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)

Comparison of the efficiency of Na+/Ca2+ exchanger or Na+/H+ exchanger inhibition and their combination in reducing coronary reperfusion-induced arrhythmias

During ischaemia/reperfusion, the rise in [Na+]i, induced by simultaneous depression of the Na+/K+-ATPase and activation of the Na+/H+ exchanger (NHE), shifts the Na+/Ca2+ exchanger (NCX) into reverse transport mode, resulting in Ca2+ i overload, which is a critical factor in enhancing the liability to cardiac arrhythmias. The inhibition of NHE, and recently NCX has been suggested to effectively protect the heart from reperfusion-induced arrhythmias. In this study, we investigated and compared the efficacy of individual or the simultaneous inhibition of the NHE and NCX against reperfusion-induced arrhythmias in Langendorff-perfused rat hearts by applying a commonly used regional ischaemiareperfusion protocol. The NHE and NCX were inhibited by cariporide and SEA0400 or the novel, more selective ORM10103, respectively. Arrhythmia diagrams calculated for the reperfusion period were analysed for the incidence and duration of extrasystoles (ESs), ventricular tachycardia (VT) and ventricular fibrillation (VF). NHE inhibition by cariporide was highly efficient in reducing the recorded reperfusion-induced arrhythmias. Following the application of SEA0400 or ORM-10103, the number and duration of arrhythmic periods were efficiently or moderately decreased. While both NCX inhibitors effectively reduced ESs, the most frequently triggered arrhythmias, they exerted limited or no effect on VTs and VFs. Of the NCX inhibitors, ORM-10103 was more effective. Surprisingly, the simultaneous inhibition of the NCX and NHE failed to significantly improve the antiarrhythmic efficacy reached by NCX blockade alone. In conclusion, although principal simultaneous NHE+NCX inhibition should be highly effective against all types of the recorded reperfusion-induced arrhythmias, NCX inhibitors, alone or in combination with cariporide, seem to be moderately suitable to provide satisfactory cardioprotection - at least in the present arrhythmia model. Since ORM10103 and SEA0400 are known to effectively inhibit after-depolarisations, it is suggested that their efficacy and that of other NCX inhibitors may be higher and more pronounced in the predominantly Ca2+ i-dependent triggered arrhythmias. © 2015, Polish Physiological Society. All rights reserved

The efficacy of cariprazine in negative symptoms of schizophrenia: Post hoc analyses of PANSS individual items and PANSS-derived factors

AbstractBackground:Negative symptoms in schizophrenia are heterogeneous and multidimensional; effective treatments are lacking. Cariprazine, a dopamine D3-preferring D3/D2 receptor partial agonist and serotonin 5-HT1A receptor partial agonist, was significantly more effective than risperidone in treating negative symptoms in a prospectively designed trial in patients with schizophrenia and persistent, predominant negative symptoms.Methods:Using post hoc analyses, we evaluated change from baseline at week 26 in individual items of the Positive and Negative Syndrome Scale (PANSS) and PANSS-derived factor models using a mixed-effects model for repeated measures (MMRM) in the intent-to-treat (ITT) population (cariprazine = 227; risperidone = 227).Results:Change from baseline was significantly different in favor of cariprazine versus risperidone on PANSS items N1-N5 (blunted affect, emotional withdrawal, poor rapport, passive/apathetic social withdrawal, difficulty in abstract thinking) (P <.05), but not on N6 (lack of spontaneity/flow of conversation) or N7 (stereotyped thinking). On all PANSS-derived negative symptom factor models evaluated (PANSS-Factor Score for Negative Symptoms, Liemburg factors, Khan factors, Pentagonal Structure Model Negative Symptom factor), statistically significant improvement was demonstrated for cariprazine versus risperidone (P <.01). Small and similar changes in positive/depressive/EPS symptoms suggested that negative symptom improvement was not pseudospecific. Change from baseline was significantly different for cariprazine versus risperidone on PANSS-based factors evaluating other relevant symptom domains (disorganized thoughts, prosocial function, cognition; P <.05).Conclusions:Since items representing different negative symptom dimensions may represent different fundamental pathophysiological mechanisms, significant improvement versus risperidone on most PANSS Negative Subscale items and across all PANSS-derived factors suggests broad-spectrum efficacy for cariprazine in treating negative symptoms of schizophrenia

Efficacy of selective NCX inhibition by ORM-10103 during simulated ischemia/reperfusion.

In this study we evaluated the effects of selective Na+/Ca2+ exchanger (NCX) inhibition by ORM-10103 on the [Ca2+]i transient (CaT), action potential (AP), and cell viability in isolated canine ventricular cardiomyocytes exposed to a simulated ischemia/reperfusion protocol performed either alone (modeling moderate low-flow ischemia) or with simultaneous strophantidine challenge (modeling more severe low-flow ischemia). CaTs were monitored using a Ca2+-sensitive fluorescent dye, APs were recorded by intracellular microelectrodes, and anaerobic shifts in cellular metabolism were verified via monitoring native NADH fluorescence. Simulated ischemia increased the NADH fluorescence, reduced the amplitudes of the AP and CaT and induced membrane depolarization. APs moderately shortened, CaTs prolonged. Diastolic [Ca2+]i ([Ca2+]iD) level increased significantly during ischemia and further elevated following strophantidine application. Reperfusion normalized the NADH level, the amplitude of the AP and duration of the [Ca2+]i transient, but only partially restored action potential triangulation and the amplitude of the CaT. [Ca2+]iD decreased in untreated, but further increased in strophantidine-treated cells. 10microM ORM-10103 significantly reduced the ischemic [Ca2+]i raise in both untreated and strophantidine-treated cells. During reperfusion ORM-10103 decreased [Ca2+]i and eliminated its diastolic elevation in untreated and strophantidine-treated cardiomyocytes. Following the application of ORM-10103 the detrimental effect of ischemia/reperfusion on cell viability and the reperfusion-induced increase in AP and CaT variabilities were substantially reduced, but ischemia-induced shifts in AP morphology were barely influenced. In conclusion, selective NCX inhibition by ORM-10103 is highly effective against ischemia/reperfusion induced pathologic alterations in [Ca2+]i homeostasis, however, it fails to normalize untoward arrhythmogenic changes in AP morphology

