggesting effects on the autonomic nervous system counteracting the cardioprotection in intact animals. © 2019 Elsevier B.V. Aarhus Universitets Forskningsfond Hjerteforeningen: 17-R116-A7616-22074 K. Corydon was supported by a scholarship from Aarhus University Research Foundation , U. Simonsen and E.R. Hedegaard were supported by the Danish Heart Foundation (grant 17-R116-A7616-22074 ). The study was also supported by Jens Anker Andersens Foundation, Helge and Peter Kornings Foundation, Direktør Kurt Bønnelycke and wife Mrs Grethe Bønnelyckes Foundation, Helge Peetz and Verner Peetz and wife Vilma Peetz grant. Appendix A

The whole-cell patch-clamp technique was used to examine the effect of gadolinium Gd3+ (a non-specific blocker of mechanically gated current IMGCh, a component of late current IL) on ionic currents in insolated rat ventricular cardiomyocytes alone and in combination with the blockers of L-type calcium currents (ICaL) nifedipine (10 μM) or verapamil (1 μM). In K+in/K+out or Cs+in/Cs+out media, blockade of ICaL produced no effect on IL at negative potentials, but inhibited IL at positive ones. In K+in/K+out medium, Gd3+ (5 μM) decreased the net persistent current (Inp) at -45 mV from 198.6±6.4 to 96.7±9.5 pA over 15 min. Gd3+ alone or in combination with ICaL blockers shifted the reversal potential of IL to more negative values. At negative potentials, Gd3+ decreased IK1 and inward current including IMGCh. At positive potentials, Gd3+ alone or in combination with ICaL blockers decreased IL. When applied for 15 min in Cs+in/Cs+out medium at -45 mV, Gd3+ produced no effect on net current and inward and outward components of IL. Thus, Gd3+ can be viewed as a specific blocker of IMGCh only in Cs+ medium.

Temperature-induced changes in cardiac output (Q̇) in fish are largely dependent on thermal modulation of heart rate ( fH), and at high temperatures Q̇ collapses due to heat-dependent depression of fH. This study tests the hypothesis that firing rate of sinoatrial pacemaker cells sets the upper thermal limit of fHin vivo. To this end, temperature dependence of action potential (AP) frequency of enzymatically isolated pacemaker cells (pacemaker rate, fPM), spontaneous beating rate of isolated sinoatrial preparations ( fSA), and in vivo fHof the cold-acclimated (4°C) brown trout ( Salmo trutta fario) were compared under acute thermal challenges. With rising temperature, fPMsteadily increased because of the acceleration of diastolic depolarization and shortening of AP duration up to the break point temperature (TBP) of 24.0 ± 0.37°C, at which point the electrical activity abruptly ceased. The maximum fPMat TBPwas much higher [193 ± 21.0 beats per minute (bpm)] than the peak fSA(94.3 ± 6.0 bpm at 24.1°C) or peak fH(76.7 ± 2.4 at 15.7 ± 0.82°C) ( P < 0.05). These findings strongly suggest that the frequency generator of the sinoatrial pacemaker cells does not limit fHat high temperatures in the brown trout in vivo.

The functioning of the heart is under the tight control of the sympathetic division of the autonomous nervous system. The terminals of the postganglionary fibers release both noradrenaline (NA), which is the major sympathetic neurotransmitter, and comediators that can contribute to the fine “tuning” of the adrenergic control of cardiac function. Purine compounds, such as diadenosine pentaphosphate (Ар5А), as well as nicotinamide adenine dinucleotide (NAD+), can act as comediators. The distinctive features of the effects of these compounds on the heart have been incompletely characterized. It is not clear whether these compounds can act as comediators, i.e., to modulate the sympathetic (adrenergic) activity in the heart. Exogenous extracellular Ар5А was shown to reduce the contractility of the ventricular myocardium and to suppress conduction in the atrioventricular (AV) junction in a Langendorff-perfused isolated rat heart. Extracellular NAD+ had a weak effect on inotropy but induced a negative dromotropic effect in the AV junction, similarly to Ар5А. We found that Ар5А and NAD+ suppress both the positive inotropic effect of noradrenaline in the ventricular myocardium and the positive dromotropic effect of NA in the AV junction. The “inhibitory” effects of both purine compounds were more pronounced in the case of combined application with NA. In addition, the influence of Ар5А and NAD+ on the effects of noradrenaline was shown to depend on the timing of the application of these compounds. Our results suggest that the role of the sympathetic comediators NAD+ and Ар5А may consist of the limitation of noradrenaline effects and/or the effects of sympathetic stimulation in the heart.

