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 atrium 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 (IC50 = 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 (IC50 = 4.7 µM) of the sodium current (INa), slowing the upstroke of the action potential in isolated cells, slowing conduction velocity across the atrium 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.

Novel Gain-of-Function Mutation in the Kv11.1 Channel Found in the Patient with Brugada Syndrome and Mild QTc Shortening.

Brugada syndrome (BrS) is an inherited disease characterized by right precordial ST-segment elevation in the right precordial leads on electrocardiograms (ECG), and high risk of life-threatening ventricular arrhythmia and sudden cardiac death (SCD). Mutations in the responsible genes have not been fully characterized in the BrS patients, except for the SCN5A gene. We identified a new genetic variant, c.1189C>T (p.R397C), in the KCNH2 gene in the asymptomatic male proband diagnosed with BrS and mild QTc shortening. We hypothesize that this variant could alter IKr-current and may be causative for the rare non-SCN5A-related form of BrS. To assess its pathogenicity, we performed patch-clamp analysis on IKr reconstituted with this KCNH2 mutation in the Chinese hamster ovary cells and compared the phenotype with the wild type. It appeared that the R397C mutation does not affect the IKr density, but facilitates activation, hampers inactivation of the hERG channels, and increases magnitude of the window current suggesting that the p.R397C is a gain-of-function mutation. In silico modeling demonstrated that this missense mutation potentially leads to the shortening of action potential in the heart.

A mutation in the cardiac KV7.1 channel possibly disrupts interaction with Yotiao protein.

Here, we characterized the p.Arg583His (R583H) Kv7.1 mutation, identified in two unrelated families suffered from LQT syndrome. This mutation is located in the HС-HD linker of the cytoplasmic portion of the Kv7.1 channel. This linker, together with HD helix are responsible for binding the A-kinase anchoring protein 9 (AKAP9), Yotiao. We studied the electrophysiological characteristics of the mutated channel expressed in CHO-K1 along with KCNE1 subunit and Yotiao protein, using the whole-cell patch-clamp technique. We found that R583H mutation, even at the heterozygous state, impedes IKs activation. Molecular modeling showed that HС and HD helixes of the C-terminal part of Kv7.1 channel are swapped along the C-terminus length of the channel and that R583 position is exposed to the outer surface of HC-HD tandem coiled-coil. Interestingly, the adenylate cyclase activator, forskolin had a smaller effect on the mutant channel comparing with the WT protein, suggesting that R583H mutation may disrupt the interaction of the channel with the adaptor protein Yotiao and, therefore, may impair phosphorylation of the KCNQ1 channel.

Characterization of hERG K+ channel inhibition by the new class III antiarrhythmic drug cavutilide.

Cavutilide (niferidil, refralon) is a new class III antiarrhythmic drug which effectively terminates persistent atrial fibrillation (AF; 84.6% of patients, mean AF duration 3 months) and demonstrates low risk of torsade de pointes (1.7%). ERG channels of rapid delayed rectifier current(IKr) are the primary target of cavutilide, but the particular reasons of higher effectiveness and lower proarrhythmic risk in comparison with other class III IKr blockers are unclear. The inhibition of hERG channels expressed in CHO-K1 cells by cavutilide was studied using whole-cell patch-clamp. The present study demonstrates high sensitivity of IhERG expressed in CHO-K1 cells to cavutilide (IC50 = 12.8 nM). Similarly to methanesulfonanilide class III agents, but unlike amiodarone and related drugs, cavutilide does not bind to hERG channels in their resting state. However, in contrast to dofetilide, cavutilide binds not only to opened, but also to inactivated channels. Moreover, at positive constantly set membrane potential (+ 60 mV) inhibition of IhERG by 100 nM cavutilide develops faster than at 0 mV and, especially, - 30 mV (τ of inhibition was 78.8, 103, and 153 ms, respectively). Thereby, cavutilide produces IhERG inhibition only when the cell is depolarized. During the same period of time, cavutilide produces greater block of IhERG when the cell is depolarized with 2 Hz frequency, if compared to 0.2 Hz. We suggest that, during the limited time after injection, cavutilide produces stronger inhibition of IKr in fibrillating atrium than in non-fibrillating ventricle. This leads to beneficial combination of antiarrhythmic effectiveness and low proarrhythmicity of cavutilide.

Global Air Pollutant Phenanthrene and Arrhythmic Outcomes in a Mouse Model.

