Proaritmiás gyógyszermellékhatások vizsgálata transzgenikus hosszú QT-szindrómás nyúlmodellek segítségével

A gyógyszer okozta ritmuszavar a különböző vegyületek súlyos és potenciálisan halálos mellékhatása. A proaritmiás mellékhatást gyakran az adott gyógyszernek a szívizom-repolarizáló ionáramait gátló hatásaival hozzák összefüggésbe, ami az EKG-n a QT-intervallum meghosszabbodásához vezet. A gyógyszerfejlesztésben alkalmazott kifinomult szűrési módszerek ellenére a proaritmia megbízható előrejelzése továbbra is jelentős kihívást jelent. Bár a gyógyszerek által kiváltott QT-idő megnyúlással összefüggő proaritmiás hatás iránt fokozottan érzékenyítenek a betegek repolarizációs tartalékát csökkentő kórfolyamatok, a legtöbb gyógyszerbiztonsági szűrésre használt modellrendszer normális, egészséges sejteket, szöveteket és állatokat használ fel. A közelmúltban több, csökkent repolarizációs tartalékkal rendelkező transzgenikus hosszú QT-szindrómás (LQTS) nyúlmodellt hoztak létre. Összefoglaló közleményünkben e modellek lehetséges felhasználását tárgyaljuk a gyógyszerindukált aritmiák előrejelzésére, összefüggésben a különböző modellekben tapasztalt repolarizáló szívizom ionáram-károsodással. Emellett áttekintjük a rendelkezésre álló transzgenikus LQTS-nyúlmodellek elektrofiziológiai jellemzőit, a modelleken végzett farmakológiai elvi bizonyító vizsgálatokat, kiemelve a transzgenikus nyúl LQTS-modellek előnyeit és hátrányait a gyógyszerindukálta ritmuszavar kutatásában

Transgenic rabbit models for cardiac disease research.

To study the pathophysiology of human cardiac diseases and to develop novel treatment strategies, complex interactions of cardiac cells on cellular, tissue and whole heart levels need to be considered. As in vitro cell-based models do not depict the complexity of the human heart, animal models are used to obtain insights that can be translated to human diseases. Mice are the most commonly used animals in cardiac research, however, differences in electrophysiological and mechanical cardiac function and a different composition of electrical and contractile proteins limit the transferability of the knowledge gained. Moreover, the small heart size and fast heart rate are major disadvantages. In contrast to rodents, electrophysiological, mechanical, and structural cardiac characteristics of rabbits resemble the human heart more closely, making them particularly suitable as an animal model for cardiac disease research. In this review, various methodological approaches for the generation of transgenic rabbits for cardiac disease research - such as pronuclear microinjection, the sleeping beauty transposon system and novel genome editing methods (ZFN and CRISPR/Cas9) - will be discussed. In the second section, we will introduce the different currently available transgenic rabbit models for monogenic cardiac diseases (such as long-QT syndrome, short-QT syndrome, and hypertrophic cardiomyopathy) in detail, especially in regards to their utility to increase the understanding of pathophysiological disease-mechanisms and novel treatment options

Mechanisms of ventricular rate adaptation as a predictor of arrhythmic risk.

Background: Protracted QT interval (QTI) adaptation to abrupt heart rate (HR) changes has been identified as a clinical arrhythmic risk marker. This study investigates the ionic mechanisms of QTI rate adaptation and its relationship to arrhythmic risk. Methods and Results: Computer simulations and experimental recordings in human and canine ventricular tissue were used to investigate the ionic basis of QTI and action potential (AP) duration (APD) to abrupt changes in HR with a protocol commonly used in clinical studies. Time for 90% QTI adaptation is 3.5 min in simulations, in agreement with experimental and clinical data in human. APD adaptation follows similar dynamics, being faster in midmyocardial cells (2.5 min) than in endocardial/epicardial cells (3.5 min). Both QTI and APD adapt in two phases following an abrupt HR change: a fast initial phase with time constant 2 min driven by [Na(+)]i dynamics. Alterations in [Na(+)]i dynamics due to Na(+)/K(+) pump (INaK) inhibition result in protracted rate adaptation, and is associated with increased proarrhythmic risk, as indicated by AP triangulation and faster ICaL recovery from inactivation, leading to formation of early afterdepolarizations (EADs). Conclusions: This study suggests that protracted QTI adaptation could be an indicator of altered [Na(+)]i dynamics following INaK inhibition as it occurs in patients with ischemia or heart failure. Increased risk of cardiac arrhythmias in patients with protracted rate adaptation may be due to increased risk of EAD formation. Key words: action potentials, ventricles, ion channels, arrhythmia