What Is the Minimum Clinically Important Change in Negative Symptoms of Schizophrenia? PANSS Based Post-hoc Analyses of a Phase III Clinical Trial

INTRODUCTION: Minimum clinically important difference (MCID) is a measure that defines the minimum amount of change in an objective score of a clinical test that must be reached for that change to be clinically noticeable. We aimed to find the MCID for patients with predominantly negative symptoms of schizophrenia at its earliest occurrence. METHODS: Data of a 26-week long, double-blind study with 454 patients [Positive and Negative Symptom Scale Negative Factor Score (PANSS-FSNS) ≥24, Positive and Negative Symptom Scale Positive Factor Score (PANSS-FSPS) ≤ 19] treated with cariprazine 4.5 mg/d or risperidone 4 mg/d were analyzed. The Clinical Global Impression—Improvement scale was used to quantify minimum improvement (CGI-I = 3) and no clinical change (CGI-I = 4) on the PANSS-FSNS, and the MCID was estimated with the following methods: as the mean PANSS-FSNS changes corresponding to the first instance of minimal improvement across all visits (MCID(1)); as the difference between the PANSS-FSNS change associated with the first instance and the PANSS-FSNS changes associated with the last recorded clinically unchanged status across all visits (MCID(2)); with the effect size approach (MCID(3)); as the Youden Index based cut-off value between no clinical change and minimal improvement (MCID(4)); as the relative likelihood of minimal improvement (MCID(5)). RESULTS: The MCID(1) and MCID(2) resulted in, respectively, a 3.8-point (18.5%) and a 1.5-point (7.3%) decrease from baseline severity on the PANSS-FSNS. Greater values were required for the MCID at later evaluation times. The cut-off between minimum improvement and no clinical change defined by the Youden Index was a−3-point (15%) change in the PANSS-FSNS. The effect size approach indicated the 1.5-point difference between minimally improved and unchanged patients to be a medium effect (ES = 0.6). CONCLUSION: Applying different methods led to different results, ranging between 7.3 and 18.5% improvement from the baseline for the MCID at its earliest occurrence in patients with predominantly negative symptoms of schizophrenia

A szívritmuszavarok és a myocardiális repolarizáció mechanizmusainak vizsgálata; antiaritmiás és proaritmiás gyógyszerhatások elemzése = Study of the mechanism of cardiac arrhythmias and repolarization, antiarrhythmic and proarrhythmic drug action

A kardiovaszkuláris betegségek és azon belül is az életet veszélyeztető kamrai és pitvari aritmiák a fő halálozási okok közé tartoznak a fejlett ipari országokban, de Magyarországon is. Ezzel összhangban, a jelen kutatási projekt is a különböző életet veszélyeztető aritmiák megelőzésének a lehetőségét, és a különböző gyógyszerek antiaritmiás és proaritmiás hatásainak a kutatását tűzte ki célul. Vizsgálataink során megállapítottuk, hogy kísérletes diabetes mellitusban mind kutyán, mind nyúlon az IKs áram és következményesen a repolarizációs rezerv csökkenése következik be amely emberben vélhetően hozzájárulhat e betegségben észlelt hirtelen szívhalál kockázat növekedéséhez. Molekuláris biológiai vizsgálatokban sikerült feltérképeznünk az emberi szívizom különféle ioncsatornáinak denzitását is. A proaritmiás gyógyszerhatások elemzésére újszerű módszert dolgoztunk ki, amelynek gyógyszerbiztonsági klinikai jelentősége és hasznosulása várható. A projekt teljesítése során további új ismereteket szereztünk a Na+/Ca2+ cseremechanizmus (NCX) repolarizációban betöltött szerepét illetően. In vivo kutya kísérletekben vizsgáltuk a peroxynitrit és gap junction csatornák szerepét az ischaemiás prekondicionálásban. Ezek az eredmények várhatóan hozzájárulnak egyrészt a szívizomzat élettani, kórélettani (aritmia mechanizmusok) ismereteinek a gyarapításában, másrészt új és biztonságos antiaritmiás terápiák kifejlesztéséhez. | Cardiovascular diseases, including life threatening ventricular and supraventricular arrhythmias, are the leading causes of mortality in industrialized countries and also in Hungary. In harmony with this, the major goal of the project was to investigate the mechanisms involved in cardiac repolarization and in antiarrhythmic and proarrhythmic drug actions. Representing important findings during the project, we established that in experimental diabetes mellitus the IKs potassium current is down-regulated resulting in the attenuation of repolarization reserve which may contribute to the increased proarrhythmic risk of diabetic patients. Using molecular biological methods we have analyzed the transmembrane ion channel densities of the human heart. To assess proarrhyhtmic drug side effects we developed a novel method which can be expected to contribute to better prediction of proarrhythmic risk in both preclinical and clinical safety pharmacology investigations. During the project, we have gained further insights regarding the role of NCX in the cardiac repolarization process. In in vivo studies we have investigated the possible role of peroxynitrite and gap junctions in ischaemic preconditioning. These results can be expected to help to better understand the physiology and pathophysiology of cardiac muscle, and arrhythmias, and should significantly contribute to the development of safer and more effective antiarrhythmic treatment modalities