An investigation into the function and mechanism of a GRK5-L41 polymorphism using human pluripotent cell-derived cardiomyocytes

A single-nucleotide polymorphism (SNP) in G protein-coupled receptor kinase 5 (termed GRK5-Q41L) was reported to be associated with a low mortality rate for patients with heart failure and cardiac ischemia (Liggett et al., 2008). An in vitro study on transfected cells and transgenic mice suggested that this GRK5-Q41L polymorphism enhances β-adrenergic receptors (β-AR) desensitisation to prevent adverse effects of chronic β-AR stimulation. An area of need is for a relevant in vitro human cellular model to further characterise phenotype and explore mechanism for the gain-of-function of GRK5-Q41L SNP. This was the aim of my thesis, wherein I used the tractability of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) to provide a valuable tool. Nickase CRISPR/Cas9 genome editing technology had previously been used in our lab to generate Q41L SNP on the GRK5 loci in HUES7 line to generate the isogenic HUES7-GRK5-Q41L model. Also, in separate experiments, human induced pluripotent stem cells (hIPSCs) harbouring homogenous GRK5-Q41 and GRK5-L41 had been generated from lymphoblastoid cells by episomal reprogramming technology; however, this hIPSC GRK5-Q41L model has heterogenic background. The isogenic HUES7-GRK5-Q41L model and heterogenic hIPSC GRK5-Q41L model were characterised for pluripotency criteria and differentiated to hPSC-CMs for further phenotype and mechanism analysis. Phenotype analysis of contractility and cell survival response of GRK5-Q41L variant exhibited preserved contractility functions and lower cell death induced during chronic catecholamine stress. Analysis for nuclear translocation of GRK5-Q41L illustrated a noncanonical function, wherein the L41 variant prevented nuclear GRK5 accumulation relative to the Q41 variant. This phenotype was also associated with the higher BNP induction in HUES7-GRK5-L41 CMs, which suggested a compensation response during chronic catecholamine stress. However, the increase of nuclear GRK5 fraction was not observed in the heterogenic hIPSC GRK5-Q41L model, which highlighted the potential effects of genetic background on phenotypic study for SNPs. This observation suggested that affects of GRK5-Q41L SNP on noncanonical functions of GRK5 maybe not the main pathway accounting for the cardioprotection phenotype of the GRK5-L41 variant Further experiments studying the mechanism of GRK5-Q41L polymorphism on the isogenic model showed that GRK5 was responsible for the activation of extracellular signal-regulated kinases (ERK1/2) during isoprenaline (ISO) stimulation. The higher phosphorylated ERK1/2 activation in GRK5-L41 suggested an association with cardioprotection phenotypes. The GRK5-L41 variant was hypothesised to exert gain-of-function during catecholamine stress by (1) accelerating GRK5-mediated β-ARs phosphorylation and desensitisation to reduce adverse effects of chronic β-AR induction; and (2) promoting higher phosphorylated ERK1/2 activation via the β-arrestin dependent pathway. Overall, the hPSC-CM model harbouring GRK5-Q41L SNP recapitulated cardioprotection phenotype in contractility and cell survival response during chronic catecholamine stress induced by ISO exposure. Molecular analysis also explored the mechanism for the gain-of-function of GRK5-Q41L SNP. Despite limitations of technique and phenotype, this thesis shows that an hPSC-CM model carrying GRK5-Q41L SNP provides a relevant human in vitro platform for the study of phenotypes and mechanisms of specific SNP