A computational model of rabbit geometry and ECG: Optimizing ventricular activation sequence and APD distribution

Computational modeling of electrophysiological properties of the rabbit heart is a commonly used way to enhance and/or complement findings from classic lab work on single cell or tissue levels. Yet, thus far, there was no possibility to extend the scope to include the resulting body surface potentials as a way of validation or to investigate the effect of certain pathologies. Based on CT imaging, we developed the first openly available computational geometrical model not only of the whole heart but also the complete torso of the rabbit. Additionally, we fabricated a 32-lead ECG-vest to record body surface potential signals of the aforementioned rabbit. Based on the developed geometrical model and the measured signals, we then optimized the activation sequence of the ventricles, recreating the functionality of the Purkinje network, and we investigated different apico-basal and transmural gradients in action potential duration. Optimization of the activation sequence resulted in an average root mean square error between measured and simulated signal of 0.074 mV/ms for all leads. The best-fit T-Wave, compared to measured data (0.038 mV/ms), resulted from incorporating an action potential duration gradient from base to apex with a respective shortening of 20 ms and a transmural gradient with a shortening of 15 ms from endocardium to epicardium. By making our model and measured data openly available, we hope to give other researchers the opportunity to verify their research, as well as to create the possibility to investigate the impact of electrophysiological alterations on body surface signals for translational research

Oxytocin exerts harmful cardiac repolarization prolonging effects in drug-induced LQTS.

Background Oxytocin is used therapeutically in psychiatric patients. Many of these also receive anti-depressant or anti-psychotic drugs causing acquired long-QT-syndrome (LQTS) by blocking HERG/IKr. We previously identified an oxytocin-induced QT-prolongation in LQT2 rabbits, indicating potential harmful effects of combined therapy. We thus aimed to analyze the effects of dual therapy with oxytocin and fluoxetine/risperidone on cardiac repolarization. Methods Effects of risperidone, fluoxetine and oxytocin on QT/QTc, short-term variability (STV) of QT, and APD were assessed in rabbits using in vivo ECG and ex vivo monophasic AP recordings in Langendorff-perfused hearts. Underlying mechanisms were assessed using patch clamp in isolated cardiomyocytes. Results Oxytocin, fluoxetine and risperidone prolonged QTc and APD in whole hearts. The combination of fluoxetine + oxytocin resulted in further QTc- and APD-prolongation, risperidone + oxytocin tended to increase QTc and APD compared to monotherapy. Temporal QT instability, STVQTc was increased by oxytocin, fluoxetine / fluoxetine + oxytocin and risperidone / risperidone + oxytocin. Similar APD-prolonging effects were confirmed in isolated cardiomyocytes due to differential effects of the compounds on repolarizing ion currents: Oxytocin reduced IKs, fluoxetine and risperidone reduced IKr, resulting in additive effects on IKtotal-tail. In addition, oxytocin reduced IK1, further reducing the repolarization reserve. Conclusion Oxytocin, risperidone and fluoxetine prolong QTc / APD. Combined treatment further prolongs QTc/APD due to differential effects on IKs and IK1 (block by oxytocin) and IKr (block by risperidone and fluoxetine), leading to pronounced impairment of repolarization reserve. Oxytocin should be used with caution in patients in the context of acquired LQTS

