Zebrafish modelling of long QT syndrome

Abstract

Long QT (LQT) syndrome is an inherited cardiac disorder affecting 1:1,000 to 1:5,000 people worldwide. It is a serious disease that affects mainly young people (early twenties to early forties), and it can cause uncontrolled arrhythmia, which can result in sudden death. The disorder is associated with mutations in any one of 12 genes, and many of these genes encode for ion channels that are vital for cardiac repolarisation, or encode for scaffolding proteins. Mouse modelling studies have been undertaken to determine the functional consequences of mutations; however, despite the mouse heart being anatomically similar to the human heart the underlying electrophysiology is very different. This difference makes it difficult to use mouse models as surrogates to understand, and ultimately treat, the human disease. The zebrafish is an emerging animal model in the biomedical research field. Recently, it has been shown that the cardiac electrophysiology of the zebrafish is similar to the human heart, and this freshwater vertebrate has been used to model several human cardiac disorders. It has also been used to test drugs for pro-arrhythmic effects. The research presented here principally concerned using zebrafish to model LQT syndrome. Two different approaches were used: to apply RNA interference (RNAi) to mediate targeted knockdown of gene expression to model LQT 2 syndrome; and to over-express a mutant cDNA to model LQT 7 syndrome. RNAi technology takes advantage of the endogenous microRNA (miRNA) pathway within eukaryotic cells to carry out targeted gene expression knockdown. As many of the mutations in LQT 2 syndrome causes haploinsufficiency, the use of miRNA provides a means to replicate this outcome as miRNA does not completely suppress gene expression. Pilot in vivo studies showed that miRNAs were effective in knocking down gene expression; however, targeting the zebrafish LQT 2 genes proved less effective. In the case of LQT 7, many mutations cause the expression of mutant proteins that interact in a dominant-negative fashion to hamper the function of unaffected LQT 7 proteins. In order to model this subtype of LQT syndrome, over-expression recombinants carrying the zebrafish LQT 7 gene transcript were engineered to carry defined mutations, and these were injected into zebrafish embryos. Some phenotypic characteristics exhibited by the zebrafish mutants were similar to those expressed by human LQT 7 patients. The research presented in this thesis show that the zebrafish contains many of the human LQT syndrome genes, and it is a species that shows limited, but still useful modelling outcomes, for some LQT syndrome subtypes. Importantly, the zebrafish appears suitable as an in vivo system to assess the impact of unclassified DNA variants identified in human LQT genes that might be suspected of pathological significance, but for which there is insufficient evidence.