Developing cellular models for hypertrophic cardiomyopathy

22/05/2020

Hypertrophic cardiomyopathy (HCM) is the most common genetic disease of the heart. It is a complex disease characterized by left ventricular hypertrophy, its anatomical hallmark, abnormal diastolic function, increase in myocyte size with distorted nuclei, myocyte disarray and increased extracellular fibrosis. Several associated molecular mechanisms and a heterogeneous set of clinical manifestations had been reported, ranging from asymptomatic mutation carriers to severe heart failure or even sudden cardiac death as the first manifestation of the disease. Unmet needs in HCM include the need for improved diagnosis since standard genetic testing often fails to identify a causative mutation, as well the need for therapies precisely targeted at the molecular cause of the disease since current treatment options only aim to alleviate symptoms. To address these needs, good disease models that can help understand the pathophysiology of HCM and test new therapeutic approaches are required. Therefore, the main objective of this thesis was to establish a new experimental approach that allows the identification and characterization of novel genetic variants that contribute to HCM phenotype, investigate the molecular mechanisms underlying it and test possible therapeutic approaches. Firstly, the performance of available computational prediction tools was benchmarked and the algorithms that show the best performance in identification of missense and splice site HCM variants were identified; moreover, this analysis showed lack of prediction power for deep intronic variants of such bioinformatics approaches. Afterwards, and to generate experimental cellular models that could be used to study the impact of HCM-associated variants, the CRISPR/Cas9 gene-editing system was applied in human induced pluripotent stem cells (hiPSCs) to create an isogenic cell line bearing an HCM-associated pathogenic variant. Lastly, a reproducible optimized strategy for generating homogeneous populations of hiPSC-derived iv cardiomyocytes capable of recapitulating HCM-specific features at the single-cell level was established. The presented work sets the foundation for an improvement in HCM diagnosis, by identifying and determining the impact of HCM-causing mutations, and the development and testing of new HCM therapeutic approaches. Keywords: hypertrophic cardiomyopathy, human induced pluripotent stem cells, cardiac differentiation, in silico prediction tools, gene editing

Autores da comunidade :

• 

  M

  Marta de Jesus Ribeiro

  ist427021

PhD in Bioengineering

- Biotecnologia Industrial

- Inglês

Instituto Superior Técnico