BACKGROUND: The three-ringed polycyclic aromatic hydrocarbon (PAH) phenanthrene (Phe) has been implicated in the cardiotoxicity of petroleum-based pollution in aquatic systems, where it disrupts the contractile and electrical function of the fish heart. Phe is also found adsorbed to particulate matter and in the gas phase of air pollution, but to date, no studies have investigated the impact of Phe on mammalian cardiac function. OBJECTIVES: Our objectives were to determine the arrhythmogenic potential of acute Phe exposure on mammalian cardiac function and define the underlying mechanisms to provide insight into the toxicity risk to humans. METHODS: Ex vivo Langendorff-perfused mouse hearts were used to test the arrhythmogenic potential of Phe on myocardial function, and voltage- and current-clamp recordings were used to define underlying cellular mechanisms in isolated cardiomyocytes. RESULTS: Mouse hearts exposed to ∼8µM Phe for 15-min exhibited a significantly slower heart rate (p=0.0006, N=10 hearts), a prolonged PR interval (p=0.036, N=8 hearts), and a slower conduction velocity (p=0.0143, N=7 hearts). Whole-cell recordings from isolated cardiomyocytes revealed action potential (AP) duration prolongation (at 80% repolarization; p=0.0408, n=9 cells) and inhibition of key murine repolarizing currents-transient outward potassium current (Ito) and ultrarapid potassium current (IKur)-following Phe exposure. A significant reduction in AP upstroke velocity (p=0.0445, n=9 cells) and inhibition of the fast sodium current (INa; p=0.001, n=8 cells) and calcium current (ICa; p=0.0001) were also observed, explaining the slowed conduction velocity in intact hearts. Finally, acute exposure to ∼8µM Phe significantly increased susceptibility to arrhythmias (p=0.0455, N=9 hearts). DISCUSSION: To the best of our knowledge, this is the first evidence of direct inhibitory effects of Phe on mammalian cardiac electrical activity at both the whole-heart and cell levels. This electrical dysfunction manifested as an increase in arrhythmia susceptibility due to impairment of both conduction and repolarization. Similar effects in humans could have serious health consequences, warranting greater regulatory attention and toxicological investigation into this ubiquitous PAH pollutant generated from fossil-fuel combustion. https://doi.org/10.1289/EHP12775.

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)

Disruption of a Conservative Motif in the C-Terminal Loop of the KCNQ1 Channel Causes LQT Syndrome.

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.

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.

The role of activation of two different sGC binding sites by NO-dependent and NO-independent mechanisms in the regulation of SACs in rat ventricular cardiomyocytes.

The mechanoelectrical feedback (MEF) mechanism in the heart that plays a significant role in the occurrence of arrhythmias, involves cation flux through cation nonselective stretch-activated channels (SACs). It is well known that nitric oxide (NO) can act as a regulator of MEF. Here we addressed the possibility of SAC's regulation along NO-dependent and NO-independent pathways, as well as the possibility of S-nitrosylation of SACs. In freshly isolated rat ventricular cardiomyocytes, using the patch-clamp method in whole-cell configuration, inward nonselective stretch-activated cation current ISAC was recorded through SACs, which occurs during dosed cell stretching. NO donor SNAP, α1-subunit of sGC activator BAY41-2272, sGC blocker ODQ, PKG blocker KT5823, PKG activator 8Br-cGMP, and S-nitrosylation blocker ascorbic acid, were employed. We concluded that the physiological concentration of NO in the cell is a necessary condition for the functioning of SACs. An increase in NO due to SNAP in an unstretched cell causes the appearance of a Gd3+ -sensitive nonselective cation current, an analog of ISAC , while in a stretched cell it eliminates ISAC . The NO-independent pathway of sGC activation of α subunit, triggered by BAY41-2272, is also important for the regulation of SACs. Since S-nitrosylation inhibitor completely abolishes ISAC , this mechanism occurs. The application of BAY41-2272 cannot induce ISAC in a nonstretched cell; however, the addition of SNAP on its background activates SACs, rather due to S-nitrosylation. ODQ eliminates ISAC , but SNAP added on the background of stretch increases ISAC in addition to ODQ. This may be a result of the lack of NO as a result of inhibition of NOS by metabolically modified ODQ. KT5823 reduces PKG activity and reduces SACs phosphorylation, leading to an increase in ISAC . 8Br-cGMP reduces ISAC by activating PKG and its phosphorylation. These results demonstrate a significant contribution of S-nitrosylation to the regulation of SACs.

Variable Clinical Appearance of the Kir2.1 Rare Variants in Russian Patients with Long QT Syndrome.

