Induction of specific mutations for dilated or hypertrophic cardiomyopathy in isogenic human induced pluripotent stem cells to model disease and to elucidate molecular and signaling pathways

• Applicant: Dr. Timon Seeger
• Subject Area: Cardiology, Angiology
• Term: from 2014 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 259402157

Cardiomyopathies are a major cause of heart disease in all age groups. Familial dilated cardiomyopathy (DCM) or hypertrophic cardiomyopathy (HCM) are leading to heart failure and sudden cardiac death and are caused by mutations in genes encoding for sarcomeric, cytoskeletal, mitochondrial and nuclear membrane proteins, as well as proteins involved in calcium metabolism. So far, most studies focused on the structural changes of the diseased cardiomyocytes, but the precise molecular mechanisms, via which the underlying mutations are leading to the devastating phenotypes, are not clarified yet. Within the complex interplay of cardiac signaling pathways, microRNAs (miRs) have important regulatory roles and were found to be specifically dysregulated in cardiomyopathies. Recently, induced pluripotent stem cells (iPSCs) have been generated from patients with cardiomyopathies and pathophysiological changes were reproduced in thereby evolved differentiated cardiomyocytes (iPSC-CMs), which offers novel opportunities to model disease. In this proposed research program, a major aim is to model cardiomyopathies by generating isogenic iPSCs and de novo introducing either DCM or HCM specific mutations by genome editing. Thereby, a strongly controlled disease model can be derived. Isogenic iPSC-CMs will be phenotypically analyzed regarding cellular size and structure, contraction force, electrophysiology, and reaction upon stress. Importantly, cellular signaling pathways known to be dysregulated in DCM and HCM, respectively, will be analyzed on RNA and protein level to test their relevance in the constructed disease model. Especially, expression of miR-145 (overexpressed in DCM and HCM), miR-208/b and miR-21/miR-129/miR-212 (upregulated in DCM), miR-1/miR-133 (controlling hypertrophy) and miR-204 as well as miR-221 (overexpression in HCM) will be analyzed. Beside these specific mechanisms, global expression (transcriptome, miRome; RNA sequencing) and broad spectrum protein analysis will be performed to discover hitherto unknown changes in DCM versus HCM associated expression and signaling profiles. Finally, the isogenic iPSC-CM edited to bear DCM versus HCM specific mutations and deduced expression/signaling profiles and phenotypic features will be employed as starting point for novel in-vitro therapeutic intervention strategies, aiming to inhibit/reverse the disease specific phenotype. In conclusion, the ultimate goal of this poposal is to generate cardiomyopathy specific isogenic iPSC-CMs to model disease for a more precise characterization of previously suggested and decipherment of novel molecular mechanisms leading to cardiac dysfunction, allowing the development of specific therapeutic strategies.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Dr. Joseph Wu, Ph.D.