Using mouse and cellular models to investigate the ACTN2 M228T variant in the heart and its link to cardiac disease

Wadmore, Kirsty (2022). Using mouse and cellular models to investigate the ACTN2 M228T variant in the heart and its link to cardiac disease. University of Birmingham. M.Sc.

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Abstract

The Z-disc acts as a protein-rich structure to tether thin filament in the contractile units also known as sarcomeres, of striated muscle cells. Numerous proteins interact in the Z-disc and are integral for maintaining the architecture of the sarcomere, while facilitating force transduction and intracellular signalling in both cardiac and skeletal muscle. Pathogenic variants in Z-disc proteins can cause diseases, e.g. cardiomyopathies. These are diseases of the heart muscle, affecting its structure and function which ultimately impede the hearts ability to pump blood around the body. They can also be associated with a high risk of sudden cardiac death. This project focused on the Z-disc sarcomeric α-actinin 2 (ACTN2) and its link to cardiomyopathies. Pathogenic variants in the gene coding for ACTN2 have been identified and linked to cardiomyopathies, although there is limited understanding about the mechanisms involved. Actn2 M228T mouse and cellular models were used to test the impact of this pathogenic variant on cardiac hypertrophy and to explore underlying disease mechanisms. Morphometrics of wild type (WT) and heterozygous (Het) adult mice were used to test for hypertrophic differences between mice of different ages and sexes. Western blots were performed on adult mouse hearts to investigate protein expression of hypertrophic markers. While increased protein expression of hypertrophic markers was observed suggesting molecular changes occurred, this was not translated into pathological signs of hypertrophy such as changes in heart weight. The homozygous (Hom) variant resulted in embryonic lethality, however no developmental delay was detected by Theiler staging at embryonic day 15.5 embryos. Both gene and protein expression were explored in hearts from homozygous embryos. An increase in Actn2 transcript expression saw a down-regulation of the corresponding Actn2 protein, suggesting degradation of the aberrant protein. Increased expression of Actn3 was not translated at the protein level and suggests Actn3 cannot compensate for reduction of Actn2. Increased expression was seen for genes involved in the foetal gene programme and supports enhancement of this programme in the presence of this genetic variant. Reduced colocalisation of Actn2 and titin seen in homozygous embryos further suggests disorganisation of sarcomeres, a feature seen in some instances of hypertrophy. Preliminary results in an induced pluripotent stem cell (iPSC) line homozygous for the variant, further suggest less mature sarcomeres in the homozygous ACTN2 M228T variant compared to the KOLFC2 parental WT. Future work should aim to phenotype this line further. Overall, these data support the pathogenic role of ACTN2 M228T in the development of cardiomyopathy and cardiac disease.

Type of Work:

Thesis (Masters by Research > M.Sc.)

Award Type:

Masters by Research > M.Sc.

Supervisor(s):