We propose to apply powerful ultrastructural techniques to in situ and isolated gap junctional membranes (GJ) from mammalian heart muscle in order to correlate changes in internal membrance structure and in GJ channel distribution with phsiological and developmental changes in GJ permeability. The structural and analytical methods to be applied to isolated GJ include low-irradiation electron microscopy of negatively stained GJ following by optical diffraction, image processing and reconstruction; X-ray diffraction; freeze fracture with rotary replication, quantitative analysis of particle size, spacing and distribution, and stereo-imaging by tilting with a goniometer stage; immunoelectron microscopy of thin-sectioned GJ using monoclonal antibodies against GJ protein determinants at the cytoplasmic face of the GJ, plus ferritin-labeled secondary antibody; and two-dimenstional electrophoresis with the Cleveland technique. Effects of incubation at different ionized Ca2+ concentrations and pH, differences between GJ isolated from atria and ventricles, and GJ differences between stages of development will be thus studied. Complementary freeze fracture and immunocytological studies on in situ GJ from atrial, ventricular, Purkinje fiber, and embryonic ventricular muscle are designed to correlate data on isolated GJ with that from relatively intact cardiac tissues. The physiological and biological issues addressed by this research are (a) change in membrane channel structure when GJ permeability changes; (b) channel dimensions, submit structure and subunit protein composition of the channel-containing units (connexons); (c) distribution, assembly, and disassembly of GJ channels and GJ precursors during cardiac development; and (d) whether GJ structure, composition, and (perhaps) permeability differ in atria, ventricles, and conduction cells, and between different stages of ventricular development. Since GJ permeability in cardiac tissues is a critical variable for both action potential conduction and the sealing off of injured cells, this research is relevant to disturbances of cardiac conduction and rhythmicity and to sealing off of heart muscle cells that are irreversibly injured by ischemia or other disease processes.
