We studied contraction in single voltage-clamped, internally perfused myocytes isolated from guinea pig ventricles . The microscopic appearance of the cell was observed and recorded with a television system, while contractile shortening was measured 1,000 times/s using a linear photodiode array. Uniform, synchronous sarcomere shortening occurred in response to depolarizations that triggered a slow inward current (I,). Changes in I,; caused by altering the amplitude of the voltage step, the extracellular [Ca"], or the holding potential were accompanied by immediate parallel changes in the extent and velocity of shortening. In particular, twitch shortening during depolarization (a) was immediately decreased when large voltage steps decreased I,;, and (b) was eliminated by depolarizations that exceeded +75 mV, the apparent reversal potential for Ca2+. In these cases, shortening was associated with the tail current during repolarization . Increases in the amplitude, duration, and the rate of the depolarizing step increased the extent and speed of sarcomere shortening over the course of four to five contractions without a simultaneous parallel increase ofIS; . Large prolonged depolarizations caused an asynchronous, nonuniform, oscillatory shortening of the cell and potentiated future twitch contractions. Increases in the duration of the depolarizing step immediately prolonged contraction; otherwise, interventions that altered the extent, velocity, and time course of shortening in intact, nonperfused cells did not affect the time course of the contraction in the internally perfused single cells. Our results provide direct support for the hypothesis that I,; both induces and grades the size of the Ca' release from the sarcoplasmic reticulum of intact cardiac muscle . In addition, a separate, depolarization-dependent process unrelated to I5; (a) grades the size of contraction, presumably by modulating Ca' accumulation in the intracellular stores, and (b) affects its time course.
SUMMARY1. Sarcomere lengths were measured during rest and throughout the time course of isometric contractions in thin, isolated rat papillary muscles using light diffraction techniques.2. Shortening of the sarcomere length occurred upon contraction at all muscle lengths, averaging 7 % at optimal length and more at shorter lengths. Relative to the narrow range of sarcomere lengths spanning the length-tension curve, this degree of shortening was considerable.3. Local changes of sarcomere length were quantitatively paralleled by local changes of tissue segment length, the latter demarcated by microspheres lodged within the muscle tissue. At all but the shortest muscle lengths, sarcomere shortening was fully accounted for by equivalent lengthening of the non-striated regions near the clamped ends of the preparation.4. It seems likely that these regions constitute the source of the large series elasticity characteristic of isolated papillary muscle preparations such as this.
We have observed the dynamics of sarcomere shortening and the diffracting action of single, functionally intact, unattached cardiac muscle cells enzymatically isolated from the ventricular tissue of adult rats. Sarcomere length was measured either (a) continuously by a light diffraction method or (b) by direct inspection of the cell's striated image as recorded on videotape or by cinemicroscopy (120-400 frames/s). At physiological levels of added CaCl2 (0.5-2.0 mM), many cells were quiescent (i.e., they did not beat spontaneously) and contracted in response to electrical stimulation (~ 1.0-ms pulse width). Sarcomere length in the quiescent, unstimulated cells (1.93 + 0.10 [SD] /~m), at peak shortening (1.57 + 0.13/~m, n = 49), and the maximum velocity of sarcomere shortening and relengthening were comparable to previous observations in intact heart muscle preparations. The dispersion of light diffracted by the cell remained narrow, and individual striations remained distinct and laterally well registered throughout the shortening-relengthening cycle. In contrast, appreciable nonuniformity and internal buckling were seen at sarcomere lengths < 1.8 /~m when the resting cell, embedded in gelatin, was longitudinally compressed. These results indicate (a) that shortening and relengthening is characterized by uniform activation between myofibrils within the cardiac cell and (b) that physiologically significant relengthening forces in living heart muscle originate at the level of the cell rather than in extracellular connections. First-order diffracted light intensity, extremely variable during sarcomere shortening, was always greatest during midrelaxation preceding the onset of a very slow and uniform phase of sarcomere relengthening.
We investigated the basis for impaired left ventricular function of hearts in which hypertrophy was produced by gradual pressure overload. We measured myoplasmic free calcium concentration ([Ca2+]i) with fura-2 and sarcomere shortening in single myocytes isolated from control hearts and hypertrophied failing hearts. Diastolic [Ca2+]i was normal, but [Ca2+]i at the peak of contraction was depressed in myocytes from failing hypertrophied hearts. Increasing drive rate from 0.20 Hz to 5.00 Hz increased both diastolic and peak [Ca2+]i. Norepinephrine (3 x 10(-6) M) increased diastolic [Ca2+]i in all cells and tended to normalize peak [Ca2+]i in myocytes from hypertrophied failing hearts during 5.00 Hz drive. Depressed peak [Ca2+]i in the hypertrophied cells was paralleled by significant decreases in both the velocity and percent of sarcomere shortening, which were measured in cells not loaded with fura-2. Sarcomere length was correlated with estimates of [Ca2+]i in intact cells and with controlled levels of [Ca2+] in chemically "skinned" myocytes. A plot of sarcomere length against [Ca2+] gave a single continuous relationship that spanned resting and peak values at all drive rates in both the control and hypertrophied myocytes. Thus heart failure in this model is reflected in impaired myocyte contraction, which is closely related to reduced levels of [Ca2+]i during systole rather than to depressed myofilament sensitivity to Ca2+.
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