Quantitative measurements on electron micrographs of heart muscle can yield information useful for cellular physiologists and at present not obtainable in other ways. These methods are subject to preparative artifact, sampling problems, and problems inherent in the mathematical description of ultrastructure. Nevertheless they provide the best available data for membrane areas of the plasmalemma and its components, as well as for membrane areas of the sarcoplasmic reticulum and mitochondria. Morphometric methods can be used to study growth of membranes. Changes in the volumes of intracellular membrane-limited subcompartments can also be measured. Quantitative analysis of freeze-fractured membrane replicas can be carried out either by a statistical approach or by optical diffraction. In this way, physiological perturbations or developmental events leading to changes in membrane permeability can be studied for correlated changes in membrane structure.

SUMMARY1. Measurements combining the techniques of point counting and line integration were performed on light and electron micrographs of Purkinje fibres from the sheep's heart. The measurements were aimed at determining membrane areas of importance for the cellular electrophysiology of this tissue.2. The mean volume fractions of the cells occupied by various constituents were: myofibrils, 0-234; mitochondria, 0-103; and nuclei, 0-009. The mean volume fraction of the fibres occupied by the interspaces between the tightly packed cells was 0-0023.3. The mean fractions ofintercellular surface area occupied by junctional specializations were: nexus, 0-17; desmosome, 0-023; and fascia adherens, 0-014.4. The mean surface to volume ratio of the Purkinje cells and fibres was 0-46 4ull which is 11-5 times the value of the surface to volume ratio of a long right circular cylinder 100 ice in diameter.5. There are two reasons for the increment in the surface to volume ratio of the fibre (when compared to that of a long right circular cylinder 100 / t t in diameter): the multicellular composition of the fibres and the extensive folding of the surface of the cells. 6. After correction for the intercellular nexal area the surface to volume ratio of a long cylindrical fibre 100 ,u in diameter was 0-39 pIt, or about 10 times the value for a long right circular cylinder 100,u in diameter. The surface to volume ratio of the tissue interspaces in the same fibre was 170#-1.7. It was concluded that the total sarcolemmal area in this tissue is great enough so that the specific membrane capacitance could be about 1 ulF/cm2 and the specific membrane resistance 20,000 Q cm2.

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Electron micrographs of osmium-fixed left ventricles from 200-g female rats were analyzed by stereological techniques. By the use of equations developed by H. Sitte it was possible to determine volume fractions of organelles and absolute membrane areas per unit cell volume for cellular membrane systems implicated in excitation-contraction coupling. The fractions of cell volume were: mitochondria 0.34, myofibrils 0.481, Tsystem 0.012, total sarcotubules 0.035, other 0.13. The membrane areas per unit cell volume (&m2/Ism8) were: external sarcolemma 0.27, external sarcolemma + T-system 0.34, total sarcotubules 1.3. Diads made up 0.08 of sarcotubular volume and 0.12 of sarcotubular membrane area. 0.14 of the external sarcolemmal membrane area was involved in diadic complexes with underlying subsarcolemmal cisterns.The ultrastructures thought to be responsible for the physiological phenomena of excitation-contraction coupling and relaxation in heart muscle have been extensively described (1-4). Nevertheless, there is as yet no quantitative information about the fractions of cell volume occupied by these structures, or about the areas of the various membranous surfaces involved in the calcium movements associated with the physiological phenomena (5-7). Although such quantitative structural estimates have been available for some time for frog skeletal muscle (8,9), the complex geometry of mammalian heart muscle has so far prevented the gathering of comparably reliable data on mammalian heart muscle. We have recently overcome this difficulty by the application of stereological techniques to electron micrographs (10). In this paper we report the results of measurements on rat ventricular myocardium, with particular emphasis on the ultrastructures implicated in excitation-contraction coupling and relaxation.Ventricles from 200-g female Sprague-Dawley rats were fixed by perfusion with isotonic Krebs-Henseleit solution (pH 7.2) modified to contain 32 mM osmium tetroxide. The fixed left ventricular myocardial wall was prepared for electron microscopic examination as previously described (11) covering them with transparent sheets of thin plastic on which a square grid (1 cm/side) had been photographically imprinted. For measurements of the sarcoplasmic reticulum we used a finer grid (0.32 cm/side, line thickness 0.080 mm) and a final magnification of X30,000; the grid and print were illuminated by-placing them horizontally on an x-ray viewing box. After completion of the initial measurements at the lower magnification, the grid was rotated 19°, the measurement was repeated, and the results obtained at the two orientations were averaged. Areas including a transverse cell boundary or a nucleus were excluded. Additional measurements were made until doubling the area counted did not significantly alter either the mean or the dispersion of the data obtained and until it was clear that the data obtained conformed to a normal distribution. The fraction of cell volume occupied by a cellular com- respectively, the number of i...

We show by confocal immunofluorescence microscopy that the water channel protein aquaporin-1, not previously identified within cardiomyocytes, localizes at 20 and 37°C to rat cardiomyocyte sarcolemmal caveolar membrane and subsarcolemmal cytoplasm of primary atrial myocyte cultures, dissociated atrial and ventricular myocytes, and in situ cardiomyocytes of atrial and ventricular frozen sections. Confocal immunofluorescence microscopy shows that the normal in situ colocalization of the quasi-muscle-specific caveolar coating protein caveolin-3 with aquaporin-1 is reversibly disrupted by exposing in situ atrial or ventricular myocytes to physiological saline made hypertonic by adding 150 mM sucrose or 75 mM NaCl to isotonic physiological saline. This causes caveolae to close off from the interstitium and swell, while aquaporin-1 is internalized reversibly. At 4°C aquaporin-1 does not colocalize with caveolin-3. We suggest that 1) in vivo, under near-isotonic conditions, caveolae may alternate frequently between brief open and closed-off states; 2) aquaporin-1-caveolin-3 colocalization may be energy dependent; and 3) while closed off from the interstitium, each caveola transiently functions as an osmometer that experiences, monitors, and reacts to net water flow from or into the subcaveolar cytosol of the myocyte.