Oxytocin exerts harmful cardiac repolarization prolonging effects in drug-induced LQTS

Background: Oxytocin is used therapeutically in psychiatric patients. Many of these also receive anti-depressant or anti-psychotic drugs causing acquired long-QT-syndrome (LQTS) by blocking HERG/IKr. We previously identified an oxytocin-induced QT-prolongation in LQT2 rabbits, indicating potential harmful effects of combined therapy. We thus aimed to analyze the effects of dual therapy with oxytocin and fluoxetine/risperidone on cardiac repolarization. Methods: Effects of risperidone, fluoxetine and oxytocin on QT/QTc, short-term variability (STV) of QT, and APD were assessed in rabbits using in vivo ECG and ex vivo monophasic AP recordings in Langendorff-perfused hearts. Underlying mechanisms were assessed using patch clamp in isolated cardiomyocytes. Results: Oxytocin, fluoxetine and risperidone prolonged QTc and APD in whole hearts. The combination of fluoxetine + oxytocin resulted in further QTc- and APD-prolongation, risperidone + oxytocin tended to increase QTc and APD compared to monotherapy. Temporal QT instability, STVQTc was increased by oxytocin, fluoxetine / fluoxetine + oxytocin and risperidone / risperidone + oxytocin. Similar APD-prolonging effects were confirmed in isolated cardiomyocytes due to differential effects of the compounds on repolarizing ion currents: Oxytocin reduced IKs, fluoxetine and risperidone reduced IKr, resulting in additive effects on IKtotal-tail. In addition, oxytocin reduced IK1, further reducing the repolarization reserve. Conclusion: Oxytocin, risperidone and fluoxetine prolong QTc / APD. Combined treatment further prolongs QTc/APD due to differential effects on IKs and IK1 (block by oxytocin) and IKr (block by risperidone and fluoxetine), leading to pronounced impairment of repolarization reserve. Oxytocin should be used with caution in patients in the context of acquired LQTS. © 2022 The Author

Transgenic LQT2, LQT5, and LQT2-5 rabbit models with decreased repolarisation reserve for prediction of drug-induced ventricular arrhythmias

Background and Purpose Reliable prediction of pro‐arrhythmic side effects of novel drug candidates is still a major challenge. Although drug‐induced pro‐arrhythmia occurs primarily in patients with pre‐existing repolarisation disturbances, healthy animals are employed for pro‐arrhythmia testing. To improve current safety screening, transgenic long QT (LQTS) rabbit models with impaired repolarisation reserve were generated by overexpressing loss‐of‐function mutations of human HERG (HERG‐G628S , loss of IKr; LQT2), KCNE1 (KCNE1‐G52R , decreased IKs; LQT5), or both transgenes (LQT2‐5) in the heart. Experimental Approach Effects of K+ channel blockers on cardiac repolarisation and arrhythmia susceptibility were assessed in healthy wild‐type (WT) and LQTS rabbits using in vivo ECG and ex vivo monophasic action potential and ECG recordings in Langendorff‐perfused hearts. Key Results LQTS models reflect patients with clinically “silent” (LQT5) or “manifest” (LQT2 and LQT2‐5) impairment in cardiac repolarisation reserve: they were more sensitive in detecting IKr‐blocking (LQT5) or IK1/IKs‐blocking (LQT2 and LQT2‐5) properties of drugs compared to healthy WT animals. Impaired QT‐shortening capacity at fast heart rates was observed due to disturbed IKs function in LQT5 and LQT2‐5. Importantly, LQTS models exhibited higher incidence, longer duration, and more malignant types of ex vivo arrhythmias than WT. Conclusion and Implications LQTS models represent patients with reduced repolarisation reserve due to different pathomechanisms. As they demonstrate increased sensitivity to different specific ion channel blockers (IKr blockade in LQT5 and IK1 and IKs blockade in LQT2 and LQT2‐5), their combined use could provide more reliable and more thorough prediction of (multichannel‐based) pro‐arrhythmic potential of novel drug candidates