This project proposes a physiological study of steps in the excitation-contraction (E-C) coupling process in vertebrate striated muscle. The overall goal is to attain a detailed understanding of the mechanisms that function in the intact fiber to control the rapid changes in intracellular calcium that in turn regulate the normal contractile response. In general, singly-dissected twitch fibers from frog skeletal muscle will be used and specific steps in the E-C coupling sequence will be studied by means of either optical, electrical or mechanical signals specific for these steps. Major emphasis will be placed on experimental determination of the amplitude and time course of the myoplasmic free [Ca] transient and its quantitative interpretation. The methodology involves intracellular injection of a calcium """"""""idicator"""""""" dye such as Antipyrylazo III, followed by optical measurement of dye-related absorbance changes that accompany electrical stimulation. In many experiments a computational model incorporating published anatomical and biochemical parameters will be used to analyze the calcium transient to obtain quantitative information about: a) the amount and time course of Ca release and re-uptake by the sarcoplasmic reticulum (SR); b) the binding of Ca to myoplasmic buffers such as these on troponin C and parvalbumin; and c) how these quantities depend on physiological variables such as membrane potential, ion substitution, sarcomere length and pharmacological agents (e.g. caffeine and valinomycin). Other experiments will make use of related optical techniques to measure (i) possible SR membrane potential changes that may control SR Ca release and/or re-uptake; (ii) possible pH and Mg transients that at early times after stimulation may reflect the movement of electrical counter-ions to Ca release and at later times may reflect the capture of Ca (and release of Mg) by buffer sites on parvalbumin; (iii) resting myoplasmic Ca levels that set the initial conditions for the quantitative interferences. By use of a microscope plus array-detector system, it should be possible to compare the amplitude of some of the dye signals along the sarcomere length (e.g. A-band vs. I-band amplitudes) and thereby gain supporting evidence as to the likely origin of the signals and the accuracy of their interpretation. Later experiments will extend the measurements to alternate fiber types, such as mammalian fast and slow twitch fibers. The methods should eventually be useful for understanding how specific steps in the normal E-C coupling process may be altered by drugs and diseases.
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