Káliumáramok vizsgálata izolált szívizom-preparátumokon és klónozott csatornákon = Study of potassium currents in isolated cardiac preparation and cloned channels

A jelen kutatás célja a kardiális betegségekhez (ritmuszavarok, szívelégtelenség és hirtelen szívhalál) kialakulásban szereplő ioncsatornák rendellenes működésének (elektromos remodeling) vizsgálata volt. A vizsgálat során feltérképeztük a kamrai repolarizációt meghatározó főbb ionáramok molekuláris összetevőit, hogy megállapítsuk melyik alegységek játszanak szerepet az adott ionáram tulajdonságaiban (kinetika, gyógyszerérzékenység, stb). Ezenfelül új vizsgálati eljárást dolgoztunk, amely magalapozza a primer szívizomsejt kultúrában történő csendesítés technikáját. A következő kutatási eredményeket értük el: a) Új herpeszvírus alapú génbeviteli eljárás dolgoztunk ki primer kutya szívizomsejt kultúrában. b) Megállapítottuk, hogy a késői egyenirányító káliumáram gyengülése játszik szerepe a repolarizáció megnyújtásában alloxán diabétesz nyúlmodellben. c) Megállapítottuk, hogy akciós potenciál fordított frekvenciafüggő megnyúlása a kamrai szívizomzat intrinszik tulajdonsága, és ebben nem játszanak közvetlen szerepet a meghatározó transzmembrán káliumionáramok frekvenciafüggő tulajdonságai. D). Megállapítottuk, hogy a befelé egyenirányító káliumáramnak kisebb a repolarizációban játszott szerepe és a proaritmia készsége kutyában mind humán kamrai szívizomzatban. | The aim of the present research project was to investigate the properties of the transmembrane ionic currents related to cardiac diseases as arrhythmias, heart failure or sudden cardiac death. During our investigation we have revealed the of the molecular structure of the main currents involved in cardiac repolarization to determine which subunits play the most important role in the main properties of the corresponding current (kinetics, drug sensitivity, etc). Moreover, we have developed a new method that can be apply in the gene silencing technique in a primer myocytes culture. We have reached the following results: a) We have performed a new herpesvirus based gene transfer technique in a primer canine ventricular myocytes culture. b) We have reported that the downregulation of the slow delayed rectifier potassium current plays an important role in the repolarization lengthening in an alloxan induced diabetes mellitus model. c) We have demonstrated that the reverse frequency dependence is an intrinsic property of the ventricular myocardium, and is independent from the frequency dependent properties of the main repolarizing potassium currents. d) We have shown that the inward rectifier potassium current have weaker role to cardiac repolarization and thereby less proarrhythmia inducing effect in dog compared to human heart

The Properties of the Transient Outward, Inward Rectifier and Acetylcholine-Sensitive Potassium Currents in Atrial Myocytes from Dogs in Sinus Rhythm and Experimentally Induced Atrial Fibrillation Dog Models

Aims: Atrial fibrillation (AF) is the most common chronic/recurrent arrhythmia, which significantly impairs quality of life and increases cardiovascular morbidity and mortality. Therefore, the aim of the present study was to investigate the properties of three repolarizing potassium currents which were shown to contribute to AF-induced electrical remodeling, i.e., the transient outward (Ito), inward rectifier (IK1) and acetylcholine-sensitive (IK,ACh) potassium currents in isolated atrial myocytes obtained from dogs either with sinus rhythm (SR) or following chronic atrial tachypacing (400/min)-induced AF. Methods: Atrial remodeling and AF were induced by chronic (4–6 weeks of) right atrial tachypacing (400/min) in dogs. Transmembrane ionic currents were measured by applying the whole-cell patch-clamp technique at 37 °C. Results: The Ito current was slightly downregulated in AF cells when compared with that recorded in SR cells. This downregulation was also associated with slowed inactivation kinetics. The IK1 current was found to be larger in AF cells; however, this upregulation was not statistically significant in the voltage range corresponding with atrial action potential (−80 mV to 0 mV). IK,ACh was activated by the cholinergic agonist carbachol (CCh; 2 µM). In SR, CCh activated a large current either in inward or outward directions. The selective IK,ACh inhibitor tertiapin (10 nM) blocked the outward CCh-induced current by 61%. In atrial cardiomyocytes isolated from dogs with AF, the presence of a constitutively active IK,ACh was observed, blocked by 59% with 10 nM tertiapin. However, in “AF atrial myocytes”, CCh activated an additional, significant ligand-dependent and tertiapin-sensitive IK,ACh current. Conclusions: In our dog AF model, Ito unlike in humans was downregulated only in a slight manner. Due to its slow inactivation kinetics, it seems that Ito may play a more significant role in atrial repolarization than in ventricular working muscle myocytes. The presence of the constitutively active IK,ACh in atrial myocytes from AF dogs shows that electrical remodeling truly developed in this model. The IK,ACh current (both ligand-dependent and constitutively active) seems to play a significant role in canine atrial electrical remodeling and may be a promising atrial selective drug target for suppressing AF

