Investigating the effects of variant calmodulin on RyR2 in generating arrhythmogenic Ca2+ release

Gendra, Aisha 2025. Investigating the effects of variant calmodulin on RyR2 in generating arrhythmogenic Ca2+ release. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

The human cardiac ryanodine receptor (hRyR2) is a Ca2+ channel located on the sarcoplasmic reticulum of cardiomyocytes. In regulating Ca2+ release, it maintains both electrical and contractile function, and in these tasks its normal function is modulated by the Ca2+-binding accessory protein calmodulin (CaM). Several CaM mutations have been linked to cardiac arrhythmias, such as Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), but their arrhythmogenic mechanisms are not fully resolved. This research aimed to investigate the effects of two arrhythmia-linked CaM mutations (D132E and Q136P) on RyR2 function (either directly, or by virtue of CaM’s regulation of its associated kinase: CaMKII) and how this might lead to aberrant Ca2+ signalling. RyR2 and CaM were recombinantly co-expressed in HEK293 cells and live-cell confocal Ca2+ imaging was used to assess recursive Ca2+ release dynamics. The effect of wild type (WT) CaM co-expression on RyR2 is inhibitory - decreasing the duration of Ca2+ release events. Co-expression of D132E and Q136P CaM was shown to diminish this inhibitory effect by increasing the duration of Ca2+ release transients. Additionally, D132E CaM significantly reduced the endoplasmic reticulum Ca2+ store load in non-oscillating HEK293 cells compared to WT CaM. It is, therefore, likely that D132E variant CaM is causing diastolic Ca2+ leakage from the channel which results in smaller Ca2+ stores. A reduction in Ca2+ sparks frequency was also observed in permeabilised mouse ventricular myocytes with both D132E and Q136P CaM variants compared to WT CaM. Additionally, Q136P CaM was found to impair CaMKII signalling, showing reduced CaMKII autophosphorylation and consequent decreased phosphorylation of RyR2 at the S2814 CaMKII phosphorylation site. Despite D132E and Q136P CaM variants showing similar RyR2-mediated Ca2+ release profiles at the cellular and spark level, these findings suggest that D132E and Q136P CaM exert their dysfunction in different ways. D132E seems to exert a greater Ca2+ leak under diastolic conditions, while Q136P CaM disrupts CaMKII-dependent RyR2 regulation, suggesting that calmodulinopathy in CPVT is likely mechanistically complex.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Pharmacy
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	26 February 2026
Last Modified:	27 Feb 2026 13:10
URI:	https://orca.cardiff.ac.uk/id/eprint/185329

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