Direct measurements of electromechanical (or excitation- contraction) coupling in single heart cells are now feasible because of three recent technological developments: viable cells can be obtained by enzymatic dissociation of tissue, the whole cell patch clamp technique permits detailed electrophysiological studies, and new, ultrasensitive force probes enable the strength of contraction to be monitored. These direct measurements offer several major advantages over previous indirect studies with multicellular preparations: first, the single cell structure is comparatively simple and uncomplicated by the three-dimensional tissue structure, and may be considered to be truly homogenous. Second, the microenvironment around the cell may be more easily and rapidly controlled. Third, the proposed research methods may eventually become applicable to human myocytes, obtained by tissue biopsies, with potential use in patient studies of cardiac hypertrophy, hypertension, dietetics or aging. The central theme of this proposal, then, is to investigate and perhaps, redefine myocardial """"""""contractility"""""""" at the cellular level using these new approaches. Using an ultrasenitive, fiber-optic based force probe recently developed in this laboratory, the twitch force and dynamic stiffness of single heart cells will be measured. The force of contraction can how be measured routinely from single frog ventricular heart cells, and efforts will be made to extend the research methods to mammalian heart cells. The experimental design will be guided by protocols developed previously for multicellular preparations and will focus on several key characteristics of excitation-contraction coupling, such as the relation of contractile force to membrane potential, frequency of stimulation, cell length, and cell stiffness. With these results, it may be possible to clarify the cloudy aspects of interpretation of previous measurements in muscle strips. In some cases, comparative studies between single cells and muscle strips will be performed to exploit the differences in anatomical structure. In other cases, parallel electrophysiological experiments will be conducted to test whether the membrane potential or ionic currents behave under a given experimental protocol in a manner consistent with that observed for contraction.
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