Abstracts Biological Sciences

An integrated approach using patient-specific induced pluripotent stem cells and protein biochemistry to study Vici syndrome associated cardiomyopathy

by Jing Qi

Vici syndrome is a rare autosomal recessive inherited multisystem disorder caused by mutations in EPG5. More than 80% of the patients with Vici syndrome suffer from cardiomyopathy. In this study, the induced pluripotent stem cell (iPSC) system was applied to model the disease, investigate the cardiac phenotype and to elucidate the underlying molecular mechanism of autophagy in cardiomyocytes derived from iPSCs. HiPSCs from the patient with Vici syndrome (Vici-iPSCs) carrying a homologous intronic mutation of EPG5 (c.4952+1G>A) were generated. These Vici-iPSC lines were pluripotent and could be differentiated into functional cardiomyocytes. Analysis of sarcomeric structure of cardiomyocytes derived from Vici-iPSCs (Vici-iPSC-CMs) showed they possessed a larger cell surface area with a higher inhomogeneity of sarcomere length compared with the cardiomyocytes derived from the Ctr-iPSCs (Ctr-iPSC-CMs). Furthermore, analysis of autophagy-related marker LC3 and lysosome associated protein 1 (LAMP1) demonstrated numerous accumulation of autophagosomes, abnormal perinuclear localization of lysosomes as well as the fusion blockade of autophagosomes with lysosomes in Vici-iPSC-CMs. Further investigation of the regulatory pathway of autophagy showed that the level of AKT phosphorylation at two activating sites (serine 473 and threonine 308) was decreased in Vici-iPSC-CMs compared to Ctr-iPSC-CMs. In addition, to figure out if these phenotypes in Vici-iPSC-CMs are caused by the EPG5 mutation, the genomic editing tool CRISPR/Cas9 was used to rescue the mutation site in EPG5. After transduction of CRISPR/Cas9-GFP and single-stranded oligonucleotides, genotype analysis of the 150 surviving Vici-iPSC colonies illustrated that non-homologous end joining (NHEJ) happens to repair the DNA double-strand break in two iPSC colonies. However, the mutation site in EPG5 was not rectified in any Vici-iPSC colonies. The data of this study illustrate that patient-specific iPSCs can be used to model Vici syndrome associated cardiomyopathy and provide a good platform to understand the function of autophagy in cardiomyocytes. Advisors/Committee Members: Guan-Schmidt, Kaomei (advisor), Guan-Schmidt, Kaomei (referee), Katschinski, Dörthe (referee).