Kardiomyozyten aus patientenspezifischen induzierbaren pluripotenten Stammzellen als Modellsystem zur Analyse des LQT2-Syndroms
Final Report Abstract
Patient-specific induced pluripotent stem cells (iPSCs) assist research on genetic cardiac maladies if the disease phenotype is recapitulated in vitro. However, genetic background variations may confound disease traits, especially for disorders with incomplete penetrance, such as long-QT syndromes (LQTS). In this project we studied the specific effects of the LQTS2-associated N996I KCNH2 mutation in human iPSC-derived cardiomyocytes (CMs). To eliminate the confounding elements that could result from individual genetic background variability, we studied this mutation under two genetically-consistent conditions by 1) correcting the N996I mutation in the LQT2-hiPSCs derived from a patient, using gene targeting; 2) introducing the same N996I mutation into an NKX2.5eGFP/w hESC reporter line, that allows the cardiac cells to be selected from a mixed differentiated population. CMs differentiated from these two pairs of isogenic human pluripotent stem cells differed only in the mutation, allowing any phenotype identified by electrophysiology to be attributed entirely to the mutation without additional confounding genetic factors. Correction of the N996I mutation normalized IKr densities and reversed the prolongation of the action potential duration (APD) in patient iPSC-derived cardiomyocytes, whereas introduction of the same point mutation reduced IKr and prolonged APD in hESCs. Most importantly, both mutated iPSC- and hESC-derived cardiomyocytes displayed a ~30-40% reduction in IKr density compared to their wild-type counterparts, while kinetic properties of the current were not altered. Moreover, further characterization of N996I-HERG pathogenesis by immunocytochemistry and western blot analysis demonstrated that a trafficking defect is the mechanism responsible for IKr reduction in both human pluripotent cell based systems. Our results demonstrated that the N996I KCNH2 mutation is the primary cause of the LQT2 phenotype. Precise genetic modification of pluripotent stem cells provided a physiologically and functionally relevant human cellular context to reveal the pathogenic mechanism underlying this specific disease phenotype.
Publications
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Isogenic Human Pluripotent Stem Cell Pairs reveal the Role of a KCNH2 Mutation in Long-QT Syndrome. The EMBO Journal, Vol. 32. 2013, Issue 24, pp. 3161-3175.
Andrea Welling, Alessandra Moretti, et al.
