Establishment of the human cardiac models using gene editing and reprogramming in Human Pluripotent Stem Cells to understand the putative functions of the G-protein coupled receptor kinase 5 polymorphism (GRK5-L41)

A nonsynonymous single polymorphism (SNP) in G protein-coupled receptor kinase 5 (GRK5) was discovered in 2008 that changes the amino acid at the position 41 from Glutamine (Q) into Leucine (L) (Liggett et al., 2008). The putative functions of the GRK5-L41 polymorphism were reported to be involved in faster desensitisation of both b1 and b2 adrenergic receptors in vitro and the cardiac protective functions by improving the survival rate of patients with heart failure conditions or transplantation in vivo. Nevertheless, the mechanisms underlying these processes are still poorly understood, which is the purpose of this thesis. This thesis presents the establishment of the first human cardiac models of GRK5- L41 polymorphism using human pluripotent stem cells (hPSCs) and their derived cardiomyocytes (CMs). The thesis is distributed into four main themes, containing (1) the formulation of monolayer cardiac differentiation protocols; (2) the establishment of human induced pluripotent stem cells from lymphoblastoid cell lines bearing the GRK5- L41 sequence; (3) the development of footprint-free and shortcut CRISRP/Nickase approach that allowed generating the gene-edited human embryonic stem cells expressing GRK5-Q41, GRK5-Q/L41, and GRK5-L41; and (4) the evaluation of GRK5- L41 functions using cardiac functional analysis assays. Relating to disease modelling and cardiovascular biomedical research, the ability to differentiate the hPSCs into CMs plays a critical role by providing an unlimited resource of human CMs for in vitro testing and experiments. Here, three main monolayer cardiac differentiation protocols, including E8-AB, mTeSR-AB, and mTeSR-CHIR, were described in details and proven to be highly consistent, efficient, robust, and reproducible. Additionally, these protocols have been ascertained to be effective in more than 27 hPSC lines routinely maintained in the lab regardless of the culture conditions (non-defined vs. defined culture conditions), cell types (human embryonic stem cells vs. human induced pluripotent stem cells), reprogramming methods, and somatic cell sources (in the case of induced pluripotent stem cells). Indeed, by using the E8-AB protocol, more than 1x107 CMs/line have been produced in this thesis, providing enough resource for functional assay analysis and mechanistic studies of GRK5-L41. Furthermore, two independent approaches were made to create the human cardiac model of GRK5-L41 polymorphism, involving the establishment of GRK5-L41 bearing hiPSCs from lymphoblastoid cell lines, and simultaneously introducing the GRK5-L41 sequence to the HUES7 genome to create the Q/L41, and L41 expressing HUES7 lines. In general, four hPSC lines were successfully generated in this thesis, including hiPSC-GRK5-L41, hiPSC-GRK5-Q41, HUES7-GRK5-Q/L41, and HUES7- GRK5-L41. The H-Fib-hiPSC, cell lines generated from HUES7-derived fibroblast, was the additional line obtained after testing the effectiveness of episomal plasmid. All cell lines were able to differentiate into CMs at high purity, approximately 85%, and were used for the development of functional assays. Four main functional experiments were developed focusing on the GRK5-related functions in the heart, consisting of contractility and hypertrophic response to catecholamine induction, especially during the chronic response. The effects of catecholamine, in this case, Isoprenaline (ISO), on the contractility of the CMs were measured by two assays, the CardioExcyte96 platform detecting the contraction rate and beating pattern in real-time, and the LANCE Ultra cAMP assay assessing the production of cAMP. These results indicated that extended culture of CMs in ISO (>30h) introduced the detrimental effects on the contractility and beating pattern of the CMs in vitro, generating the arrhythmias in GRK5-Q41 CMs. Interestingly, the GRK5-L41 CMs exhibited a high level of the beat rate in response to ISO and maintained it constantly during prolonged exposure to ISO similar to that of b-blocker treatments in GRK5-Q41 CMs. The Western Blot analysis of the cellular distribution of GRK5 spotted the localisation of GRK5 during ISO treatment for 72h. Further characterisation using immunofluorescence analysis of chronic exposure to ISO demonstrated the elevation of BNP level, a hypertrophic marker, indicating that ISO treatment duration (>30h) induced the hypertrophic response of hPSC-CMs in vitro. Taken together, the findings within this thesis has been the first step in a discovery process of the cardiac protective functions of GRK5-L41 polymorphism during heart failure. Despite the presence of limitation and difficulty, it manages to provide sufficient information to explore further the interrelationship between nuclear accumulation of GRK5, hypertrophic response, and contractility regulation mediated by either GRK5-Q41 and GRK5-L41 in hPSC-CMs in vitro