Elucidating ryanodine receptor domain interactions in sudden cardiac death: towards the development of novel therapeutic strategies

Jundi, Hala 2009. Elucidating ryanodine receptor domain interactions in sudden cardiac death: towards the development of novel therapeutic strategies. PhD Thesis, Cardiff University.

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Abstract

Interdomain Interactions within the complex three-dimensional architecture of the cardiac ryanodine receptor (RyR2) are pivotal in channel regulation. Acquired or genetic abnormalities that perturb these stabilising intra-molecular interactions are pathogenic. This laboratory identified the interacting- or l-Domain of human RyR2 that mediated interaction between cytoplasmic and transmembrane (TM) assemblies. To further elucidate the precise roles of functional motifs within the l-Domain, three contiguous fragments spanning RyR2 amino acid residues 3722-4610 were synthesised using a cell-free system. One fragment termed IDB (amino acid residues 4353-4499) profoundly modulated cellular Ca2+ cycling and resulted in the remarkable normalisation of intercellular synchrony following its microinjection into ouabain-treated cardiomyocyte monolayers. These phenomena were linked to IDB- mediated stabilisation of RyR2 and were fully corroborated using IDB purified from a bacterial expression system. Bioinformatic analysis revealed striking structural homology between sub-fragments of the RyR2 l-Domain and l-Domain-like regions of inositol 1,4,5- trisphosphate receptors (IP3R). Recombinant expression of l-domain sub-fragments in RyR- null human embryonic kidney (HEK) cells remodelled carbachol-evoked Ca2+-responses and suppressed homeostatic Ca2+ signalling events indicating that IDB also modulated IP3R signalling mechanisms. In both HL-1 and HEK cells, IDB-dependent Ca2+ modulation extended to surrounding cells that were not microinjected with recombinant protein. This so- called 'bystander effect' was mediated by the transfer of signalling molecules via direct cell- to-cell coupling (gap junctions) and also by the extracellular transmission of diffusible effectors. This thesis supports the concept that RyR2 stabilisation rescues pathogenic Ca2+ dysregulation and suggests that there is substantial merit in developing further epitope-targeting strategies for the therapeutic normalisation of Ca2+ cycling in cardiac disease.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Medicine
Subjects:	R Medicine > R Medicine (General)
ISBN:	9781303222702
Date of First Compliant Deposit:	30 March 2016
Last Modified:	19 Oct 2019 02:38
URI:	https://orca.cardiff.ac.uk/id/eprint/55106

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