Gomes, John; (2022) Investigation of arrhythmogenesis in the desmoplakin knockout mouse: A model of arrhythmogenic cardiomyopathy. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Arrhythmogenic cardiomyopathy (ACM), in contrast with other cardiomyopathies, often presents with lethal ventricular arrhythmias with athletes affected more severely. It has the characteristic feature of fibrofatty replacement of the right ventricular myocardium, although left ventricular variants have been reported. It has been associated with desmosomal protein mutations – structural proteins involved in cell-cell adhesion at the intercalated disk between cardiomyocytes. Arrhythmias are often noted to occur in a ‘concealed phase’ with minimal or no evidence of structural change. There is a need for better understanding of the mechanisms promoting arrhythmogenesis in order to improve arrhythmic risk prediction. A cardiac restricted heterozygous desmoplakin (DSP) knockout mouse was developed using the Cre-lox system with the cardiac restricted αMHC promoter (αMHC-Cre DSP flox/+) as a model of ‘concealed phase’ ACM and was studied with ECG, electrophysiology study and histology. This model recapitulated the ventricular arrhythmias seen in patients with evidence of conduction delay at electrophysiology study. This was no evidence of fibrofatty replacement of the myocardium on histology. Immunohistochemistry, however, revealed connexin 43 (Cx43) mislocalization away from the intercalated disk and a reduction in mRNA expression. Cx43 is a protein that makes up gap junctions which are involved in allowing rapid electrical conduction in the heart. The sodium channel is also located at the intercalated disk, but no change in its distribution, mRNA expression or change in the sodium current was noted. This suggests that interactions between Cx43 and desmosomal proteins are a key driver of arrhythmogenesis in ACM. In order to assess the effect of exercise on the arrhythmic phenotype, the mice were allowed to exercise freely before electrophysiology study. One group had slow release β blocker pellets implanted prior to exercise. Exercise made the mice more prone to arrhythmia, consistent with human studies. β blockers significantly reduced the numbers of mice developing ventricular arrhythmia as well as reducing the conduction delay observed at electrophysiology study. Cx43 showed less mislocalization in the β blocker treated mice, suggesting a role in slowing disease progression. Using the CreER promoter, which knocks DSP out in the adult mouse, the effect of DSP loss in adulthood was investigated. Mice with a complete knockout of DSP in adulthood became rapidly unwell and died, with bradycardia the only notable arrhythmia. However, heterozygous CreER knockout mice did not develop arrhythmia. The αMHC promoter is maximally expressed in early postnatal life. This suggests that this period, when desmosomes and adherens junctions are forming the mixed cell-cell junctions called the area composita, is significant in forming functional gap junctions to allow normal conduction. This mechanism may be relevant to arrhythmogenesis in other inherited cardiomyopathies. HL-1 cells were used as a cellular cardiomyocyte model to express DSP mutations identified in our ACM patient cohort. These two mutations (R1113X and T586fsX594) were both nonsense mutations at the N terminus. Cx43 was also found to be mislocalised in this model and shows similarity between the heterozygous knockout murine model and a cellular m0del of disease causing mutations. Sodium channel localisation was variable and showed less membrane localisation with the R1113X mutation. This may account for differences in the arrhythmia burden amongst ACM patients and shows the complex nature of the interactions at the intercalated disk. Plakoglobin was found to be localised at the nucleus with mutant DSP. This shows it is a key binding partner for desmoplakin at the intercalated disk and may also promote arrhythmogenesis by alterations in nuclear signalling. In conclusion, this work has established the heterozygous DSP knockout mouse and HL-1 cells as useful models for investigating the mechanisms of arrhythmogenesis in ACM. The key mechanism is interaction of desmoplakin and Cx43 at the intercalated disk. Restriction on exercise and treatment with β blockers for ACM patients is supported by this model. Further investigation of the mechanisms of interaction of DSP with other desmosomal proteins, the sodium channel and Cx43 may allow better prediction of arrhythmic risk and targeted therapies for ACM patients.

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