Role of RAF1 in human cardiogenesis and congenital heart defects

项目总结

Project Summary The goal of this project is to discover fundamental RAF1-dependent mechanisms that regulate early steps of cardiogenesis to further delineate the molecular basis of congenital heart defects (CHDs) and cardiomyopathies. RAF1 (or CRAF) is a serine/threonine-specific protein kinase that is ubiquitously expressed and controls cell proliferation, apoptosis, cell differentiation, and oncogenic transformation. Newborns with RAF1 germline loss- of-function have been recently identified and present with severe CHDs. Moreover, infants with Noonan syndrome (NS) associated RAF1 mutations present with obstructive hypertrophic cardiomyopathy and a variety of CHDs. Currently, no specific treatment exists for NS children with CHDs or cardiomyopathy, therefore there is an urgent need to understand the molecular mechanisms underlying cardiac developmental defects to identify specific therapeutic strategies. Using hiPSCs and cardiac-directed differentiation as a developmental model, we discovered that RAF1 was required for human cardiogenesis by regulating cardiac mesoderm specification and showed that NS RAF1 mutations impacted this process. How RAF1 regulates early human cardiogenesis programs and how NS mutations, such as those found in the RAF1 gene, impair cardiac development remains elusive. Hence, the overall goal of this proposal is to discover signaling, genetic and epigenetic networks modulated by RAF1 and impacted by NS RAF1 mutations during early cardiogenesis. We seek to perform an unprecedented in-depth investigation into the signaling and epigenetic networks altered by loss of RAF1 or NS RAF1 mutations at early stages of human cardiac differentiation. Toward that goal, we have compiled a “toolbox” of hiPSC lines generated by genome editing (CRISPR-Cas9) to enable a comprehensive analysis of gain-and loss-of-function phenotypes, with a solid track record for such analyses. Our proposal will break ground beyond current knowledge by achieving the following aims: Aim 1: Discover the function of RAF1 in early human cardiogenesis. Aim 2: To interrogate the impact of Noonan syndrome RAF1 mutations on human cardiac development. Aim 3: Delineate the role of nuclear RAF1 in early human cardiogenesis. Successful completion of our proposal will illuminate the fundamental role of RAF1 in human cardiogenesis and the molecular mechanisms underlying CHDs and cardiomyopathy in NS RAF1 at an unprecedented resolution. It will also provide fundamental new knowledge regarding the transcriptional, epigenetic and protein networks controlling human cardiac development and will pave the way for follow up studies to design new therapies for NS children with heart defects. Finally, we anticipate that our study will open new avenues of investigation of other developmental defects observed in NS and will highlight hiPSCs as a powerful model system to decipher the molecular mechanisms underlying CHDs.