3-methyl-phenanthrene (3-MP) disrupts the electrical and contractile activity of the heart of the polar fish, navaga cod (Eleginus nawaga).

Alkylated polycyclic aromatic hydrocarbons are abundant in crude oil and are enriched during petroleum refinement but knowledge of their cardiotoxicity remains limited. Polycyclic aromatic hydrocarbons (PAHs) are considered the main hazardous components in crude oil and the tricyclic PAH phenanthrene, has been singled out for its direct effects on cardiac tissue in mammals and fish. Here we test the impact of the monomethylated phenanthrene, 3-methylphenanthrene (3-MP), on the contractile and electrical function of the atria and ventricle of a polar fish, the navaga cod (Eleginus nawaga). Using patch-clamp electrophysiology in atrial and ventricular cardiomyocytes we show that 3-MP is a potent inhibitor of the delayed rectifier current IKr (EC50=0.25 µM) and prolongs ventricular action potential duration. Unlike the parent compound phenanthrene, 3-MP did not reduce the amplitude of the L-type Ca2+ current (ICa) but it accelerated current inactivation thus reducing charge transfer across the myocyte membrane and compromising pressure development of the whole heart. 3-MP was a potent inhibitor (EC50=4.7 µM) of the sodium current (INa), slowing the upstroke of the action potential in isolated cells, slowing conduction velocity across the atria measured with optical mapping, and increasing atrio-ventricular delay in a working whole heart preparation. Together, these findings reveal the strong cardiotoxic potential of this phenanthrene derivative on the fish heart. As 3-MP and other alkylated phenanthrenes comprise a large fraction of the PAHs in crude oil mixtures, these findings are worrisome for Arctic species facing increasing incidence of spills and leaks from the petroleum industry. 3-MP is also a major component of polluted air but is not routinely measured. This is also of concern if the hearts of humans and other terrestrial animals respond to this PAH in a similar manner to fish.

Organization of the blood system of rhynchonellid brachiopod Hemithiris psittacea (Brachiopoda: Rhynchonelliformea).

The brachiopods are sessile invertebrates with an unusual blood system, which consists of a long-branched dorsal vessel. It is still unknown how blood circulates in this system. In the present study, for the first time we propose the circulation of blood in brachiopod Hemithiris psittacea based on morphological and experimental data. The main heart is located on the dorsal side of the stomach and divides the dorsal vessel into anterior and posterior parts. The anterior part enters the lophophore, where it gives off blind branches to each tentacle. The posterior part passes by the funnels of the nephridia and forms a blindly closed network in the gonads. We suggest that the circulation of blood includes three successive stages. During the first phase of systole of the main heart, blood flows through the anterior dorsal vessel. During the second phase of systole, blood flows through the posterior dorsal vessel. During diastole, blood flows from the anterior and posterior vessels and fills the main heart. The origin of a peculiar blood system in brachiopods can be explained by reduction of the ventral vessel, which is probably correlates with the reduction of the ventral side of the brachiopod ancestor's body. Another peculiarity of brachiopod blood system is the presence of an ampullar heart, which functions as a blood depot and allows blood to move in the vessels in two directions in an oscillatory mode. The brachiopod blood system contains vessels lacking true endothelium and can be classified as an "incompletely closed" type.

Tricyclic hydrocarbon fluorene attenuates ventricular ionic currents and pressure development in the navaga cod.

The release of polycyclic aromatic hydrocarbons (PAHs) into the environment due to oil and diesel fuel spills is a serious threat to Arctic fish populations. PAHs produce multiple toxic effects in fish, but disturbance of electrical and contractile activity of the heart seems to be the most negative effect. Our study focused on the effects of fluorene, a tricyclic PAH resembling the well-investigated tricyclic phenanthrene, on major ionic currents and action potential (AP) waveform in isolated ventricular myocytes and on contractile activity in isolated whole hearts of polar navaga cod (Eleginus nawaga). Among the studied currents, the repolarizing rapid delayed rectifier K+ current IKr demonstrated the highest sensitivity to fluorene with IC50 of 0.54 µM. The depolarizing inward currents, INa and ICaL, were inhibited with 10 µM fluorene by 20.2 ± 2.8 % and 27.9 ± 8.4 %, respectively, thereby being much less sensitive to fluorene than IKr. Inward rectifier IK1 current was insensitive to fluorene (up to 10 µM). While 3 µM fluorene prolonged APs, 10 µM also slowed the AP upstroke. Resting membrane potential was not affected by any tested concentrations. In isolated heart experiments 10 µM fluorene caused modest depression of ventricular contractile activity. Thus, we have demonstrated that fluorene, a tricyclic PAH present in high quantities in crude oil, strongly impacts electrical activity with only slight effects on contractile activity in the heart of the polar fish, the navaga cod.

