Parasympathetic stimulation exerts significant influence on heart rate and contractility through the release of the neurotransmitter acetylcholine, which then activates muscarinic receptors found on all cardiac myocytes. These effects are mediated in whole or in part by direct and indirect signaling mechanisms affecting cardiac ion channel activity. The indirect effects involve modulation of cAMP-dependent beta-adrenergic responses. In fact, muscarinic receptor activation inhibits as well as facilitates beta-adrenergic responses, suggesting that the net result is a balance between these inhibitory and stimulatory effects. Recent evidence suggests that in atrial preparations, both the inhibitory and stimulatory effects are mediated solely by the production of nitric oxide (NO). However, this signaling mechanism may not be as important in mediating muscarinic responses in adult ventricular myocytes. Our working hypothesis is that there is developmental regulation of the signaling pathway coupling muscarinic receptors to modulation of beta-adrenergic responses, which may lead to cell type specific signaling mechanisms in the adult heart. If this is the case, then what is responsible for muscarinic responses in adult ventricular myocytes? There is uncontested evidence that the inhibitory effects can be explained by direct G-dependent inhibition of adenylyl cyclase types V and/or VI (AC5 and AC6). However, we propose the novel idea that the stimulatory effects are due to direct G-dependent stimulation of AC4 and/or AC7. Therefore the specific aims of this proposal are to test the following four hypotheses: 1) NO does not contribute to either muscannic inhibitory or muscarinic stimulatory responses in adult ventricular myocytes; 2) muscarinic stimulatory responses in adult ventricular myocytes are due to direct G protein dependent activation of specific AC isoforms; 3) NO does contribute to muscarinic inhibitory and muscannic stimulatory signaling mechanisms in atrial myocytes; and 4) NO is also involved in muscarinic signaling mechanisms in neonatal ventricular myocytes. We will use control and genetically altered animal models to identify the signaling pathways involved in mediating muscarinic responses by recording CAMP regulated cardiac ion channel activity in conjunction with direct fluorescence measurements of cAMP activity in isolated cardiac myocytes.
