| Cardiovascular disease is the leading cause of mortality in the United States and congenital heart defects are the most common form of lethal birth defect. In spite of this, the molecular etiology of these problems remains poorly defined. The goal of this work is to advance our understanding of the fundamental cellular and molecular mechanisms that guide cardiovascular development, structure, and function. Toward this goal, I have focused on characterizing the role of the transcription factor Serum Response Factor (SRF), in regulating the genetic program underlying cardiac differentiation. SRF is enriched in muscle tissue, and previous studies have implicated SRF as important for mesoderm differentiation and myogenic development.;In this present study, I adopted a loss-of-function strategy to investigate the role of SRF in regulating cardiomyocyte-specific gene expression. Using cultured ventricular myocytes containing floxed-SRF alleles, the SRF gene was specifically excised by infection with Adeno-viral CRE-recombinase. The resulting SRF-null cardiomyocytes have dramatic defects in contractile apparatus structure, including Z-disc and stress fiber formation. Consistent with this phenotype, gene array and RT-PCR analyses reveal the attenuation of sarcomeric and cytoskeletal genes in addition to other cardiac transcription factors. Examination of these genes by chromatin immunoprecipitation and informatics analysis reveals that at least 23 of these proteins are likely to be directly regulated by SRF. Interestingly, these experiments also support the hypothesis that SRF is required for the induction of genes associated with the progression of pathologic cellular cardiac hypertrophy.;To characterize the role of SRF in cardiac developmental morphology and gene expression, I also employed a tissue-specific knockout approach. Selective excision of SRF in the embryonic mouse heart is necessary because the complete knockout is embryonic lethal prior to the onset of heart development. Excision mediated by MLC2v-CRE yields variable phenotypes resulting from the mosaic deletion of SRF. In contrast, SM22-CRE mediates excision of SRF in the cardiovascular system sufficient to cause embryonic lethality at or before E11.5. By E11.0 in development these mutant embryos reveal growth retardation, pericardial effusion, and morphological defects in trabeculation resulting from the absence of SRF in cardiovascular tissue during development.;Together, these results suggest that SRF regulates cardiomyocyte function by virtue of its ability to control expression of genes involved in multiple levels of sarcomeric structure and function. Further, this data suggests a new paradigm for SRF as a central regulator of a complex hierarchical network of transcription factors in cardiac myocytes. This work will contribute toward the development of novel diagnostic and therapeutic options for cardiovascular disease. |
