Excitation-Contraction Coupling of Isolated Cardiac Fibers with Disrupted or Closed Sarcolemmas

Single cells of adult rat ventricle were separated with 0.1% trypsin and 0.05—0.1% collagenase. Ten percent of these cells were tubular and striated, contracted spontaneously in the presence of 0.05 mM CaCl2 or after addition of 0.1-0.5 mM CaCl2, and developed con fracture in 1 mM CaCl2. A resting potential of —30 to —50 mv and an action potential more than 100 msec in duration were recorded in some of the cells. These relatively intact cells were contrasted with myocardial fibers (10-lOOyu,) obtained by homogenization. In the presence of 0.025 mM ethyleneglycol bis(y3-aminoethylether)-N, N'tetraacetic acid (EGTA), localized cyclic contractions with intracellular asynchrony were observed under the phase microscope in obviously disrupted areas of these mechanically separated fibers. These contractions were insensitive to the ratio of Na + to K + , and neither resting potentials nor action potentials were observed. Thus, these disrupted fibers were considered skinned fibers of cardiac muscle; this assumption was further supported by the absence of modification of fiber sensitivity to Ca 2+ by simultaneous skinning with EDTA. Effects of Ca 2+ were studied with control of pCa in the medium by EGTA-Ca buffers, either directly on the homogenate or after depletion of Ca 2+ content of the tissue. At 22°C, pH 7.0, and in the presence of 4 mM MgCL, 5 mM adenosine triphosphate, and 2 mM EGTA, cyclic contractions were inhibited and a tonic contraction was induced at a pCa of 6.25—6.5, which represented the threshold for activation of myofilaments. With the same medium but with low EGTA (less than 0.2 mM), cyclic contractions were obtained if pCa was equal to or lower than 7.4—7.6. Therefore, these contractions corresponded to a cyclic release of Ca 2+ from cellular stores, yielding a transient activation of myofilaments. This large release of Ca 2 + within the fiber was induced by a small addition of Ca 2+ to the medium. It was concluded that a regenerative release of Ca 2+ could be demonstrated in heart muscle under these conditions. Localized cyclic contractions were not observed in disrupted fibers of frog heart, in which spontaneously beating cells could be separated with enzymes.