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

Thomas, Nia Lowri 2005. Molecular mechanisms underlying cardiac ryanodine receptor dysfunction in sudden cardiac death. PhD Thesis, Cardiff University.

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Ca<sup>2+</sup> 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 Ca<sup>2+</sup> that may result in sudden death. GFP-tagged RyR2 mutants (R<sup>176</sup>Q/T<sup>2504</sup>M, L<sup>433</sup>P and N<sup>2386</sup>I) were generated and expressed in a human embryonic kidney (HEK) cell model, enabling profiling of the amplitude and temporal characteristics of caffeine-evoked Ca<sup>2+ </sup>release through homotetrameric channels using confocal microscopy. Mutants were functionally heterogeneous and demonstrated profound differences in Ca<sup>2+</sup> release when compared with WT channels, including the novel observation that one of the mutants (L<sup>433</sup>P) exhibited reduced sensitivity to caffeine activation. The molecular basis of this heterogeneity was investigated by determining the sensitivity of the mutant channels to cytoplasmic Ca<sup>2+</sup>. This was achieved by evaluation of caffeine-induced Ca<sup>2+</sup> release from WT or mutants channels in streptolysin-O permeabilised HEK cells, where the cytoplasmic Ca<sup>2+</sup> concentration was clamped. Although resting ER Ca<sup>2+ </sup>store and cytoplasmic Ca<sup>2+</sup> levels were comparable in all cells, RyR2 mutants were characterised by a profound loss of Ca<sup>2+</sup>-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 N<sup>2386</sup>I 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 Ca<sup>2+</sup> 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: 04 Jun 2017 05:50

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