During mammalian development, the heart is the first organ to develop;the product of exquisite spatial and temporal control over cardiogenic mesodermal cell fate specification. From heart looping to cardiomyocyte cell fate determination, multiple transcription factors play critical roles during the development of the heart. Genetic silencing of Nkx2.5 in mice, the mammalian homologue of D. melanogaster tinman, results in embryonic lethality between 9.0 and 10.0 days post coitus due to abnormal heart morphogenesis. Clinically, missense and nonsense mutation in Nkx2.5 are highly conserved across multiple generations of families with high incidences of congenital heart defects. In Nkx2.5-/- mice, the cardiac specific isoform of the DEAD-box RNA helicase Mov10l1 (Csm) was found to be absent. DEAD-box RNA helicases are involved in virtually all biological processes involving RNA;specifically the nuclear DEAD-box RNA helicases p68 and p72 have been shown to be required for the processing of microRNA primary transcripts. MicroRNAs are critical for normal developmental processes are disregulated under pathological conditions. We demonstrate that Csm physically interacts with argonaute-2, a member of the RNA induced silencing complex, at P-bodies which are known to be sites of mRNA degradation. In addition, morpholino knockdown of Csm in the Danio rerio model system completely abrogates the heart looping process inhibiting cellular proliferation, whereas alpha-MHC promoter FLAG- Csm transgenic mice exhibit a hyperplastic heart. Transgenic animals have a significantly reduced survival rate. FLAG-Csm was found to co-immunoprecipitate a subset of cardiac microRNAs suggesting that it is actively involved in microRNA regulatory pathway. The overall hypothesis is that Csm is critical to both the normal development and function of the heart by facilitating the maturation of mircroRNAs throughout development and into adulthood. To test our hypothesis, we will determine the biochemical function of Csm and its interacting partners. Finally we will determine the biological and physiological effects Csm overexpression, and in conjunction with our biochemical and molecular analyses, determine the functional significance of Csm in the regulation of cardiac gene expression and the maintenance and function of the normal myocardium.
Over the last 20 years, great advancements have been made in Molecular Medicine, offering the hope that genetic defects can be cured by genetically based treatments. The discovery of cardiac transcription factors that regulate heart formation during development and the recent discovery that microRNAs can regulate the heart's response to stress, such as a myocardial infarction, have advanced our understanding of the molecular signature that defines a healthy heart. Only with the understanding of this heart specific molecular signature can we begin to design novel genetic therapeutics to treat congenital heart defects. Additional research in the areas linking microRNAs, cardiac transcription factors, and the cardiac specific RNA helicase Csm, the subject of this grant application, to cardiovascular development and disease will further our understanding of the molecular signature of a healthy heart, and aid in the development of future therapeutics.
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