Application) In the heart and its constituent muscle cells, stretch is a crucial physiological stimulus and regulator for both normal muscle contraction and the secretion of atrial natriuretic peptide (ANP) hormone and angiotensin II. Stretch is also a major determinant in the development of cardiac hypertrophy, cardiac arrhythmias, and heart failure, three serious abnormalities which occur frequently in many common heart diseases. While stretch and its normal and abnormal consequences can be measured, the structures which act as sensors and transducers for stretch in normal heart muscle cells have not been identified or characterized. Recent research on noncardiac tissues suggests that plasma membrane-associated vesicles called caveolae function as novel signal transduction systems for multiple stimuli including stretch. The long-term goal of this project is to characterize the structural, functional and biochemical properties of cardiac myocyte caveolae that enable caveolae to act as combined stretch sensors and stretch transducers in intact heart muscle, and to relate these properties to the structure, protein composition, and phosphorylation of caveolae and caveolae-associated proteins isolated from primary cultures of atrial myocytes from adult rats.
The specific aims are (1) to test in intact atria and cultured atrial myocytes the hypothesis that atrial myocyte caveolae are implicated in variations in the stretch-dependence of ANP secretion and that such variations correlate with (a) presence of amount of atrial peptide in caveolae, and (b) with changes in phosphorylation of the caveolar coat protein caveolin, and/or other caveolae-associated proteins including cytoskeletal proteins that interact with caveolae; (2) to use experimental techniques that cause caveolae to be either open or closed to the interstitial space, or. alternatively, open or closed to the cytosol, or that promote either the insertion or withdrawal of caveolar necks into or out of the plasmalemmal lipid bilayer, in order to test hypotheses about the regulation, involvement of cytoskeletal proteins, and physiological determinants of these events; (3) (a) to identify, isolate, and localize in situ (by a combination of biochemical, immunofluorescence, and immunoelectron microscopic techniques) the glycophosphatidylinositol-linked proteins and other proteins inside cardiac myocyte caveolae, as well as proteins associated with the cytosolic surface of these caveolae; and (b) to test whether stretch, stimulation of specific plasma membrane receptors, stimulation or inhibition of relevant second messenger pathways, or changes in external or cytosolic Ca2+ concentration release intracaveolar proteins from cardiac myocyte caveolae.
