Anoxia of the heart causes failure of contraction prior to any irreversible injury. The mechanism by which anoxia blocks cardiac excitation-contraction coupling is unknown. Studies, in whole muscle are confounded by heterogeneity; however it has not previously been possible to achieve the low oxygen tensions required to study anoxia in single myocytes during electrophysiology recordings. Guided by calculations of oxygen transport, we developed a system in which myocytes in an open dish are insulated from oxygen by a laminar counterflowing column of argon, permitting free access by microelectrodes while maintaining a P02 less than 0.02 torr. In the absence of glucose, the amplitude of stimulated contraction of anoxic ventricular myocytes fell to zero over two minutes, after a lag period attributable to consumption of endogenous glycogen. The cytosolic calcium transient, measured by Indo-1 fluorescence, fell to zero simultaneously with the contraction. After the twitch had failed, microinjection of caffeine around the cell still caused a large calcium release and contraction, indicating that sarcoplasmic reticulum calcium stores were not depleted. Failure of the twitch was accompanied by shortening and then failure of the action potential; under voltage clamp, large outward currents, reversing at the resting potential, developed during contractile failure. After failure of action potential-mediated contraction, voltage clamp depolarization, using a large command voltage to compensate for the series resistance error due to the outward currents restored a normal twitch contraction. We conclude that anoxic contractile failure in the rat myocyte is due to alteration of the action potential, and the distal pathways of excitation-contraction coupling remain essentially intact.
