The long-term goal of this research is to elucidate the mechanisms involved in the regulation, development, and function of muscarinic acetylcholine receptors in the embryonic chick heart. The chick embryo represents an attractive system for the study of cardiac mAChR both in vivo and in cell culture. Recent work from Dr. Nathanson's laboratory has demonstrated that treatment of chick heart cells with muscarinic agonists and transforming growth factor-beta regulates the expression of mRNAs encoding the two main mAChR subtypes expressed in chick heart, cm2 and cm4. The promoter for the cm2 receptor has also been isolated and shown to drive expression when transfected into cardiac cells. This proposal will determine the cellular and molecular mechanisms responsible for the regulation of mAChR expression by muscarinic agonists and TGF-beta. Muscarinic receptors activate a G protein regulated inwardly rectifying potassium (GIRK) channel. Nathanson's laboratory has found that treatment of chick embryos in ovo with muscarinic agonists causes a large decrease in the levels of mRNA encoding the inward-rectifying potassium channel subunits GIRK1 and GIRK4 in atria, while the level of GIRK1 mRNA in ventricle is not changed. This proposal will determine the mechanism responsible for the regulation of potassium channel expression by mAChR activation and the basis for the tissue-specific differences in regulation of GIRK expression by agonist. Transcription factors of the GATA-family have been shown to be involved in the regulation of expression of a number of cardiac genes. This laboratory has found that coexpression of GATA-6 in cells which do not normally express the m2 receptor with a luciferase reporter gene under the transcriptional control of the cm2 promoter causes a 100- to 600- fold increase in transcriptional activity. This proposal will determine factors in the regulation of m2 receptor expression in cardiac cells. This research will provide valuable new information on the basic mechanisms regulating the expression and function of mAChR in the heart. In addition, this research may aid in understanding the etiology of a variety of cardiac abnormalities.
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