BACKGROUND: The KCNJ2 gene encodes inward rectifier Kir2.1 channels, maintaining resting potential and cell excitability. Presumably, clinical phenotypes of mutation carriers correlate with ion permeability defects. Loss-of-function mutations lead to QTc prolongation with variable dysmorphic features, whereas gain-of-function mutations cause short QT syndrome and/or atrial fibrillation. METHODS: We screened 210 probands with Long QT syndrome for mutations in the KCNJ2 gene. The electrophysiological study was performed for the p.Val93Ile variant in the transfected CHO-K1 cells. RESULTS: We found three rare genetic variants, p.Arg67Trp, p.Val93Ile, and p.R218Q, in three unrelated LQTS probands. Probands with p.Arg67Trp and p.R218Q had a phenotype typical for Andersen-Tawil (ATS), and the p.Val93Ile carrier had lone QTc prolongation. Variant p.Val93Ile was initially described as a gain-of-function pathogenic mutation causing familial atrial fibrillation. We validated electrophysiological features of this variant in CHO-K1 cells, but no family members of these patients had atrial fibrillation. Using ACMG (2015) criteria, we re-assessed this variant as a variant of unknown significance (class III). CONCLUSIONS: LQT7 is a rare form of LQTS in Russia, and accounts for 1% of the LQTS cohort. Variant p.Val93Ile leads to a gain-of-function effect in the different cell lines, but its clinical appearance is not so consistent. The clinical significance of this variant might be overestimated.

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.

Migraine-Associated Mutation in the Na,K-ATPase Leads to Disturbances in Cardiac Metabolism and Reduced Cardiac Function.

Background Mutations in ATP1A2 gene encoding the Na,K-ATPase α2 isoform are associated with familial hemiplegic migraine type 2. Migraine with aura is a known risk factor for heart disease. The Na,K-ATPase is important for cardiac function, but its role for heart disease remains unknown. We hypothesized that ATP1A2 is a susceptibility gene for heart disease and aimed to assess the underlying disease mechanism. Methods and Results Mice heterozygous for the familial hemiplegic migraine type 2-associated G301R mutation in the Atp1a2 gene (α2+/G301R mice) and matching wild-type controls were compared. Reduced expression of the Na,K-ATPase α2 isoform and increased expression of the α1 isoform were observed in hearts from α2+/G301R mice (Western blot). Left ventricular dilation and reduced ejection fraction were shown in hearts from 8-month-old α2+/G301R mice (cardiac magnetic resonance imaging), and this was associated with reduced nocturnal blood pressure (radiotelemetry). Cardiac function and blood pressure of 3-month-old α2+/G301R mice were similar to wild-type mice. Amplified Na,K-ATPase-dependent Src kinase/Ras/Erk1/2 (p44/42 mitogen-activated protein kinase) signaling was observed in hearts from 8-month-old α2+/G301R mice, and this was associated with mitochondrial uncoupling (respirometry), increased oxidative stress (malondialdehyde measurements), and a heart failure-associated metabolic shift (hyperpolarized magnetic resonance). Mitochondrial membrane potential (5,5´,6,6´-tetrachloro-1,1´,3,3´-tetraethylbenzimidazolocarbocyanine iodide dye assay) and mitochondrial ultrastructure (transmission electron microscopy) were similar between the groups. Proteomics of heart tissue further suggested amplified Src/Ras/Erk1/2 signaling and increased oxidative stress and provided the molecular basis for systolic dysfunction in 8-month-old α2+/G301R mice. Conclusions Our findings suggest that ATP1A2 mutation leads to disturbed cardiac metabolism and reduced cardiac function mediated via Na,K-ATPase-dependent reactive oxygen species signaling through the Src/Ras/Erk1/2 pathway.

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.

The role of M3 receptors in regulation of electrical activity deteriorates in the rat heart during ageing.

Ageing is a complex process which affects all systems of the organism and therefore changes the environment where the heart is working. In this study we demonstrate the ageing-related changes in the mechanisms of parasympathetic regulation of mammalian heart. Electrophysiological effects produced by selective activation of M3-cholinoreceptors were compared in isolated cardiac preparations from young adult (4 months), adult (1 year) and ageing (2 years) rats using sharp glass microelectrode technique. M3-receptors were activated with muscarinic agonist pilocarpine (10-5M) in the presence of selective M2 antagonist AQ-RA741 (10-7M). In atrial and ventricular myocardium from young rats M3 stimulation induced shortening of action potentials(APs), while no significant effect was observed in both elder groups. The main mechanism of M3-induced AP shortening is inhibition of L-type Ca2+ current, estimated using whole-cell patch-clamp. It was negligible in atrial myocytes from ageing animals in comparison with young rats. The loss of sensitivity to stimulation of M3-receptors is due to decrease in M3 gene expression, shown by RT-PCR both in atrial and ventricular samples from ageing rats. Thus, in ageing rat heart M3-receptors are down-regulated and not involved in regulation of electrical activity.

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.