Bioengineering and Characterization of Troponin Peptides for use as Therapeutic Reagents to Modulate Muscle Contractility

Abstract

Steady state myoplasmic ion concentration and slow ion transport across the sarcolemma can be quantitatively studied by means of ion sensing electrodes (ISE). ISE's readout represents the sum of the electrochemical potential of the ion of interest and the resting membrane potential (Vm). Vm is measured either by using an independent standard microelectrode or a double-barreled capillary. These approaches have known limitations. Ion transport and membrane potential are intricately connected, the resting potential may be unstable or be far from the desired value, ion transport can be electrically silent, and membrane currents may be carried by more than one ion. Thus, an electrophysiological method able to simultaneously measure the concentration of more than one ion, and to measure and control Vm and membrane current (Im) is desirable for physiological and pathophysiological studies. For skeletal fibers, a triple-barreled electrode has been used in the past allowing for current injection to modify Vm under current clamp conditions. Here we describe a novel four-microelectrode system composed of a two-microelectrode voltage-clamp amplifier (TEV-200A, Dagan) and a two channel high impedance electrometer (FD223a, WPI). This approach allows for potentiometric measurements of the concentration and transport of up to two ions in short murine skeletal muscle fibers subjected to either current-or voltage-clamp conditions. Ion translocation by primary and secondary active transport mechanisms, ion exchangers or passive ion diffusion can be studied. The system performance is exemplified by using Na, K and H selective electrodes made with commercially available ionophores and fibers enzymatically dissociated from the flexor digitorum brevis muscles.