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Functional characterisation of arrhythmia and cardiomyopathy-linked mutations at R1051 in the cardiac ryanodine receptor: effects at different organisational levels of calcium release

Harris, Tessa 2025. Functional characterisation of arrhythmia and cardiomyopathy-linked mutations at R1051 in the cardiac ryanodine receptor: effects at different organisational levels of calcium release. PhD Thesis, Cardiff University.
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Abstract

The human cardiac ryanodine receptor (hRyR2) is a calcium (Ca2+) ion channel found on the sarcoplasmic reticulum of cardiomyocytes. It plays a key role in excitation-contraction coupling, and dysfunction of hRyR2 due to mutation is associated with arrhythmias and cardiomyopathies. Clustering of hRyR2 via inter-channel interactions is proposed to regulate its function, preventing uncontrolled Ca2+ release. This study aimed to characterise three arrhythmia and cardiomyopathy-associated mutations – R1051P, R1051C, and R1051H – that are found in a domain thought to be involved in the inter-channel interaction, to determine whether they cause dysfunctional Ca2+ release from hRyR2 and if this is due to changes to the clustering interaction. The research comprised three main parts to assess hRyR2 function at the whole cell, population (i.e. cluster), and single channel level. Spontaneous whole cell Ca2+ release from human embryonic kidney cells expressing R1051-mutant hRyR2 was significantly different for each mutant compared to the Wild Type (WT) and showed slowed Ca2+ release kinetics and increased abnormalities that are indicative of dysfunction. All mutants also showed significantly reduced endoplasmic reticulum Ca2+ store loads, likely caused by Ca2+ leak; this is strongly associated with channel gain of function and arrhythmia. Droplet interface bilayers and total internal reflection fluorescence microscopy were used to assess Ca2+ release from populations of hRyR2. This revealed increased activity and increased cooperativity of Ca2+ release from R1051P populations, and R1051C / R1051H appeared to form larger clusters than the WT. Single channel analysis of R1051P gating compared to the WT revealed no differences at sub-activating and saturating Ca2+ concentrations. This indicates that dysfunction of R1051-mutants most likely arises at the population level possibly due to altered inter-channel clustering, which contributes to abnormal Ca2+ handling in whole cells. This highlights a potentially novel mechanism that may contribute to the development of arrhythmia and cardiomyopathy.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Schools > Music
Subjects: Q Science > Q Science (General)
Date of First Compliant Deposit: 8 October 2025
Last Modified: 08 Oct 2025 13:16
URI: https://orca.cardiff.ac.uk/id/eprint/181554

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