Electrical Restitution and Its Modifications by Antiarrhythmic Drugs in Undiseased Human Ventricular Muscle

Introduction: Re-entry is a basic mechanism of ventricular fibrillation, which can be elicited by extrasystolic activity, but the timing of an extrasystole can be critical. The action potential duration (APD) of an extrasystole depends on the proximity of the preceding beat, and the relation between its timing and its APD is called electrical restitution. The aim of the present work was to study and compare the effect of several antiarrhythmic drugs on restitution in preparations from undiseased human ventricular muscle, and other mammalian species. Methods: Action potentials were recorded in preparations obtained from rat, guinea pig, rabbit, and dog hearts; and from undiseased human donor hearts using the conventional microelectrode technique. Preparations were stimulated with different basic cycle lengths (BCLs) ranging from 300 to 5,000 ms. To study restitution, single test pulses were applied at every 20th beat while the preparation was driven at 1,000 ms BCL. Results: Marked differences were found between the animal and human preparations regarding restitution and steady-state frequency dependent curves. In human ventricular muscle, restitution kinetics were slower in preparations with large phase 1 repolarization with shorter APDs at 1000 ms BCL compared to preparations with small phase 1. Preparations having APD longer than 300 ms at 1000 ms BCL had slower restitution kinetics than those having APD shorter than 250 ms. The selective IKr inhibitors E-4031 and sotalol increased overall APD and slowed the restitution kinetics, while IKs inhibition did not influence APD and electrical restitution. Mexiletine and nisoldipine shortened APD, but only mexiletine slowed restitution kinetics. Discussion: Frequency dependent APD changes, including electrical restitution, were partly determined by the APD at the BCL. Small phase 1 associated with slower restitution suggests a role of Ito in restitution. APD prolonging drugs slowed restitution, while mexiletine, a known inhibitor of INa, shortened basic APD but also slowed restitution

A szív repolarizációs folyamatának celluláris szintű élettani, kórélettani és farmakológiai vizsgálata = Physiological, pathophysiological and pharmacological study of the cardiac repolarization

Kísérleteink során a szívizom repolarizációjával és a repolarizációs rezerv szerepével foglalkoztunk. Vizsgálataink szerint az IKs kulcsfontosságú szerepe van a repolarizációs rezerv kialakításában. Bizonyítottuk, hogy experimentális diabéteszben az IKs és Ito káliumáramok downregulációja miatt a repolarizációs rezerv beszűkül, és ennek vélhetően megnövekedett proaritmiás kockázat lehet a következménye. Új in vivo módszert dolgoztunk ki, amely lehetővé teszi a csökkent repolarizációs rezerv megítélést és alkalmas lehet a kórfolyamatok és gyógyszer proaritmiás hatásainak előrejelzésére. Molekuláris biológiai kísérleteink szerint emberi szívizomban az Ito áram kialakításában az eddigi vélekedésektől eltérően más alfa egységek is meghatározzák. Ezen munkánkat 9 in extenzo angol nyelvű közleményben foglaltuk össze, melyek kummulatív impakt faktora 34.61. | We studied the nature of cardiac repolarization and the function of the repolarization reserve in cellular. In spite of IKs plays little role on normal repolarization it has a key role establishing the repolarization reserve. In experimental diabetes due to downregulation of IKs and Ito the repolarization reserve decreased which probably is associated with increased proarrhythmic risk. We developed a new in vivo method which is suitable to investigate the repolarization reserve and to predict the possible increased proarrhythmic risk in pathophysiological situation or after drug applications. Our molecular biological experiments rereated that in addition to the knew proteins other previously unrecognized alfa subunits contribute to the transmembrane ion channels conducting Ito. The results obtained during the granting period was published in 9 English papers (IF= 34.61)

Antiaritmiás és proaritmiás mechanizmusok elemzése = Analysis of antiarrhythmic and proarrhythmic mechanism

