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Abstract.-The rate constants for the turnover of cross-bridges during frog muscle contraction were determined from an analysis of the motion that follows step decreases in load. For a given projection from the myosin filament, there appears to be a range of about 100 X along the length of the filament over which the projection can attach to the actin filament and form a cross-bridge. The site of attachment is then displaced by a distance of this same order before the link is broken. The values of the rate constants also imply that a cross-bridge is formed each time an actin site comes within range of a myosin projection, so that the turnover of cross-bridges for a given contraction distance is independent of the speed of the motion.There is considerable evidence that the contractile force of striated muscle fibers is developed by cross-bridges that form between the interdigitating myofilaments when the cell is activated. 1 The cross-bridges, which appear to consist of projections from the myosin-containing filaments that have attached to sites on the actin-containing filaments, break and reform when the myofilaments slide past each other and the cell shortens. This type of cyclic contraction mechanism was analyzed by A. F. Huxley2 in terms of (1) rate constants f and g which characterize the making and breaking of cross-bridges, respectively, and (2) the force function for a cross-bridge, k; it was shown that the steady forcevelocity and force-energy relations for frog muscle could be closely fitted when these functions were properly chosen and energy output was associated with cross-bridge turnover.Additional constraints are imposed on the model parameters if non-steady as well as steady motions are accounted for. In the present study we have determined values of f, g, and k that produce isotonic velocity transients very much like those seen in living muscle fibers after step changes in load from Po to P < Po. 4 These functions provide quantitative information about the force-generating process in muscle at the molecular level and thereby place real limits on specific models of the contraction mechanism.' Calculation technique.-The influence of f, g, and k on the response of the model to step changes in load was found by simulating the operation of the model, with various values of the parameters, on a digital computer.' Following the treatment of Huxley,2 actin sites were taken to be randomly distributed with respect to the projections on the myosin filament.7 The state of the system at a given time is then characterized by a distribution function, n(x), defined as the fraction of the actin sites at x that are attached to cross-bridges; the origin of the coordinate system is the position of the actin site at which the cross-bridge exerts zero force (Fig. la). The contractile force developed by the cross-bridges is 504

The contraction kinetics of calcium-activated skinned muscle fibers were studied after step decreases in load by means of a quickly responding recording system. The steady velocity at a given relative load was close to that found in electrically stimulated, intact muscle fibers. The presteady motion had the same shape as that of intact fibers, but the time scale of the transient was nearly two times slower. The duration of the initial phase of the motion, where the velocity was greater than the steady value, and the time at which the subsequent low velocity phase ended, were both stretched out to the same extent. Changing the temperature had the same effect on the length of these two phases of the transient. The results indicate that both phases of the transient are produced by the same underlying factors and can be taken as evidence that the entire transient originates in the crossbridge mechanism. In this case the experimental technique described here provides a basis for distinguishing between chemical parameters that affect contractility by (a) controlling the number of sites at which crossbridges can be formed, as opposed to (b) changing the kinetic properties of a given number of sites.

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The development of cellular injury in the rat left ventricle resulting from left coronary artery occlusion was examined by immunofluorescence after intravenous injection of monoclonal antimyosin. Cardiac muscle cells that bound antimyosin during ischemia were localized by staining sections with fluorescein-conjugated anti-mouse IgG. Fluorescent staining was detectable within the ischemic region of the left ventricle 3 hr after occlusion and injection of antimyosin. After 6 hr of ischemia, the highly irregular margin of the ischemic zone was clearly outlined by fluorescent cells. At 3-6 hr after occlusion, marked heterogeneity in cellular staining was observed in the epicardial half of the ischemic area, with intensely fluorescent cells intermixed with cells of markedly lower fluorescence. By 24 hr, a homogeneous pattern of staining was observed throughout the ischemic zone. In nonischemic regions of the heart and in rats treated for 24 hr with antimyosin without occlusion, there were only background levels of staining. We conclude that: (i) visualization of ischemic cells via antimyosin provides a sensitive means for examining developing patterns of injury; (ii) the heterogeneity of staining during early ischemia may reflect variation in cellular resistance to deprivation; and (ini) the pattern of fluorescence at the margin of the occluded region indicates that the "border zone" is composed of interdigitating ischemic and nonischemic tissues.The temporal and spatial distribution of myocardial injury developing in response to coronary occlusion has been the subject of many investigations. In contrast to emerging techniques such as scintiscans, positron emission tomography, and NMR, which provide assessment of ischemic zones, methods have not been exploited by which damaged cells would be individually labeled and directly visualized microscopically. A suitable method would unequivocally identify injured cells and permit large areas to be surveyed quickly. Such a method requires the development of a probe that penetrates cell membranes and remains confined to the cells. Defects in the plasma membrane of ischemic myocytes were observed by Jennings et al., occasionally after 1 hr, commonly after 2 hr of coronary occlusion, and were considered a manifestation of irreversible injury (1, 2). In work by Shell and colleagues, creatine phosphokinase appeared in the circulation in proportion to its depletion in ischemic myocardium (3). The loss of this enzyme is a manifestation of the increased permeability of ischemic cell membranes. Haber and co-workers demonstrated the preferential uptake of isotopically labeled specific antibodies to myosin in ischemic heart muscle (4-6). Apparently, analogous to the egress of macromolecular enzymes, the antibodies penetrated defective cell membranes to bind to the essentially insoluble intracellular protein, myosin.In this report, we describe the use of fluorescein-labeled monoclonal anticardiac myosin to examine the patterns of developing ischemic injury at the cellular level...