Sodium current preserves electrical excitability in the heart of hibernating ground squirrel (Citellus undulatus).

Hibernating mammals are capable of maintaining normal cardiac function at low temperatures. Excitability of cardiac myocytes crucially depends on the fast sodium current (INa), which is decreased in hypothermia due to both depolarization of resting membrane potential and direct negative effect of low temperature. Therefore, INa in hibernating mammals should have specific features allowing to maintain excitability of myocardium at low temperatures. The current-voltage dependence of INa, its steady-state inactivation and activation and recovery from inactivation were studied in winter hibernating (WH) and summer active (SA) ground squirrels and in rats using whole-cell patch clamp at 10 °C and 20 °C. INa peak amplitude and the parameters of steady-state activation and inactivation curves did not differ between SA and WH ground squirrels at both temperatures. However, at both temperatures strong positive shift of activation and inactivation curves by 5-12 mV was observed in both WH and SA ground squirrels if compared to rats. This peculiarity of cardiac INa in ground squirrels helps to maintain excitability in conditions of depolarized resting membrane potential. The time course of INa recovery from inactivation at 10 °C was faster in WH than in SA ground squirrels, which could ensure normal activation of myocardium during hibernation.

High temperature and hyperkalemia cause exit block of action potentials at the atrioventricular junction of rainbow trout (Oncorhynchus mykiss) heart.

At critically high temperatures, atrioventricular (AV) block causes ventricular bradycardia and collapse of cardiac output in fish. Here, the possible role of the AV canal in high temperature-induced heart failure was examined. To this end, optical mapping was used to measure action potential (AP) conduction in isolated AV junction preparations of the rainbow trout (Oncorhynchus mykiss) heart during acute warming/cooling in the presence of 4 or 8 mM external K+ concentration. The preparation included the AV canal and some atrial and ventricular tissue at its edges, and it was paced either from atrial or ventricular side at a frequency of 0.67 Hz (40 beats min-1) to trigger forward (anterograde) and backward (retrograde) conduction, respectively. The propagation of AP was fast in atrial and ventricular tissues, but much slower in the AV canal, causing an AV delay. Acute warming from 15 °C to 27 °C or cooling from 15 °C to 5 °C did not impair AP conduction in the AV canal, as both anterograde and retrograde excitations propagated regularly through the AV canal. In contrast, anterograde conduction through the AV canal did not trigger ventricular excitation at the boundary zone between the AV canal and the ventricle when extracellular K+ concentration was raised from 4 mM to 8 mM at 27 °C. Also, the retrograde conduction was blocked at the border between the AV canal and the atrium in high K+ at 27 °C. These findings suggest that the AV canal is resistant against high temperatures (and high K+), but the ventricular muscle cannot be excited by APs coming from the AV canal when temperature and external K+ concentration are simultaneously elevated. Therefore, bradycardia at high temperatures in fish may occur due to inability of AP of the AV canal to trigger ventricular AP at the junctional zone between the AV canal and the proximal part of the ventricle.

a1-adrenergic receptors accompanied by GATA4 expression are related to proarrhythmic conduction and automaticity in rat interatrial septum

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 (10−5 M) and phenylephrine (10−5 M) induced spontaneous action potentials, which could be abolished by α1-blocker prazosin (10−5 M), but not β1-blocker atenolol (10−5 M). Optical mapping showed drastic phenylephrine-induced slowing of conduction in adult rat IAS. The α1-dependent ectopic automaticity of IAS myocardium might be explained by immunohistochemical data indicating the presence of transcription factor GATA4 and abundant α1A-adrenoreceptors in myocytes from adult rat IAS. An elevated sensitivity to adrenergic stimulation due to involvement of α1-adrenergic pathways may underlie increased proarrhythmic potential of adult IAS at least in rats. (AU)

α1-adrenergic receptors accompanied by GATA4 expression are related to proarrhythmic conduction and automaticity in rat interatrial septum.

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 (10-5 M) and phenylephrine (10-5 M) induced spontaneous action potentials, which could be abolished by α1-blocker prazosin (10-5 M), but not ß1-blocker atenolol (10-5 M). Optical mapping showed drastic phenylephrine-induced slowing of conduction in adult rat IAS. The α1-dependent ectopic automaticity of IAS myocardium might be explained by immunohistochemical data indicating the presence of transcription factor GATA4 and abundant α1A-adrenoreceptors in myocytes from adult rat IAS. An elevated sensitivity to adrenergic stimulation due to involvement of α1-adrenergic pathways may underlie increased proarrhythmic potential of adult IAS at least in rats.