Diadenosine pentaphosphate (Ap5A) belongs to the family of diadenosine polyphosphates, endogenously produced compounds that affect vascular tone and cardiac performance when released from platelets. The previous findings indicate that Ap5A shortens action potentials (APs) in rat myocardium via activation of purine P2 receptors. The present study demonstrates alternative mechanism of Ap5A electrophysiological effects found in guinea pig myocardium. Ap5A (10−4 M) shortens APs in guinea pig working atrial myocardium and slows down pacemaker activity in the sinoatrial node. P1 receptors antagonist DPCPX (10−7 M) or selective GIRK channels blocker tertiapin (10−6 M) completely abolished all Ap5A effects, while P2 blocker PPADS (10−4 M) was ineffective. Patch-clamp experiments revealed potassium inward rectifier current activated by Ap5A in guinea pig atrial myocytes. The current was abolished by DPCPX or tertiapin and therefore was considered as potassium acetylcholine-dependent inward rectifier (I KACh). Thus, unlike rat, in guinea pig atrium Ap5A produces activation of P1 receptors and subsequent opening of KACh channels leading to negative effects on cardiac electrical activity.

Electrophysiological effects produced by selective activation of M3 cholinoreceptors were studied in isolated left atrium preparations from rat using the standard sharp glass microelectrode technique. The stimulation of M3 receptors was obtained by application of muscarinic agonist pilocarpine (10−5 M) in the presence of selective M2 antagonist methoctramine (10−7 M). Stimulation of M3 receptors induced marked reduction of action potential duration by 14.4 ± 2.4% and 16.1 ± 2.5% of control duration measured at 50 and 90% of repolarization, respectively. This effect was completely abolished by selective M3 blocker 4-DAMP (10−8 M). In isolated myocytes obtained from the rat left atrium, similar pharmacological stimulation of M3 receptors led to suppression of peak L-type calcium current by 13.9 ± 2.6% of control amplitude (measured at +10 mV), but failed to affect K+ currents Ito, IKur, and IKir. In the absence of M2 blocker methoctramine, pilocarpine (10−5 M) produced stronger attenuation of ICaL and induced an increase in IKir. This additive inward rectifier current could be abolished by highly selective blocker of Kir3.1/3.4 channels tertiapin-Q (10−6 M) and therefore was identified as IKACh. Thus, in the rat atrial myocardium activation of M3 receptors leads to shortening of action potentials via suppression of ICaL, but does not enhance the major potassium currents involved in repolarization. Joint stimulation of M2 and M3 receptors produces stronger action potential shortening due to M2-mediated activation of IKACh.

Fishes of north-temperate latitudes exhibit marked seasonal changes in electrical excitability of the heart partly as an outcome of temperature-dependent changes in the density of major K+ ion currents: delayed rectifiers (IKr, IKs) and background inward rectifier (IK1). In the arctic teleost, navaga cod (Eleginus navaga), IKr and IK1 are strongly up-regulated in winter. The current study tests the hypothesis that the ligand-gated K+ current, the acetylcholine-activated inward rectifier, IKACh, is also modified by seasonal acclimatization in atrial myocytes of navaga. In sinoatrial preparations of the summer-acclimatized (SA) navaga, 10−6 M carbamylcholine chloride (CCh) caused slowing of heart rate, shortening of atrial action potential (AP) duration and a drastic reduction of AP amplitude, eventually resulting in inexcitability. In winter-acclimatized (WA) atria CCh slowed HR and reduced AP duration, but reduction of AP amplitude was modest and never resulted in inexcitability. The difference in cholinergic response between SA and WA navaga is explained by seasonal changes in IKACh density. The peak density of IKACh, induced by 10−5 M CCh, at the common experimental temperature (+6 °C) was 0.97 ± 0.28 pA/pF in SA navaga but only 0.183 ± 0.013 pA/pF in WA navaga (a 5.3-fold difference, P

Type 3 muscarinic receptors (M3 receptors) participate in the mediation of cholinergic effects in mammalian myocardium, along with M2 receptors. However, myocardium of adult mammals demonstrates only modest electrophysiological effects in response to selective stimulation of M3 receptors which are hardly comparable to the effects produced by M2 stimulation. In the present study, the effects of selective M3 stimulation induced by application of the muscarinic agonist pilocarpine (10 M) in the presence of the selective M2 blocker methoctramine (100 nM) on the action potential (AP) waveform were investigated in isolated atrial and ventricular preparations from newborn and 3-week-old rats and compared to those in preparations from adult rats. In the atrial myocardium, stimulation of M3 receptors produced a comparable reduction of AP duration in newborn and adult rats, while in 3-week-old rats the effect was negligible. In ventricular myocardial preparations from newborn rats, the effect of M3 stimulation was more than 3 times stronger compared to that from adult rats, while preparations from 3-week old rats demonstrated no definite effect, similarly to atrial preparations. In all studied types of cardiac preparations, the effects of M3 stimulation were eliminated by the selective M3 antagonist 4-DAMP (10 nM). The results of RT-PCR show that the amount of product of the M3 receptor gene decreases with the maturation of animals both in atrial and ventricular myocardium. We concluded that the contribution of M3 receptors to the mediation of cardiac cholinergic responses decreases during postnatal ontogenesis. These age-related changes may be associated with downregulation of M3 receptor gene expression.

