Molecular mechanisms underlying cardiac ryanodine receptor dysfunction in sudden cardiac death.

Thomas, Nia Lowri ORCID: https://orcid.org/0000-0001-8822-8576 2005. Molecular mechanisms underlying cardiac ryanodine receptor dysfunction in sudden cardiac death. PhD Thesis, Cardiff University.

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Abstract

Ca²⁺ release via the cardiac ryanodine receptor (RyR2) is a fundamental event in excitation-contraction coupling. Point mutations in the gene encoding RyR2 are associated with arrhythmogenic right ventricular dysplasia type 2 (ARVD2), a disease likely characterised by abnormal release of Ca²⁺ that may result in sudden death. GFP-tagged RyR2 mutants (R¹⁷⁶Q/T²⁵⁰⁴M, L⁴³³P and N²³⁸⁶I) were generated and expressed in a human embryonic kidney (HEK) cell model, enabling profiling of the amplitude and temporal characteristics of caffeine-evoked Ca²⁺release through homotetrameric channels using confocal microscopy. Mutants were functionally heterogeneous and demonstrated profound differences in Ca²⁺ release when compared with WT channels, including the novel observation that one of the mutants (L⁴³³P) exhibited reduced sensitivity to caffeine activation. The molecular basis of this heterogeneity was investigated by determining the sensitivity of the mutant channels to cytoplasmic Ca²⁺. This was achieved by evaluation of caffeine-induced Ca²⁺ release from WT or mutants channels in streptolysin-O permeabilised HEK cells, where the cytoplasmic Ca²⁺ concentration was clamped. Although resting ER Ca²⁺store and cytoplasmic Ca²⁺ levels were comparable in all cells, RyR2 mutants were characterised by a profound loss of Ca²⁺-dependent inactivation. We also investigated whether these mutations disrupted the interaction between RyR2 and accessory proteins involved in normal channel function. cDNA encoding mutation susceptible regions were constructed and screened against a human cardiac cDNA library using a yeast two hybrid system. The N²³⁸⁶I mutation abolished association of the RyR2 domain with two cardiac proteins, which robustly occurred with the corresponding WT domain. These findings demonstrate that ARVD2-linked RyR2 mutations critically affect channel activation and suggest that differential sensitivity to cytoplasmic Ca²⁺ may be a causative mechanism in the pathogenesis of this disease.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Medicine
Subjects:	R Medicine > R Medicine (General)
Date of First Compliant Deposit:	30 March 2016
Last Modified:	05 Jul 2023 12:19
URI:	https://orca.cardiff.ac.uk/id/eprint/54084

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