MODULATION OF THE ENDOTHELIAL NITRIC-OXIDE SYNTHASE CAVEOLIN INTERACTION IN CARDIAC MYOCYTES - IMPLICATIONS FOR THE AUTONOMIC REGULATION OFHEART-RATE

The endothelial isoform of nitric oxide synthase (eNOS) is dually acyl ated and thereby targeted to signal transducing microdomains termed ca veolae. In endothelial cells, eNOS interacts with caveolin-1, which re presses eNOS enzyme activity. In cardiac myocytes, eNOS associates wit h the muscle-specific caveolin-3 isoform, but whether this interaction affects NO production and regulates myocyte function is unknown. We i solated neonatal cardiac myocytes from mutant mice with targeted disru ption of the eNOS gene and transfected them with wild-type (WT) eNOS o r myristoylation-deficient (myr(-)) eNOS mutant cDNA. In myocytes expr essing WT eNOS, the muscarinic cholinergic agonist carbachol completel y abrogated the spontaneous beating rate and induced a 4-fold elevatio n of the cGMP level. By contrast, in the myr(-) eNOS myocytes, carbach ol failed to exert its negative chronotropic effect and to increase cG MP levels. We then used a reversible permeabilization protocol to load intact neonatal rat myocytes with an oligopeptide corresponding to th e caveolin-3 scaffolding domain. This peptide completely and specifica lly inhibited the carbachol-induced negative chronotropic effect and t he accompanying cGMP elevation. Thus, our results suggest that acylate d eNOS may couple muscarinic receptor activation to heart rate control and indicate a key role for eNOS/caveolin interactions in the choline rgic modulation of cardiac myocyte function.