Melatonin treatment improves ventricular conduction via upregulation of Nav1.5 channel proteins and sodium current in the normal rat heart.

Melatonin treatment was reported to reduce the risk of cardiac arrhythmias, and crucial for this antiarrhythmic action was the effect of melatonin on activation spread. The aim of the present study was evaluation of the mechanisms of this activation enhancement. Experiments were performed in a total of 123 control and melatonin-treated (10 mg/kg, daily, for 7 days) male Wistar rats. In epicardial mapping studies (64 leads, interlead distance 0.5 mm) in the anesthetized animals, activation times (ATs) were determined in each lead as dV/dt minimum during QRS complex under sinus rhythm. Epicardial pacing was performed to measure conduction velocity (CV) across the mapped area. Average left ventricular ATs were shorter in the treated animals as compared to the controls, whereas the minimal epicardial ATs indicating the duration of activation propagation via the ventricular conduction system did not differ between the groups. CV was higher in the treated groups indicating that melatonin affected conduction via contractile myocardium The area of Cx43-derived fluorescence, as well as the expression of Cx43 protein, was similar in ventricles in the control and melatonin-treated groups. Expression of Gja1 gene transcripts encoding Cx43, was increased in the last group. An uncoupling agent octanol modified myocardial conduction properties (time of activation, action potential upstroke velocity, passive electrotonic phase duration) similarly in both groups. On the other hand, the expression of both Scn5a gene transcripts encoding Nav1.5 proteins, as well as peak density of transmembrane sodium current were increased in the ventricular myocytes from the melatonin-treated animals. Thus, a week-long melatonin treatment caused the increase of conduction velocity via enhancement of sodium channel proteins expression and increase of sodium current in the ventricular myocytes.

Ionic currents underlying different patterns of electrical activity in working cardiac myocytes of mammals and non-mammalian vertebrates.

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+ and Ca2+ currents, and outward K+ currents. This review compares atrial and ventricular APs and underlying ion currents between different taxa of vertebrates. We have collected literature data and attempted to find common electrophysiological features for two or more vertebrate groups, show differences between taxa and cardiac chambers, and indicate gaps in the existing data. Although electrical excitability of the heart in all vertebrates is based on the same superfamily of channels, there is a vast variability of AP waveforms between atrial and ventricular myocytes, between different species of the same vertebrate class and between endothermic and ectothermic animals. The wide variability of AP shapes is related to species-specific differences in animal size, heart rate, stage of ontogenetic development, excitation-contraction coupling, temperature and oxygen availability. Some of the differences between taxa are related to evolutionary development of genomes, which appear e.g. in the expression of different Na+ and K+ channel orthologues in cardiomyocytes of vertebrates. There is a wonderful variability of AP shapes and underlying ion currents with which electrical excitability of vertebrate heart can be generated depending on the intrinsic and extrinsic conditions of animal body. This multitude of ionic mechanisms provides excellent material for studying how the function of the vertebrate heart can adapt or acclimate to prevailing physiological and environmental conditions.

Adrenergic prolongation of action potential duration in rainbow trout myocardium via inhibition of the delayed rectifier potassium current, IKr.

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 Ca2+ influx on the L-type Ca current (ICaL) during the cardiac action potential (AP). Here, we report a K+- based mechanism that prolongs AP duration (APD) in fish hearts following adrenergic stimulation. We show that adrenergic stimulation inhibits the delayed rectifier K+ current (IKr) in rainbow trout (Oncorhynchus mykiss) cardiomyocytes. This slows repolarization and prolongs APD which may contribute to positive inotropy following adrenergic stimulation in fish hearts. The endogenous ligand, adrenaline (1 µM), which activates both α- and ß-ARs reduced maximal IKr tail current to 61.4 ± 3.9% of control in atrial and ventricular myocytes resulting in an APD prolongation of ~20% at both 50 and 90% repolarization. This effect was reproduced by the α-specific adrenergic agonist, phenylephrine (1 µM), but not the ß-specific adrenergic agonist isoproterenol (1 µM). Adrenaline (1 µM) in the presence of ß1 and ß2-blockers (1 µM atenolol and 1 µM ICI-118551, respectively) also inhibited IKr. Thus, IKr suppression following α-adrenergic stimulation leads to APD prolongation in the rainbow trout heart. This is the first time this mechanism has been identified in fish and may act in unison with the well-known enhancement of ICaL following adrenergic stimulation to prolong APD and increase cardiac inotropy.

