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Suitability of hSC and hiPSC derived cardiomyocytes as in vitro cell model that represents the physiological conditions for cardiotoxicity screening

Boek - Dissertatie

Cardiotoxicity is one of the most prevalent unwanted side effects of new drugs in development. To improve the early detection of these side effects, an in vitro model that captures the in vivo physiology is desired in the non-clinical safety screening of new drug compounds. A promising model are human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM). Starting from differentiated cells of a human donor, the cells are de-differentiated into a pluripotent state. The hiPSC cells can then be differentiated again into specific cell types such as cardiomyocytes (CM). These hiPSC-CMs express a broader repertoire of cardiac proteins than expected for heterologous cell expression systems, which overexpress one protein of interest. Therefore, a more physiological model is created. However, the model is still too immature compared to native CM as their morphology and electrophysiological parameters correspond more to an embryonic like state. The resting membrane potential (RMP) is for example more depolarised compared to mature CM, which can influence ion channel characteristics and possibly their pharmacological profile. Depolarization of the RMP is caused by a decreased expression of Kir2.1, as reported in literature, and/or due to a technical issue when recording AP data in the patch-clamp setup when large leak currents are present. Other factors that might affect ion channel pharmacology are auxiliary subunits that interact with the ion channel subunits or by changes in cellular conditions such as membrane cholesterol. In this thesis the Nav1.5 channel, responsible for the fast depolarization of the action potential (AP), was researched. Nav1.5 availability for activation was found to be dependent on the RMP as at membrane potentials more depolarized than -60 mV all channels were inactivated. RMP values, determined with the patch-clamp technique, were in isolated cells around -50 mV while hiPSC-CMs in syncytium had a RMP of approximately -70 mV, which is closer to the physiological RMP. The more hyperpolarised RMP reflected in a faster upstroke of the AP as more Nav1.5 channels were available. This indicates that pharmacological screening of drugs, targeting Nav1.5, will not be detected during AP recordings if the RMP is too depolarised. We also researched if the heterologous expression systems, expressing Nav1.5 alone or in co-expression with β1, can be compared to the hiPSC-CM INa and if the pharmacological profile is changed. From our data we concluded that the hiPSC-CM model compared the most to the heterologous model expressing both Nav1.5 and its β1 subunit and both had a similar pharmacological profile. Afterwards, the effect of membrane cholesterol on the repolarizing cardiac ion channels and their pharmacological profile for the drug quinidine was evaluated, in order to research the possible need for a hypercholesterolemia model in cardiotoxicity screening. Cholesterol is known to have an effect on ion channels and is elevated in hypercholesterolemia patients. However, 9 due to the broad effects of cholesterol on AP data it is difficult to translate the obtained and reported data from heterologous cell models to the hiPSC-CMs model. Perspectives are suggested for future research and to make a conclusion on the need for a hypercholesterolemia model in cardiotoxicity screening. In conclusion, the hiPSCCM model is a suitable model for cardiotoxicity screening when being precautious for its current limitations e.g. the RMP. It is therefore advised to use hiPSC-CM in a syncytium when wanting to analyse RMP values and AP data. High throughput systems which do not need access to the cell can be performed on isolated cells or monolayers but determination of the upstroke velocity can be used as quality control, as it is correlated to the RMP. It has to be noted, however, for researching the mechanisms of how an ion channel is modulated, due to a compound or cellular changes, that it is still advised to use heterologous expression models. The hiPSC-CM model is, at present, still immature and too variable but maturation studies are being performed to solve these limitations.
Aantal pagina's: 190
Jaar van publicatie:2021
Trefwoorden:Doctoral thesis
Toegankelijkheid:Open