CARDIOMYOCYTE CHIRALITY DEFECTS IN CONGENITAL HEART DISEASE

Topic Area: Congenital Heart DiseaseArea of Interest: Research to improve the understanding of the causes of congenital heart defects. Congenital heart disease (CHD) is a structural malformation of the heart at birth that usually results in some form of aberrant cardiopulmonary physiology. It is the most common birth defect, occurring in 1% of all newborns. Advances in surgical technique and medical therapy have resulted in a large and quickly expanding population of patients with CHD surviving into adulthood, increasing around 5% yearly. Underlying most forms of CHD disease is a developmental error of the normal heart during embryogenesis, varying significantly in impact and severity. Early failure to pattern right and left correctly results in a number of defects termed laterality diseases. A classic example of this is heterotaxy, in which complex cardiovascular malformations are accompanied usually by aberrations in visceral organ situs. Linkage analysis in an inherited X-linked form of heterotaxy has implicated loss of function mutations in the transcription factor ZIC3. Loss of Zic3 in mice and other organisms has recapitulated the complex cardiovascular malformations; however, ZIC3's function and role in generating left-right asymmetry remains unclear, and studies in mammals have been hampered by embryonic lethality.The goal of this proposal is to generate a human cardiomyocyte model of a CHD that recapitulates the early error in left-right patterning resulting laterality defects and to validate a functional assay to measure left-right patterning at the cellular level. In the first aim, the X-linked form of heterotaxy will be modeled by targeting ZIC3 expression in iPSCs using CRISPR interference. In this approach, an endonuclease-dead mutant of Cas9 will be expressed under an inducible tetracycline responsive element and will be targeted to bind the transcriptional region upstream of the start site of ZIC3, sterically inhibiting its transcription. In the second aim, a phenotype driven approach will be undertaken and iPS cells will be derived directly from heterotaxy patients. Gene expression and electromechanical properties of these cells will be characterized. Importantly, here we will establish a functional output of cellular chirality in iPSC-CM using specialized ring shaped micropatterned culture system. This system has been shown to induce left-right polarization of numerous cell types and will be used to follow iPS cells in real time as they differentiate into cardiomyocytes. From this, we will be able to measure left versus rightward bias in iPS cells that have lost ZIC3 as well as in iPS cells that were derived from a laterality defect disease. We expect these cells to exhibit aberrant polarization compared to normal cells and to be able to capture that phenotype using our system as described.This is a highly innovative project on three fronts. First, this would be the first described use of iPS cells to recapitulate a chirality defect at the cellular level that is central to CHD. Second, the use of an inducible CRISPRi will allow for the suppression of genes with temporal control enabling stage-specific interrogation of gene function through a developmental process. It would also enable experimental systems to study of genes in human cells whose loss of function is lethal in development of other mammals. Third, the characterization of the chirality of iPS cells as they differentiate into cardiomyocytes and the cardiomyocytes themselves is wholly novel and would provide an incredibly meaningful functional output for the interrogation of genes and pathways responsible for correct patterning in development. These studies would provide the underpinning for future studies, including (1) genetic and mechanistic interrogation of ZIC3 and heterotaxy in these patterning defects; (2) investigation of the electromechanical consequence of chirality defects on engineered heart tissue; (3) exploring complex genotypes that lead to laterality disease in humans; and (4) investigating the causation of laterality defects by exposures such as medication and drugs by using an iPSCCM model of left right polarization.