DIFFERENT METHODS TO MODEL CARDIAC ARRHYTHMOGENIC DISEASES: FROM TRANSFECTED CELLS TO CARDIOMYOCYTES DERIVED FROM HUMAN INDUCED PLURIPOTENT STEM CELLS

Cardiomyopathies are a heterogeneous group of diseases of the heart muscle, associated to alterations of cardiac currents and ions handling that cause the impairment of the cardiac function leading to the insurgence of arrhythmias, heart failure and sudden death. In this project, we focus our attention on ion channel diseases. I will use different models, from expression system, to human adult cardiomyocytes isolated from septal samples, until human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM) to design cardiomyopathies and used it to test the efficacy and safeness of “old” drugs in restore cardiac function. I analysed the electrophysiological feature of those models, by calcium and sodium fluorescence and by patch clamp technique to better investigate the cardiac impairment. In the cell-based model of a mixed phenotype between BrS and LQT3s obtained in HEK293 cells transfected with the wt-SCN5A gene or the 1795insD-SCN5A, together with the β-subunits, we tested the potential efficacy of Mexiletine in increase the altered peak sodium current (INa) and in reducing the late sodium current (INaL) (that were respectively reduce and increase by the mutation on the cardiac sodium channel). The double efficacy make it a possible effective treatment for the overlap syndrome. Our results confirmed this hypothesis, Mexiletine (500µM) in chronic treatment (48h incubation, washed before recordings) is able to increase INa. In cardiomyocytes isolated from human septal samples of obstructive HCM (OHCM) patients undergoing surgical myectomy, we analysed the efficacy and safeness of Disopyramide. Less is known about the complexity of its action, beside the sodium channel block, so we investigated its effects in reducing arrhythmic event by measuring action potential (AP) and cardiac ion currents by patch clamp technique and Calcium transient features, using calcium sensing fluorescent dye. Our results show that it is able to reduced AP duration, the upstroke velocity and the incidence of EAD and DAD reducing the intensity of INa-L and ICa-L and inhibiting Ik and calcium sparks from RYR. It’s action on multiple ion channel make Disopyramide a safe drug for OHCM, more than the actual treatment suggested by the guidelines. hiPSCs are a novel tool to obtain cells with preserved genetic background of healthy individuals or patients. The limitations of this model consist in the maturity of the cardiomyocytes, that are consistently different from an adult ventricular cardiomyocytes (for both morphological and electrophysiological features). To improve the maturation level of our cells we used long term culture (until 90 days post differentiation) associated to patterned surfaces. By measuring calcium handling and action potential profile at different time points (30, 60 and 90 days), we tried to understand how the electrophysiological features of these cardiomyocytes evolve in the control and DMD line. hiPSC-CM could be the future of disease modelling but the limits in the maturation protocols need to be overpass, our methods shows how is possible do that using a long term culture associated with patterned surfaces and how it could give us the chance to understand the disease progression during cardiomyocytes development and which pathway target with drugs to inhibits disease progression. Pay attention in choosing the right model for our purpose is essential, this could let our work to successfully and reliable result with high translational value. The troubles in modelling cardiomyopathy associated with elevated arrhythmic risk make the decision even harder and probably the combination of different model will be a valid option. Our results also highlight the importance of a personalized therapy for those patients, in which every subject has a different background and different phenotype manifestation; studying, not the disease, but the patient’s phenotype will help us to identify specific drug treatment suitable for that subject.