Saturday, May 4, 2024
5/4/2024
Project Summary Hypoplastic left heart syndrome (HLHS) is a fatal form of congenital heart disease characterized by the underdevelopment of left-sided structures, including the left ventricle (LV), mitral and aortic valves, and ascending aorta. These three structural deficiencies result in diminished circulation that invariably leads to morbidity without surgical intervention. It is currently thought that HLHS is an oligogenic disease of co- occurring structural defects caused by different genetic lesions. In some cases, however, one structural defect might be primary and the others secondary. As HLHS is clinically heterogeneous, there are likely multiple paths to disease pathogenesis caused by diverse, and often unknown, genetic etiologies. To begin studying the affected genes in the context of heart development, I mined published databases for de novo and rare inherited variants in HLHS probands for follow-up in zebrafish. I initially selected RBFOX2, a highly conserved RNA binding protein (RBP), based on its strong statistical link to HLHS and lack of study in cardiac development. I published that rbfox2 mutant zebrafish display all three HLHS-like structural heart defects that arise secondary to compromised pump function (Huang et al. Nat Commun 2022). However, unlike patients that are heterozygous for RBFOX2, heterozygous zebrafish and mice are healthy. Therefore, no appropriate model exists to investigate how halving the dose of RBFOX2 affects CM biology. Moreover, many variants statistically linked to HLHS remain unvalidated or unstudied in whole animal models. To address these knowledge gaps, I engineered and characterized RBFOX2 het and null human (h) iPSC-CMs. Both genetic cohorts show dose-dependent reductions in cell size, cell adhesion, calcium handling, and oxygen consumption rates. Myofibril alignment is also perturbed. Based on this phenotypic characterization and integration of several sequencing datasets, I propose to test the hypothesis that RBFOX2 directly regulates CM cell adhesion and growth by controlling transcript stability and splicing of an ECM-cytoskeletal-myofibril gene network in Aim 1. To identify additional HLHS-linked variants that cause cardiac phenotypes in zebrafish, I conducted a pilot CRISPR screen and recovered an ortholog of FOXC2, which encodes a transcription factor expressed in the endocardium and myocardium. Preliminary phenotyping revealed HLHS-like cardiac phenotypes in foxc2 global knock-outs that also fail to activate a Notch reporter in the endocardium. In Aim 2, I propose to test the hypothesis that endocardial-specific FOXC2-deficiency leads to primary valve and secondary myocardial defects caused by defective Notch signaling. Overall, I anticipate learning that both myocardial- and endocardial-intrinsic defects recapitulate specific aspects of HLHS. More broadly, implicating human variants as causal for HLHS pathogenesis and deciphering their mechanism of action in whole animal and human iPSC models will ultimately lead to better predictors of heart failure following surgical intervention and new therapeutic inroads for patient-specific clinical management of the disease.
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