Thiopental alters contraction, intracellular Ca2+, and pH in rat ventricular myocytes

Background: Myocardial contractility is regulated by intracellular concentration of free Ca2+ ([Ca2+](i)) and myofilament Ca2+ sensitivity. The objective of this study was to elucidate the direct effects of thiopental on cardiac excitation-contraction coupling using individual, field-stimulated ventricular myocytes, Methods: Freshly isolated rat ventricular myocytes were loaded with the Ca2+ indicator, fura-2, and placed on the stage of an inverted fluorescence microscope in a temperature-regulated bath. [Ca2+](i) (340/380 ratio) and myocyte shortening (video-edge detection) were monitored simultaneously in individual cells field-stimulated at 0.3 Hz. Amplitude and timing of myocyte shortening and [Ca2+](i) were compared before and after addition of thiopental. Intracellular pH was measured with the pH indicator, BCECF (500/440 ratio). Real-time uptake of Ca2+ into isolated sarcoplasmic reticulum vesicles was measured using fura-2 free acid in the extravesicular compartment. One hundred thirty-two cells were studied. Results: Field stimulation increased [Ca2+](i) from 85 +/- 10 nM to 355 +/- 22 nM (mean +/- SEM), Myocytes shortened by 10% of resting cell length (127 +/- 5 mu m). Times to peak [Ca2+](i) and shortening were 139 +/- 6 and 173 +/- 7 msec, respectively, Times to 50% recovery for [Ca2+](i) and shortening were 296 +/- 6 and 290 +/- 6 ms, respectively. Addition of thiopental (30-1,000 mu M) resulted in dose-dependent decreases In peak [Ca2+](i) and myocyte shortening. Thiopental altered time to peak and time to 50% recovery for [Ca2+](i) and myocyte shortening and inhibited the rate of uptake of Ca2+ into isolated sarcoplasmic reticulum vesicles. Thiopental did not, however, alter the amount of Ca2+ released in response to caffeine in sarcoplasmic reticulum vesicles or intact cells. Thiopental (100 mu M) increased intracellular pH and caused an upward shift in the dose-response curve to extracellular Ca2+ for shortening, with no concomitant effect on peak [Ca2+](i). These effects were abolished by ethylisopropyl amiloride, an inhibitor of Na+-H+ exchange. Conclusion: Thiopental has a direct negative inotropic effect on cardiac excitation-contraction coupling at the cellular level, which is mediated by a decrease in [Ca2+](i). Thiopental also increases myofilament Ca2+ sensitivity via alkalinization of the cell, which may partially offset its negative inotropic effect.