The Dependence of the Electrophysiological Effects of Class III Antiarrhythmic Drug Refralon on the Frequency of Myocardium Activation.

We studied the frequency dependence of the effects of the novel Russian class III antiarrhythmic drug refralon on the duration of action potentials (AP) in rabbit ventricular myocardium. The absence of an inverse frequency dependence of AP prolongation was demonstrated: the effects of refralon at stimulation frequency of 1 Hz were stronger than at 0.1 Hz. The patch-clamp experiments with recording of rapid delayed rectifier potassium current IKr in a heterologous expression system showed that the blocking effect of refralon developed significantly faster at 2 Hz depolarization frequency than at 0.2 Hz. This feature of refralon distinguishes it among the majority of other class III drugs (sotalol, dofetilide, E-4031) and explains the relatively high safety of this drug together with its high efficacy.

MicroRNA miR-133a-3p Facilitates Adrenergic Proarrhythmic Ectopy in Rat Pulmonary Vein Myocardium by Increasing cAMP Content.

Cardiac-specific microRNA miR-133a-3p modulates adrenergic signaling. Adrenergic receptors and their intracellular pathways are the key players in proarrhythmic ectopy derived from the myocardial sleeves of the pulmonary veins. We studied the effect of miR-133a-3p on ectopy induced by norepinephrine in myocardial tissue of rat pulmonary veins. Using microelectrode technique, we revealed facilitation of proarrhythmic pattern of spontaneous bursts of action potentials induced by norepinephrine in tissue preparations of the pulmonary veins isolated from rats in 24 h after injection of a transfection mixture containing miR-133a-3p (1 mg/kg) in vivo. According to ELISA data, the cAMP level in the pulmonary vein myocardium of rats receiving miR-133a-3p was 2-fold higher than in control animals. Bioinformatic analysis showed that mRNA of protein phosphatases and some phosphodiesterases are most probable targets of miR-133a-3p. The proarrhythmic effect of miR-133a-3p can be related to inhibition of the expression of phosphodiesterases accompanied by cAMP accumulation and increased intracellular ß-adrenergic signaling.

Regulation of NaV1.5 Sodium Channels by Small G-Proteins of the Rho Family in a Heterologous Expression System.

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.

Gadolinium as an Inhibitor of Ionic Currents in Isolated Rat Ventricular Cardiomyocytes.

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.

Diadenosine Polyphosphates Suppress the Effects of Sympathetic Nerve Stimulation in Rabbit Heart Pacemaker.

The modulatory influence of diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A) on the effect of intramural autonomic nerve stimulation in isolated rabbit sinoatrial node were examined. Electrical activity of the sinoatrial node was recorded intracellularly. Against the background of blockade of adrenergic effects with propranolol (3×10-6 M) or in preparations isolated 2 h after injection of reserpine (2 mg/kg), nerve stimulation induced short-term membrane hyperpolarization and diminished the sinus node firing rate. These phenomena were not affected by Ap4A or Ap5A (10-5 M). Under the action of atropine (3×10-6 M) that completely eliminated the cholinergic influences, nerve stimulation enhanced the sinus node firing rate by 17.30±3.45% from the initial rate. Both Ap4A and Ap5A moderated the stimulation-induced elevation of firing rate to 9.9±2.8 and 10.5±2.9%, respectively. The data suggest that diadenosine polyphosphates significantly modulate the sympathetic influences on the heart rhythm, but have no effect on the parasympathetic control over activity of sinoatrial node.

Effect of Purine Co-Transmitters on Automatic Activity Caused by Norepinephrine in Myocardial Sleeves of Pulmonary Veins.

We studied the effect of extracellular purine nucleotides (NAD+ and ATP) on spontaneous arrhythmogenic activity caused by norepinephrine in myocardial sleeves of pulmonary veins. In pulmonary veins, NAD+ and ATP reduced the frequency of action potentials and their duration at regular type of spontaneous activity caused by norepinephrine. NAD+ and ATP lengthened the intervals between spike bursts at periodic (burst) type of spontaneous activity. In addition, ATP shortened the duration of spike bursts and the number of action potentials in the "bursts" caused by norepinephrine in the pulmonary veins. It was hypothesized that NAD+ and ATP attenuate the effects of sympathetic stimulation and when released together with norepinephrine from sympathetic endings in vivo, probably, reduce arrhythmogenic activity in myocardial sleeves of pulmonary veins.

Effects of Nicotinamide Adenine Dinucleotide (NAD(+)) and Diadenosine Tetraphosphate (Ap4A) on Electrical Activity of Working and Pacemaker Atrial Myocardium in Guinea Pigs.

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.

Different Myocardial Sensitivity in Newborn and Mature Rats to Selective Stimulation of M3 Cholinoreceptors.

