Kálium ion-csatornák szerepének vizsgálata szívizomsejtekben a génkifejeződés RNS interferencia révén történő blokkolásával = Investigation of the role of potassium ion channels of heart muscle cells by means of RNA interference-mediated blocking of gene expression

Kutatási témánk a szívizom kálium ioncsatornáinak vizsgálata RNS interferencia technikával. Az RNS interferenciát kiváltó expressziós kazettát Aujeszky-féle vírus (AyV) vektorral juttattuk be a szívizomsejtekbe. A konkrét feladat megoldásához több olyan általános problémát is meg kellett oldanunk, amelyhez modellként egy másik posztmitotikus sejttípust, az idegsejteket is felhasználtuk. A munka első fázisában fluoreszcens markereket kifejező AyV törzseket állítottunk elő. E kísérletek célja a vírus génbeviteli hatékonyságának tesztelése volt. Különböző színű markereket vittünk be posztmitotikus sejtekbe (szívizom, idegsejtek). Tenyésztett felnőtt kutya szívizomsejtekben a génbeviteli hatékonyság, magas vírus titer alkalmazásával, közel 100%-os volt. Megvizsgáltuk azt is, hogy a PRV által bevitt gének működőképesek-e a célsejtekben, ill. hogy a vírus hatással van-e ezekre a sejtekre. Modellként egy fluoreszcens kalcium szenzort, a troponeont használtuk. A troponeon mind szívizomsejtekben, mind neuronokban kiválóan működött. A virulencia csökkentése végett különféle PRV mutánsokat állítottunk elő. A korai fehérje 0, a ribonukleotid reduktáz gének kiütése és az ún. antiszensz promóter inaktiválása olyan vírust eredményezett, amely epitél sejteken (PK-15 sejtvonal, ezen szaporítjuk a vírust) megfelelően szaporodott, szívizomsejtekben azonban avirulensnek bizonyult. Továbbá, kutya Kv4.3 gént csendesítő RNS interferenciát közvetítő vírusokat állítottunk elő. | Our research project is the analysis of cardiac potassium ion channels using RNA interference technique. We used pseudorabies virus (PRV) vectors for the delivery of expression cassettes evoking RNA interference. In the first phase of our work we constructed various fluorescence proteins expressing recombinant viruses. The aim of this work was to test the efficiency of gene delivery by the virus. We delivered fluorescence markers with various colors to cardiomyocytes and neurons. The efficiency of PRV-based gene delivery to canine cardiomyocytes was close to 100% in high titer virus infection. The fluorescent markers were delivered to neurons in vivo using mouse and rat models. The next step was the examination whether the delivered gene retains its functionality in a virus-based system. We have used troponeon, a genetically encoded fluorescence activity marker, as model to test this problem. According to our examinations, troponeon, delivered to both cardiomyocytes and neurons, performed very well in both cell types. We have constructed various mutant PRV strains by the deletion of early protein 0 and ribonucleotide reductase genes, as well as the deleted the putative antisense promoter region of the virus. PRVs containing these triple mutations proved to be ideal gene delivery vectors to cardiomyocytes. Furthermore, we have generated viruses expressing the hairpin RNAs for knocking down Kv4.3 gene expression of the dog cardiomyocytes

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)

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

Herpesvirus-mediated delivery of a genetically encoded fluorescent Ca2+ sensor to canine cardiomyocytes

We report the development and application of a pseudorabies virus-based system for delivery of troponeon, a fluorescent Ca2+ sensor to adult canine cardiomyocytes. The efficacy of transduction was assessed by calculating the ratio of fluorescently labelled and nonlabelled cells in cell culture. Interaction of the virus vector with electrophysiological properties of cardiomyocytes was evaluated by the analysis of transient outward current (Ito), kinetics of the intracellular Ca2+ transients, and cell shortening. Functionality of transferred troponeon was verified by FRET analysis. We demonstrated that the transfer efficiency of troponeon to cultured adult cardiac myocytes was virtually 100%. We showed that even after four days neither the amplitude nor the kinetics of the Ito current was significantly changed and no major shifts occurred in parameters of [Ca2+]i transients. Furthermore, we demonstrated that infection of cardiomyocytes with the virus did not affect the morphology, viability, and physiological attributes of cells

Self-restoration of cardiac excitation rhythm by anti-arrhythmic ion channel gating

Homeostatic regulation protects organisms against hazardous physiological changes. However, such regulation is limited in certain organs and associated biological processes. For example, the heart fails to self-restore its normal electrical activity once disturbed, as with sustained arrhythmias. Here we present proof-of-concept of a biological self-restoring system that allows automatic detection and correction of such abnormal excitation rhythms. For the heart, its realization involves the integration of ion channels with newly designed gating properties into cardiomyocytes. This allows cardiac tissue to i) discriminate between normal rhythm and arrhythmia based on frequency-dependent gating and ii) generate an ionic current for termination of the detected arrhythmia. We show in silico, that for both human atrial and ventricular arrhythmias, activation of these channels leads to rapid and repeated restoration of normal excitation rhythm. Experimental validation is provided by injecting the designed channel current for arrhythmia termination in human atrial myocytes using dynamic clamp

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