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 t...
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| Format: | Thesis (University of Nottingham only) |
| Language: | English |
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2021
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| Online Access: | https://eprints.nottingham.ac.uk/65137/ |
| _version_ | 1848800191631589376 |
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| author | Vo, Nguyen T. N. |
| author_facet | Vo, Nguyen T. N. |
| author_sort | Vo, Nguyen T. N. |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | 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 SNPs |
| first_indexed | 2025-11-14T20:47:38Z |
| format | Thesis (University of Nottingham only) |
| id | nottingham-65137 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T20:47:38Z |
| publishDate | 2021 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-651372023-08-04T04:30:27Z https://eprints.nottingham.ac.uk/65137/ An investigation into the function and mechanism of a GRK5-L41 polymorphism using human pluripotent cell-derived cardiomyocytes Vo, Nguyen T. N. 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 SNPs 2021-08-04 Thesis (University of Nottingham only) NonPeerReviewed application/pdf en cc_by https://eprints.nottingham.ac.uk/65137/1/Nguyen%20Vo%20PhD%20Thesis%20An%20investigation%20into%20the%20function%20and%20mechanism%20of%20a%20GRK5-L41%20polymorphism%20using%20hPSC-CMs.pdf Vo, Nguyen T. N. (2021) An investigation into the function and mechanism of a GRK5-L41 polymorphism using human pluripotent cell-derived cardiomyocytes. PhD thesis, University of Nottingham. GRK5-L41 polymorphism; G proteins; Pluripotent Stem Cells; Heart cells; Cardiac model |
| spellingShingle | GRK5-L41 polymorphism; G proteins; Pluripotent Stem Cells; Heart cells; Cardiac model Vo, Nguyen T. N. An investigation into the function and mechanism of a GRK5-L41 polymorphism using human pluripotent cell-derived cardiomyocytes |
| title | An investigation into the function and mechanism of a GRK5-L41 polymorphism using human pluripotent cell-derived cardiomyocytes |
| title_full | An investigation into the function and mechanism of a GRK5-L41 polymorphism using human pluripotent cell-derived cardiomyocytes |
| title_fullStr | An investigation into the function and mechanism of a GRK5-L41 polymorphism using human pluripotent cell-derived cardiomyocytes |
| title_full_unstemmed | An investigation into the function and mechanism of a GRK5-L41 polymorphism using human pluripotent cell-derived cardiomyocytes |
| title_short | An investigation into the function and mechanism of a GRK5-L41 polymorphism using human pluripotent cell-derived cardiomyocytes |
| title_sort | investigation into the function and mechanism of a grk5-l41 polymorphism using human pluripotent cell-derived cardiomyocytes |
| topic | GRK5-L41 polymorphism; G proteins; Pluripotent Stem Cells; Heart cells; Cardiac model |
| url | https://eprints.nottingham.ac.uk/65137/ |