The effects of selective activation of subtype 3 muscarinic (M3) receptors on electrical activity of isolated preparations of the atrial and ventricular myocardium of the newborn and 4-month-old rats were examined. Application of muscarinic receptor agonist pilocarpine (10(-5) M) in preparations with M2 cholinoreceptors blocked by selective antagonist methoctramine (10(-7) M) decreased the duration of action potentials in the atrial and ventricular myocardium. Selective blocker of M3 cholinoreceptors 4-DAMP (10(-8) M) prevented this effect. While stimulation of ventricular M3 cholinoreceptors with pilocarpine was significantly stronger in newborn pups than in mature rats, similar stimulation of atrial receptors revealed no significant difference in both groups.

Changes in electrical activity of working myocardium under condition of if current inhibition.

The study examined the effect of ZD7288, a blocker of hyperpolarization-activated "funny" current If, on electrical activity in working atrial and ventricular myocardium in rats. In concentrations range from 3×10(-6) to 3×10(-5) M, the agent significantly increased the duration of action potentials at 50 and 90% repolarization levels in both atrial and ventricular myocardium at the fixed stimulation rate of 5 Hz. The blocker affected neither resting potential nor the upstroke velocity of action potential. In patch-clamp experiments, ZD7288 selectively inhibited If current, but produced no effect on delayed rectifier potassium currents that determine the rate of repolarization. The described effects of ZD7288 are not related to its non-specific effects on the ionic currents except If.

[Effect of adaptation to hypoxia on expression of NO synthase isoforms in rat myocardium].

Previously we have shown that adaptation to hypoxia (AH) is cardio- and vasoprotective in myocardial ischemic and reperfusion injury and this protection is associated with restriction of nitrosative stress. The present study was focused on further elucidation of NO-dependent mechanisms of AH by identifying specific NO synthases (NOS) that could play the major role in AH protection. AH was performed in a normobaric hypoxic chamber by breathing hypoxic gas mixture (9.5-10% O2) for 5-10 min with intervening 4 min normoxia (5-8 cycles daily for 21 days). Expression of neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) protein was measured in the left ventricular myocardium using Western blot analysis with respective antibodies. AH educed iNOS protein expression by 71% (p < 0.05) whereas eNOS protein expression tended to be reduced by 41% compared to control (p < 0.05). nNOS protein expression remained unchanged after AH. Selective iNOS inhibition can mimic the AH-induced protection. Therefore protective effects of AH could be at least partially due to restriction of iNOS and, probably, eNOS expression.

Effects of interleukin-18 on bioelectric activity of rat atrial cardiomyocytes under normal conditions and during gradual stretching of the tissue.

Effects of IL-18 (50 ng/ml) on bioelectric activity of rat atrial cardiomyocytes under normal conditions and after gradual stretching of the tissue was studied using microelectrode technique. It was shown that in 85% experiments, IL-18 increased the duration of action potential at the level of 25, 50, and 90% repolarization without changing the magnitude of the resting potential, amplitude and repetition rate of action potentials, and cardiomyocyte contraction force. In addition, IL-18 abolished mechanically induced changes in the shape of action potentials during stretching.

Effect of selective stimulation of muscarinic M3 cholinoceptors on electrical and contractile activity of rat ventricular myocardium.

We studied the effect of selective activation of muscarinic M3receptors on electrical activity in the isolated preparation of rat ventricular myocardium as well as contractile activity of the left ventricle of Langendorff-perfused isolated heart. Application of muscarinic agonist pilocarpine (10(-5)M) against the background of selective blockade of subtype 2 muscarinic receptors with methoctramine (10(-7)M) markedly shortened the duration of action potentials in the isolated ventricular myocardium and reduced the amplitude and maximum rates of left-ventricular pressure rise and decay in the isolated heart paced at a fixed rate. All these effects were significantly suppressed by selective M3receptor blocker 4-DAMP (10(-8)M), which attested to the involvement of M3muscarinic receptors.

[Vasoprotective effect of adaptation to hypoxia in myocardial ischemia and reperfusion injury].

Adaptation to hypoxia is known to be cardioprotective in ischemic and reperfusion (IR) injury of the myocardium. This study was focused on investigating a possibility for prevention of endothelial dysfunction in IR injury of the rat heart using adaptation to intermittent hypoxia, which was performed in a cyclic mode (5-10 min of hypoxia interspersed with 4 min of normoxia, 5-8 cycles daily) for 21 days. Endothelial function of coronary blood vessels was evaluated after the in vitro IR of isolated heart (15 min of ischemia and 10 min of reperfusion) by the increment of coronary flow rate in response to acetylcholine. Endothelium-dependent relaxation of isolated rat aorta was evaluated after the IR myocardial injury in situ (30 min of ischemia and 60 min of reperfusion) by a relaxation response of noradrenaline-precontracted vessel rings to acetylcholine. The following major results were obtained in this study: 1) IR myocardial injury induced endothelial dysfunction of coronary blood vessels and the aorta, a non-coronary blood vessel, remote from the IR injury area; and 2) adaptation to hypoxia prevented the endothelial dysfunction of both coronary and non-coronary blood vessels associated with the IR injury. Therefore, adaptation to hypoxia is not only cardioprotective but also vasoprotective in myocardial IR injury.

Inotropic effects of gaseous transmitters in isolated rat heart preparation.

