The long range objective of these studies is to elucidate the mechanisms of regulation of gene expression in cardiac tissue which determine the level of responsiveness of the heart to autonomic stimulation. We will test the hypothesis that innervation of the embryonic chick heart results in the coordinate regulation of the expression of members of several families of genes including isoforms of muscarinic receptors, G-proteins, and myosin heavy chains. At 3 1/2 - 5 days in ovo, many biochemical and physiologic processes characteristic of the atrium are not present. We have demonstrated that the development of these processes in ovo is associated with a 6-8 fold increase in mRNA coding for alphai2. Co-culture of heart cells 3 1/2 days in ovo with ciliary ganglia, with medium conditioned by growth of heart cells and ciliary ganglia or with TGF-beta appears to induce the development of cholinergic responsiveness and an increase in alphao and alphai. Using this model system, we will test the hypotheses that during co-culture of embryonic chick heart cells and ciliary ganglia, muscarinic stimulation becomes coupled to inhibition of adenylate cyclase activity and to stimulation of an atrial-specific pertussis toxin sensitive diacylglycerol formation, and that these changes take place in parallel with the appearance of mRNAs coding for isoforms of G-proteins and muscarinic receptors either previously not expressed or expressed at low levels. We will test the hypothesis that TGF-beta is the soluble factor in conditioned medium responsible for the development of a muscarinic response and that a unique TGF-beta family member is induced during co-culture. To determine whether expression of G-protein alpha-subunits and/or muscarinic receptor subtypes which appear during co-culture with ciliary ganglia regulates the development of a muscarinic response, heart cells 3 1/2 days in ovo will be co-transfected with genes coding for these receptors and G- proteins and the effect on chronotropic response to muscarinic stimulation and an acetylcholine dependent K+ current determined. Genomic clones of these G-protein alpha-subunits and receptors will be used to determine the transcription start site an 5' upstream response elements. Deletions of the 5' upstream regulatory region ligated to a luciferase reporter gene transfected into heart cells 3 1/2 days in ovo will be used to determine whether responsiveness to condition medium and TGF-beta depend on common response elements. These studies should help our understanding of the role of innervation in the development and regulation of autonomic responsiveness and may aid in our understanding of the genesis of arrhythmias and control of contractile state.
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