Transport across the nuclear envelope is vital in the processing of genetic information during cell differentiation. Deficiency in the nuclear localization of transcription factors has been related to congenital abnormalities including cardiac defects. A case in point is the lethal defect in heart development described recently in embryos lacking calreticulin, a multifunctional calcium-binding chaperone protein. However, the mechanisms responsible for calreticulin-mediated regulation of nuclear function and the contribution of calreticulin in cardiogenesis is poorly understood. To address this question, we established a stem cell- based model of calreticulin gene knockout (crt-/-) and obtained preliminary data suggesting a contribution of calreticulin in securing the structural and functional competence of nuclear pores.
In Aim #1, the requirement for calreticulin in nuclear translocation of cardiac transcription factors will be determined by laser confocal microscopy, and the microanatomy of nuclear pores will be evaluated by mapping the architecture and composition of individual nuclear pore complexes using atomic force microscopy and immunocytochemistry. Proteomic analysis will be performed to define the molecular basis of the calreticulin effect. To further define the mechanism underlying regulation of pore structure and function, in Aim #2, we will evaluate the contribution of calreticulin in determining the calcium filling state of the nuclear cisterna by fluorescence resonance energy transfer-based probing. In addition, the gene profile of wildtype versus crt-/- stem cell- derived cardiomyocytes will be established by genomic scans to map genes affected by calreticulin deficiency including genes involved in the regulation of the nuclear transport machinery, calcium dependent pathways and cellular metabolism.
In Aim #3, rescue of nuclear pore competence and restoration of cardiogenesis will be tested by transfection of calreticulin constructs that recapitulate specialized functions of this protein. This proposal will thus provide novel mechanistic insight into molecular events associated with nucleocytoplasmic communication critical for cardiac differentiation. As abnormal function of the nuclear envelope has been linked to a growing number of cardiac congenital defects, this proposal will establish a framework for targeted strategies aimed at supporting reparative cardiogenesis.