We studied the effects of carbon monoxide and sodium hydrosulfide, hydrogen sulfide donor, on contractile activity of the left ventricle in Langendorf-perfused isolated rat heart. Carbon monoxide 5×10(-5) M significantly accelerated sinus rhythm and left-ventricular pressure wave growth and decay. To the contrary, negative inotropic and chronotropic effects were observed at higher concentrations of carbon monoxide (10(-4), 3×10(-4) M). Sodium hydrosulfide (10(-4)-4×10(-4) M) decreased all the parameters of left-ventricular contractive activity and reduced contraction rate. Carbon monoxide and hydrogen sulfide, which together with nitrogen oxide are qualified as a new class of gaseous signal compounds, may substantially modulate pumping function of the heart.

Effect of nitric oxide on mechanoelectric feedback in rat right atrium.

In situ microelectrode examination of rat right atrium showed that in physiologically prestretched tissue, NO donor SNAP modifies the repolarization phase of cardiomyocyte AP in a "hump-like" way provoking the development of arrhythmia. Gadolinium both prevents and eliminates this effect attesting to involvement of stretch-activated channels in the development of NO-induced abnormalities. Elevation of SNAP concentration or further stretch of the tissue (presumably, it increases NO concentration) eliminated the hump depolarization induced by moderate SNAP stimulation. Thus, low NO opens the stretch-activated channels while high NO inactivates them.

[Investigation of pacemaker shift in the rabbit sinoatrial node using the optical mapping technique].

Changes of the activation sequence in the rabbit sinoatrial node under the influence of low temperature and I(f) selective blocker ivabradine have been studied using the optical mapping technique. Both factors caused a shift of the pacemaker within the sinoatrial node region. These results are compared with the data obtained recently in the investigation of pacemaker shift under the influence of cholinergic and adrenergic factors. Possible mechanisms of the pacemaker shift are discussed. The suppression of electric activity in the central part of the sinoatrial node during the action of acetylcholine, which is called cholinergic inexcitability, may be considered as one of the mechanisms of the pacemaker shift. It is shown that the main cause of cholinergic inexcitability is the activation of potassium acetylcholine-dependent current I(KACh).

The effect of hydrogen sulfide on electrical activity of rat atrial myocardium.

Changes in the configuration of action potentials and in the frequency of pacemaker discharges in a preparation of isolated rat right atrium under the effect of sodium hydrosulfide degrading in water solution with hydrogen sulfide release were studied by intracellular recording of action potentials in the myocardium. Sodium hydrosulfide in concentrations of 100-500 microM markedly reduced the duration of action potentials at the level of 50 and 90% repolarization and decelerated the sinus rhythm. Moreover, sodium hydrosulfide shortened action potentials in the preparations working in the forced rhythm. Glybenclamide (potassium ATP-dependent channel blocker; 10 microM) reduced the effect sodium hydrosulfide (200 microM) by more than 60%, which suggested the involvement of potassium ATP-dependent current in the realization of the effect of hydrogen sulfide on configuration of action potentials. Hence, hydrogen sulfide, recently described as a signal compound, modulates many electrophysiological parameters of the myocardium.

[M3-cholinoreceptors in mammalian heart].

Experimental facts, proving the presence of M3-cholinoreceptros in parasympathetic regulation of heart, are reviewed. Results of molecular biological and biochemical studies, showing existence of M3-cholinoreceptors in myocardium, are discussed. Physiological data, that demonstrates changes of action potential configuration due to M3-receptors activation, specific potassium current, linked with M3-receptors stimulation, and other evidence of functional importance of M3-receptros in myocardium, is analyzed. Studies dealing with cardioprotective role of M3-receptors are reviewed with special attention. The review widens our knowledge about molecular mechanisms of parasympathetic heart regulation.

[Mechanisms of functioning and regulation of mammalian sinoatrial node].

Actual data concerning mechanisms of automaticity in sinoatrial node, which acts as a primary pacemaker in mammalian heart, is reviewed. Studies dealing with ionic currents, maintaining automatic generation of excitation in the sinoatrial cells, and possible role of intracellular calcium turnover are discussed. Special attention is given to the differences between the central and peripheral parts of sinoatrial node, phenomenon of intranodal pacemaker shift resulting from that differences and possible role of pacemaker shift in the modulation of the sinus rhythm. Mechanisms of sinus rhythm regulation under the action of acetylcholine and noradrenalin are also discussed in detail.

[Changes in the activation sequence in the rabbit sinoatrial node induced by adrenergic stimulation].

We used high resolution optical mapping for the study of changes of activation sequence of a rabbit sinoatrial node induced by adrenergic stimulation during natural and paced rhythm. Activation of adrenoreceptors with isoproterenol (10, 100 nanoM, 1 microM) as well as stimulation of intramural postganglionic sympathetic nerves caused pacemaker migration within sinoatrial node and increase of the rate of generation of excitation. Pacemaker migration in cases of pronounced acceleration of rhythm could proceed in two stages. Termination of adrenergic influences has been followed by restoration of initial chronotopography of excitation of sinoatrial node.