Intrauterine L-NAME Exposure Weakens the Development of Sympathetic Innervation and Induces the Remodeling of Arterial Vessels in Two-Week-Old Rats.

Nitric oxide (NO) has been shown to stimulate differentiation and increase the survival of ganglionic sympathetic neurons. The proportion of neuronal NOS-immunoreactive sympathetic preganglionic neurons is particularly high in newborn rats and decreases with maturation. However, the role of NO in the development of vascular sympathetic innervation has never been studied before. We tested the hypothesis that intrauterine NO deficiency weakened the development of vascular sympathetic innervation and thereby changed the contractility of peripheral arteries and blood pressure level in two-week-old offspring. Pregnant rats consumed NOS inhibitor L-NAME (250 mg/L in drinking water) from gestational day 10 until delivery. Pups in the L-NAME group had a reduced body weight and blood level of NO metabolites at 1-2 postnatal days. Saphenous arteries from two-week-old L-NAME offspring demonstrated a lower density of sympathetic innervation, a smaller inner diameter, reduced maximal active force and decreased α-actin/ß-actin mRNA expression ratio compared to the controls. Importantly, pups in the L-NAME group exhibited decreased blood pressure levels before, but not after, ganglionic blockade with chlorisondamine. In conclusion, intrauterine L-NAME exposure is followed by the impaired development of the sympathetic nervous system in early postnatal life, which is accompanied by the structural and functional remodeling of arterial blood vessels.

The snake heart pacemaker is localized near the sinoatrial valve.

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.

Micro-RNA 133a-3p induces repolarization abnormalities in atrial myocardium and modulates ventricular electrophysiology affecting ICa,L and Ito currents.

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 ß-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.

Further insights into the molecular complexity of the human sinus node - The role of 'novel' transcription factors and microRNAs.

RESEARCH PURPOSE: The sinus node (SN) is the heart's primary pacemaker. Key ion channels (mainly the funny channel, HCN4) and Ca2+-handling proteins in the SN are responsible for its function. Transcription factors (TFs) regulate gene expression through inhibition or activation and microRNAs (miRs) do this through inhibition. There is high expression of macrophages and mast cells within the SN connective tissue. 'Novel'/unexplored TFs and miRs in the regulation of ion channels and immune cells in the SN are not well understood. Using RNAseq and bioinformatics, the expression profile and predicted interaction of key TFs and cell markers with key miRs in the adult human SN vs. right atrial tissue (RA) were determined. PRINCIPAL RESULTS: 68 and 60 TFs significantly more or less expressed in the SN vs. RA respectively. Among those more expressed were ISL1 and TBX3 (involved in embryonic development of the SN) and 'novel' RUNX1-2, CEBPA, GLI1-2 and SOX2. These TFs were predicted to regulate HCN4 expression in the SN. Markers for different cells: fibroblasts (COL1A1), fat (FABP4), macrophages (CSF1R and CD209), natural killer (GZMA) and mast (TPSAB1) were significantly more expressed in the SN vs. RA. Interestingly, RUNX1-3, CEBPA and GLI1 also regulate expression of these cells. MiR-486-3p inhibits HCN4 and markers involved in immune response. MAJOR CONCLUSIONS: In conclusion, RUNX1-2, CSF1R, TPSAB1, COL1A1 and HCN4 are highly expressed in the SN but not miR-486-3p. Their complex interactions can be used to treat SN dysfunction such as bradycardia. Interestingly, another research group recently reported miR-486-3p is upregulated in blood samples from severe COVID-19 patients who suffer from bradycardia.

Repolarizing potassium currents in working myocardium of Japanese quail: a novel translational model for cardiac electrophysiology.

Birds developed endothermy and four-chambered high-performance heart independently from mammals. Though avian embryos are extensively studied and widely used as various models for heart research, little is known about cardiac physiology of adult birds. Meanwhile, cardiac electrophysiology is in search for easily accessible and relevant model objects which resemble human myocardium in the pattern of repolarizing currents (IKr, IKs, IKur and Ito). This study focuses on the configuration of electrical activity and electrophysiological phenotype of working myocardium in adult Japanese quails (Coturnix japonica). The resting membrane potential and action potential (AP) waveform in quail atrial myocardium were similar to that in working myocardium of rodents. Using whole-cell patch clamp and sharp glass microelectrodes, we demonstrated that the repolarization of quail atrial and ventricular myocardium is determined by voltage-dependent potassium currents IKr, IKs and Ito - the latter was previously considered as an exclusive evolutionary feature of mammals. The specific blockers of these currents, dofetilide (3 µmol l-1), HMR 1556 (30 µmol l-1) and 4-aminopyridine (3 mmol l-1), prolonged AP in both ventricular and atrial myocardial preparations. The expression of the corresponding channels responsible for these currents in quail myocardium was investigated with quantitative RT-PCR and western blotting. In conclusion, the described pattern of repolarizing ionic currents and channels in quail myocardium makes this species a novel and suitable experimental model for translational cardiac research and reveals new information related to the evolution of cardiac electrophysiology in vertebrates.