Effects of nucleotide polyphosphate compounds (nicotinamide adenine dinucleotide, NAD(+); diadenosine tetraphosphate, Ap4A) on the confi guration of action potentials were studied in isolated preparations of guinea pig sinoatrial node and right atrial appendage (auricle). In the working myocardium, NAD(+) and Ap4A in concentrations of 10(-5) and 10(-4) M had no effect on resting potential, but significantly reduced the duration of action potentials; the most pronounced decrease was found at 25% repolarization. In the primary pacemaker of the sinoatrial node, both concentrations of NAD(+) and Ap4A induced hyperpolarization and reduction in the rate of slow diastolic depolarization, but significant slowing of the sinus rhythm was produced by these substances only in the concentration of 10(-4) M. Moreover, AP shortening and marked acceleration of AP upstroke were observed in the pacemaker myocardium after application of polyphosphates. Comparative analysis of the effects of NAD(+) and Ap4A in the working and pacemaker myocardium drove us to a hypothesis on inhibitory effects of these substances on L-type calcium current accompanied by stimulation of one or several potassium currents, which induce enhancement of repolarization and hyperpolarization of membranes probably mediated by the activation of purine receptors.

Seasonal thermal remodelling (acclimatization) and laboratory thermal remodelling (acclimation) can induce different physiological changes in ectothermic animals. As global temperatures are changing at an increasing rate, there is urgency to understand the compensatory abilities of key organs such as the heart to adjust under natural conditions. Thus, the aim of the present study was to directly compare the acclimatization and acclimatory response within a single eurythermal fish species, the European shorthorn sculpin (Myoxocephalus scorpio). We used current- and voltage-clamp to measure ionic current densities in both isolated atrial and ventricular myocytes from three groups of fish: (1) summer-caught fish kept at 12°C (‘summer-acclimated’); (2) summer-caught fish kept at 3°C (‘cold acclimated’); and (3) fish caught in March (‘winter-acclimatized’). At a common test temperature of 7.5°C, action potential (AP) was shortened by both winter acclimatization and cold acclimation compared with summer acclimation; however, winter acclimatization caused a greater shortening than did cold acclimation. Shortening of AP was achieved mostly by a significant increase in repolarizing current density (IKr and IK1) following winter acclimatization, with cold acclimation having only minor effects. Compared with summer acclimation, the depolarizing L-type calcium current (ICa) was larger following winter acclimatization, but again, there was no effect of cold acclimation on ICa. Interestingly, the other depolarizing current, INa, was downregulated at low temperatures. Our further analysis shows that ionic current remodelling is primarily due to changes in ion channel density rather than current kinetics. In summary, acclimatization profoundly modified the electrical activity of the sculpin heart while acclimation to the same temperature for >1.5 months produced very limited remodelling effects. © 2019. Published by The Company of Biologists Ltd. Russian Foundation for Basic Research, RFBR: 18-315-20049 The study was supported by the Russian Foundation for Basic Research (18-315-20049 to D.V.A.).

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.

Electrical excitability (EE) is vital for cardiac function and strongly modulated by temperature and external K+ concentration ([K+]o), as formulated in the hypothesis of temperature-dependent deterioration of electrical excitability (TDEE). As little is known about EE of arctic stenothermic fishes, we tested the TDEE hypothesis on ventricular myocytes of polar cod (Boreogadus saida) and navaga (Eleginus nawaga) of the Arctic Ocean and those of temperate freshwater burbot (Lota lota). Ventricular action potentials (APs) were elicited in current-clamp experiments at 3, 9 and 15°C, and AP characteristics and the current needed to elicit APs were examined. At 3°C, ventricular APs of polar cod and navaga were similar but differed from those of burbot in having a lower rate of AP upstroke and a higher rate of repolarization. EE of ventricular myocytes – defined as the ease with which all-or-none APs are triggered – was little affected by acute temperature changes between 3 and 15°C in any species. However, AP duration (APD50) was drastically reduced at higher temperatures. Elevation of [K+]o from 3 to 5.4 mmol l−1 and further to 8 mmol l−1 at 3, 9 and 15°C strongly affected EE and AP characteristics in polar cod and navaga, but had a lesser effect in burbot. In all species, ventricular excitation was resistant to acute temperature elevations, while small increases in [K+]o severely compromised EE, in particular in the marine stenotherms. This suggests that EE of the heart in these Gadiformes species is resistant against acute warming, but less so against the simultaneous temperature and exercise stresses.

International audience; Styrene-maleic acid (SMA) copolymers are used to extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding SMA lipid particles (SMALPs). SMALPs can serve as stable water-soluble nanocontainers for structural and functional studies of membrane proteins. Here, we used SMA copolymers to study full-length pore-forming α-subunits hKCNH5 and hKCNQ1 of human neuronal and cardiac voltage-gated potassium (Kv) channels, as well as the fusion construct comprising of an α-subunit hKCNQ1 and its regulatory transmembrane KCNE1 β-subunit (hKCNE1-hKCNQ1) with added affinity tags, expressed in mammalian COS-1 cells. All these recombinant proteins were shown to be functionally active. Treatment with the SMA copolymer, followed by purification on the affinity column, enabled extraction of all three channels. A DLS experiment demonstrated that Negative stain electron microscopy and single particle image analysis revealed a four-fold symmetry within channel-containing SMALPs, which indicates that purified hKCNH5 and hKCNQ1 channels, as well as the hKCNE1-hKCNQ1 fusion construct, retained their structural integrity as tetramers.