Szívizomsejtben a K+-áramok antiaritmiás ''repolarizációs tartalékot'' tartanak fenn: Az IKr, IKs és IK1 akár egyikének gátlása súlyos proaritmiás repolarizáció megnyúlást okoz és letális kamrafibrillációhoz vezethet. - Alloxán-diabeteses kutyán az Ito és IKs csökkenése ugyanilyen hatású, amit a Kv4.3 és Mink csatorna fehérje expresszió egyidejű gátoltsága kisér. - Szívbillyentyű átültetés céljából használt, egészséges donor szivekből izolált humán kamrai miocitákon az IKs csökkenése szintén növeli a proaritmia és a hirtelen szívhalál rizikóját. - A K+-csatorna alegység expresszió mértékének sorrendje: KvLQT1 és ERG1-nél: ember>nyúl>tengerimalad, Mink-nél: tengerimalac>ember>nyúl. - Az INa+/Ca2+ csereáram gátlása kutyán antiaritmiás hatású, csökkenti az utódepolarizációt és a triggerelt aktivitást. - A leghatékonyabb új antiaritmikumok ''többszörös (tedisamil) ill. ''hibrid'' (azimilid, terikalant) ioncsatorna-gátlók. A pitvarszelektiv antiaritmiás szerek hatásmódja: az IKAch, IKur és IK1 izolált blokkolása. - A prekondicionálás antiaritmiás hatásában kutyán a NO trigger és mediátor, prosztaciklin- és szabadgyök-képződés viszont nem vesz részt ebben. | In the myocardial cell, there is a 'repolarization reserve' maintained by K+ currents: inhibition any of the IKr, IKs and IK1 results in severe proarrhythmic prolongation of repolarization and may cause lethal ventricular fibrillation. - The decrease of IKs and also Ito exhibits the same effect in alloxan-diabetic dogs which is accompanied by concomitant retardation of the expression of the K+ channel proteins Kv4.3 and Mink. - In undiseased human ventricular myocites obtained from donors for valve transplant surgery diminution of IKs also increases the risk of proarrhythmia and suddenc cardiac death. The extent of the order of expression of K+ channel subunits: human>rabbit>guinea pig with KvLQT1 and ERG1, and guinea pig>human>rabbit with Mink. - Inhibition of the INa+/Ca2+ exchange current is antiarrhythmic in dogs; it decreases the afterdepolarization and triggered activity. - The most efficacious new antiarrhythmics are ''multiple'' (tedisamil) or 'hybrid' (azimilid, terikalant) ion-channel blockers. - The mode of action of atrial-selective antiarrhythmics: isolated block of IKAch or IKur or IKs. - In the antiarrhythmic action of preconditioning in dogs nitrogen oxide acts as trigger and mediator, whereas formation of prostacyclin and free radicals are not involved in this effect

A Comparative Study of the Rapid (IKr) and Slow (IKs) Delayed Rectifier Potassium Currents in Undiseased Human, Dog, Rabbit, and Guinea Pig Cardiac Ventricular Preparations

To understand the large inter-species variations in drug effects on repolarization, the properties of the rapid (IKr) and the slow (IKs) components of the delayed rectifier potassium currents were compared in myocytes isolated from undiseased human donor (HM), dog (DM), rabbit (RM) and guinea pig (GM) ventricles by applying the patch clamp and conventional microelectrode techniques at 37 °C. The amplitude of the E-4031-sensitive IKr tail current measured at −40 mV after a 1 s long test pulse of 20 mV, which was very similar in HM and DM but significant larger in RM and GM. The L-735,821-sensitive IKs tail current was considerably larger in GM than in RM. In HM, the IKs tail was even smaller than in DM. At 30 mV, the IKr component was activated extremely rapidly and monoexponentially in each studied species. The deactivation of the IKr component in HM, DM, and RM measured at −40 mV. After a 30 mV pulse, it was slow and biexponential, while in GM, the IKr tail current was best fitted triexponentially. At 30 mV, the IKs component activated slowly and had an apparent monoxponential time course in HM, DM, and RM. In contrast, in GM, the activation was clearly biexponential. In HM, DM, and RM, IKs component deactivation measured at −40 mV was fast and monoexponential, while in GM, in addition to the fast component, another slower component was also revealed. These results suggest that the IK in HM resembles that measured in DM and RM and considerably differs from that observed in GM. These findings suggest that the dog and rabbit are more appropriate species than the guinea pig for preclinical evaluation of new potential drugs expected to affect cardiac repolarization

A Possible Explanation for the Low Penetrance of Pathogenic KCNE1 Variants in Long QT Syndrome Type 5

Long QT syndrome (LQTS) is an inherited cardiac rhythm disorder associated with increased incidence of cardiac arrhythmias and sudden death. LQTS type 5 (LQT5) is caused by dominant mutant variants of KCNE1, a regulatory subunit of the voltage-gated ion channels generating the cardiac potassium current IKs. While mutant LQT5 KCNE1 variants are known to inhibit IKs amplitudes in heterologous expression systems, cardiomyocytes from a transgenic rabbit LQT5 model displayed unchanged IKs amplitudes, pointing towards the critical role of additional factors in the development of the LQT5 phenotype in vivo. In this study, we demonstrate that KCNE3, a candidate regulatory subunit of IKs channels minimizes the inhibitory effects of LQT5 KCNE1 variants on IKs amplitudes, while current deactivation is accelerated. Such changes recapitulate IKs properties observed in LQT5 transgenic rabbits. We show that KCNE3 accomplishes this by displacing the KCNE1 subunit within the IKs ion channel complex, as evidenced by a dedicated biophysical assay. These findings depict KCNE3 as an integral part of the IKs channel complex that regulates IKs function in cardiomyocytes and modifies the development of the LQT5 phenotype