Attenuation of inward rectifier potassium current contributes to the α1-adrenergic receptor-induced proarrhythmicity in the caval vein myocardium.

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.

Electrophysiological differences in cholinergic signaling between the hearts of summer and winter frogs (Rana temporaria).

The striking seasonal difference in sensitivity of frog cardiac muscle to acetylcholine or stimulation of parasympathetic nervous fibers has been noted almost a century ago, although its electrophysiological basis has never been revealed. The present study compares the effects of the muscarinic agonist carbamylcholine chloride (CCh 10-8-10-5 M) on electrical activity in isolated atrial and ventricular preparations from dormant frogs (Rana temporaria) caught in January (winter-acclimatized, WA) and from active frogs caught in July (summer-acclimatized, SA). Seasonal differences in the density of potassium acetylcholine-dependent current (IKACh) were also studied in atrial and ventricular myocytes from both summer and winter groups. In atrial myocardium, CCh produced concentration-dependent shortening of action potentials (APs). CCh concentration producing a 50% reduction of AP duration was lower in WA (1.03 × 10-7 M) than SA atria (2.7 × 10-7 M). 10-6 M CCh induced drastic reduction of AP amplitude rendering the tissue unexcitable in both WA or SA atrial preparations. Ventricular preparations showed greater seasonal difference in CCh sensitivity. While 10-6 M induced inexcitability in 50% of tested WA preparations, in SA preparations even 10-5 M CCh was without effect. This striking difference between cholinergic effects in SA and WA frog ventricle could be explained by seasonal changes in the IKACh density. The density of IKACh induced by 10-5 M CCh and measured at 0 mV was 14.4 ± 3.45 pA/pF in WA, but only 1.5 ± 0.4 pA/pF in SA atrial cells. In ventricular cells, the respective values were 2.61 ± 0.56 and 0.71 ± 0.09 pA/pF. Thus, hibernating winter frog has a much greater electrophysiological cholinergic response than active summer frog due to up-regulation of IKACh.

Negative inotropic effects of diadenosine tetraphosphate are mediated by protein kinase C and phosphodiesterases stimulation in the rat heart.

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 Prolongs Action Potentials in Guinea Pig Atrial Myocardium via Inhibition of Rapid Delayed Rectifier.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Niferidil prolongs APs in guinea pig atrial myocardium via inhibition of I Kr.

Effects of new antiarrhythmic agent SS-68 on excitation conduction, electrical activity in Purkinje fibers and pulmonary veins: Assessment of safety and side effects risk.

The compound SS-68 has been selected among numerous new derivatives of indole and demonstrated antiarrhythmic effects in animal models. The present study concerns several aspects of SS-68 safety and efficacy as a potential antiarrhythmic drug. The first estimation of atrioventricular conduction in mammalian heart under SS-68 has been carried out; effects of SS-68 in Purkinje fibers and myocardium of pulmonary veins have been investigated. The drug weakly affects cardiac atrioventricular conduction: only high concentrations of SS-68 (≥10 µmol/L) significantly decrease this parameter. Also, the drug weakly affects Purkinje fibers automaticity, but effectively alters action potential waveform in Purkinje fibers in a concentration-dependent manner. SS-68 (0.1-100 µmol/L) failed to induce any early or delayed afterdepolarizations in Purkinje fibers both in basal conditions and under provocation of proarrhythmic activity by norepinephrine (NE). Moreover, 10 µmol/L SS-68 suppressed NE-induced extra-beats and rapid firing in Purkinje fibers. In pulmonary veins only high concentrations of SS-68 significantly increased action potential duration, while lower concentrations (0.1-1 µmol/L) were ineffective. Also, 0.1-100 µmol/L SS-68 was unable to elicit arrhythmogenic alternations of action potential waveform in pulmonary veins. In conclusion, SS-68 has no proarrhythmic effects, such as afterdepolarizations or abnormal automaticity in used experimental models.