Duncan, Gary
(2017)
Using human induced pluripotent stem cells to study the in vitro phenotype of the cardiac channelopathies.
PhD thesis, University of Nottingham.
Abstract
Long QT Syndrome 2 (LQTS2) and Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) are two prevalent cardiac channelopathies. LQTS2 is the second most common form of LQTS and accounts for up to 45% of cases (Schwartz et al, 2012). CPVT1 is the most common form of CPVT and relates to dysfunction of the RYR2, mutations in RYR2 are thought to account for up to 50% of CPVT cases (Nyegaard et al, 2012). Treatment for these conditions is vital as mortality rates for untreated individuals can range from 21% to potentially as high as 50% (Leenhardt et al, 1995; Schwartz et al, 2012). Discovering novel compounds which treat these conditions is beset by suboptimal drug discovery methods which utilise animal models, which can be inaccurate due to inter-species variability in the cardiovascular system (Rajamohan et al, 2013), and heterologous overexpression systems which test the effect of a drug on specific ion channels which are limited by the inability of these systems to model the dynamic interaction between different ion channels in vivo (Kramer et al, 2013). Using human induced pluripotent stem cells (hiPSC) it is possible to generate cardiomyocytes which may enable the creation of humanised models of disease. Such models may enable the discovery of efficacious compounds more readily than current drug development techniques and they may also allow the study of disease pathogenesis in more detail than is currently available in human systems.
In this thesis different tissue sample types were investigated for their ability to robustly derive cell lines in vitro for use in reprogramming to hiPSCs. Furthermore in this thesis three different non-integrative methods of hiPSC generation, Sendai virus, episomal reprogramming using nucleofection and episomal reprogramming using GET peptide mediated transduction, were tested to assess which was able to most robustly generate hiPSCs. These hiPSCs were ultimately differentiated into cardiomyocytes (hiPSC-CMs) and used in downstream phenotyping assays. An LQTS2-hiPSC model was used as part of an in vitro drug assay to test the effectiveness of Ikr, Ikatp, PPARδ and Iks agonists in reducing the action potential duration (APD) of the LQTS2-hiPSC-CMs which would thereby indicate a role in enhancing repolarisation. The effects of these new candidate drugs were compared to the effect observed by the traditional LQTS2 therapy, β-blockers. This work was carried out using the CellOPTIQ platform and voltage was analysed by labelling the hiPSC-CMs with voltage sensitive cell dye FluoVolt. Furthermore in this thesis a CPVT-hiPSC model was generated and genetically corrected using CRISPR/Cas9 genome engineering technology. These hiPSCs were then differentiated to cardiomyocytes and analysed for their calcium handling properties using fluorescent labelling dye Fura2-AM.
Assessment of the voltage properties of the LQTS2-hiPSC-CMs indicated that the LQTS2-hiPSC-CMs exhibit increased APD in comparison to healthy-hiPSC-CMs and an increase in triangularisation time which is indicative of repolarisation time therefore indicating a faithful recapitulation of the patient’s phenotype in vitro. Moreover the APD of LQT2-hiPSC-CMs was found to decrease in response to treatment with Ikatp, Ikr, PPARδ and Iks agonists. These data indicate that the drugs tested in this thesis may provide a beneficial effect to LQTS2 patients and should be investigated further.
Assessment of the calcium handling mechanics of CPVT-hiPSC-CMs and genetically corrected CPVT-WT-hiPSC-CMs indicated that differences exist in the way in which these hiPSC-CMs handle calcium in vitro. The genetically corrected CPVT-WT-hiPSC-CMs were found to show enhanced functionality of RYR2 and SERCA when challenged with relevant pharmacological blockade, potentially indicating a corrected phenotype. However assessment of whether the CPVT-hiPSC-CMs exhibited a “normal” phenotype was inconclusive. There exist differences in the calcium handling properties of CPVT-WT-hiPSC-CMs and unrelated healthy-hiPSC-CMs.
To summarise, this thesis assesses the effectiveness of multiple tissue samples to elucidate a robust method to derive starting cell populations from patients. Moreover it goes on to establish a robust protocol for generating hiPSCs from these samples using a non-integrative reprogramming method. The generation of an LQTS2 disease model enabled an in vitro drug assay which identified 9 compounds which were able to reduce the APD in LQTS2-hiPSC-CMs, of which 7 showed utility in reducing the repolarisation time of the LQT2-hiPSC-CMs. These results indicate that these compounds may exert beneficial effects in the treatment of patients with LQTS2. In addition to this, preliminary results in the CPVT disease model created seem to indicate an increase in functionality of both RYR2 and SERCA after CRISPR/Cas9 genetic correction of the S2246L mutation. Attempts to establish if CRISPR/Cas9 genetic correction generated “normal” calcium handling properties were inconclusive. There remains a large amount of variability within genetically distinct hiPSC-CMs with regards to their calcium handling properties, likely owing to the highly spatial and temporal nature of calcium movement in cardiomyocytes and this should be investigated further.
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