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The roles of HSP20 in cardiac hypertrophy

Sin, Yuan Yan (Angie) (2012) The roles of HSP20 in cardiac hypertrophy. PhD thesis, University of Glasgow.

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Abstract

Cardiac hypertrophy often develops to compensate for hemodynamic overload and is associated with heart failure. Recent studies have revealed that overexpression and PKA-mediated phosphorylation of heat shock protein 20 (HSP20) at Ser16 can attenuate hypertrophic growth of cardiomyocytes and trigger cardioprotective functions following sustained β-adrenergic stimulation (Fan et al., 2004, 2005, 2006). However, the molecular mechanism of HSP20 induced cardioprotection remains to be fully elucidated. In order to gain insight into the protective mode of action of HSP20, I attempted to (1) investigate the spatiotemporal control of PKA-mediated phosphorylation of HSP20, as well as (2) identifying novel protein binding partners for HSP20 utilising cutting edge ProtoArray technology. Initially, I set up an in vitro hypertrophy model using sustained isoprenaline (ISO)-stimulated neonatal rat cardiomyocytes. Cell size, protein synthesis and fetal gene expression were assessed as parameters of hypertrophic growth. In the first section of my studies, members of the cAMP-specific PDE4 family were shown to form signalling complexes with HSP20, and that the PKA-mediated phosphorylation of HSP20 could be modulated by PDE4. Based on peptide array data, a cell-permeable peptide ‘bs906’ was developed to inhibit the interaction of PDE4 with HSP20. Interestingly, the disruption of the PDE4-HSP20 complex was shown to induce PKA-mediated phosphorylation of HSP20 and trigger cardioprotection against the hypertrophic response measured in neonatal cardiomyocytes upon chronic β-adrenergic stimulation. In the second part of my studies, protein kinase D1 (PKD1) was identified as one interacting partner that robustly associated with HSP20. This interaction was confirmed by biochemical and immunocytochemical means. Using similar approaches to those used for the PDE4-HSP20 interaction, a cell-permeable peptide ‘HJL09’ was generated to promote disruption of the PKD1-HSP20 complex. Experimentation using the peptide concluded that the disruption of the PKD1-HSP20 complex reduced HSP20 phosphorylation and attenuated the hypertrophic response in cultured cardiomyocytes as shown by reduced increases in cell size, protein content and actin reorganisation. In undertaking this work, I also defined a novel PKD phosphorylation site (Ser16) on HSP20 that conforms to the PKD phosphorylation motif of RxxS (also a PKA site). My biochemical data suggested that PKD1 may regulate the cardioprotective function of HSP20 via phosphorylation at Ser16. In situ proximity ligation assay (PLA) further revealed a role of HSP20 as ‘molecular escort’ in targeting the nuclear translocation of PKD1. This function, in part, may be responsible for the induction of fetal gene reexpression as selective disruption of PKD1-HSP20 complex using ‘HJL09’ hindered the nuclear influx of the complex, thereby attenuating hypertrophic signalling. In summary, these studies describe some exciting findings which provide further insight into novel signalling mechanism of cardiac hypertrophy in neonatal rat cardiomyocytes. I have shown that PKA and PKD1 exhibiting opposite functions despite sharing the phosphorylation site on HSP20. In this regard, HSP20 functions as a molecular nexus for the opposing actions of the PKA and PKD1 signalling pathways in hypertrophy, suggesting that crosstalk may occur between anti-hypertrophic and pro-hypertrophic pathways. The identification and characterisation of these complexes should help to build a better understanding of the hypertrophic signalling pathway, and may provide novel therapeutic strategies for the treatment of cardiac hypertrophy.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: HSP20, hypertrophy, phosphodiesterase, cAMP-dependent PKA, PKD1, phosphorylation
Subjects: R Medicine > R Medicine (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
Supervisor's Name: Baillie, Dr. George
Date of Award: 2012
Depositing User: Dr Yuan Yan (Angie) Sin
Unique ID: glathesis:2012-3581
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 17 Oct 2012
Last Modified: 10 Dec 2012 14:08
URI: http://theses.gla.ac.uk/id/eprint/3581

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