ROLE OF CA2+ CHANNEL IN CARDIAC EXCITATION-CONTRACTION COUPLING IN THE RAT - EVIDENCE FROM CA2+ TRANSIENTS AND CONTRACTION

1. Optical methods were used to measure simultaneously unloaded cell shortening and intracellular Ca2+ transients in whole-cell voltage clamped rat ventricular myocytes. Red light ( > 670 nm) was used to measure cell shortening with a linear photodiode array. The dyes Fura-2 (K(d) = 140 nM) and Mag-Fura-2 (K(d) = 44-mu-M) were used as Ca2+ indicators with fluorescence excitation at 340 and 410 nm and emission at 510 nm. 2. Repeated measurements at 6 s intervals as 0.4 mM-Fura-2 diffused into the cell from the tip of the voltage clamp pipette showed no decrease in the rate of rise and peak value of the intracellular Ca2+ transient and only a small suppression of cell shortening, suggesting that the molecular mechanisms regulating the Ca2+ release were not significantly altered by the buffering capacity of the Fura-2. 3. Experiments in which the sarcoplasmic reticulum (SR) was depleted of Ca2+ either by exposure to caffeine or by repeated brief (20 ms) voltage clamp depolarizations confirm that the SR is the major source of activator Ca2+. 4. Mag-Fura-2 (1 or 5 mM) was used to register the initial rapid development of the [Ca2+]i transient but the later time course of the Ca2+ transients measured with this dye was obscured by motion artifacts resulting from cell shortening. 5. Both Fura-2 and Mag-Fura-2 showed that depolarization to 0 mV from a holding potential of -80 mV resulted in a [Ca2+]i transient which developed with a delay of 3-9 ms and approached its peak value in an additional 8-19 ms. Both Ca2+ indicators also showed that the Ca2+ transient approached its peak value more slowly as the clamped membrane potential was made increasingly more positive. 6. The voltage dependencies of the Ca2+ signal (Fura-2) and cell shortening were both bell-shaped and were qualitatively similar to the voltage dependence of Ca2+ current simultaneously measured. This was observed with holding potentials of both -40 and -80 mV. 7. Comparison of the temporal relation of the Ca2+ current, I(Ca), and intracellular Ca2+ transient (Fura-2) and cell shortening at different membrane potentials showed that Ca2+ transient measured 25 ms into the depolarization correlated closely to the integral of the Ca2+ current measured prior to this time. Cell shortening, on the other hand, peaked about 100 ms later and correlated with measurements of the Ca2+ activity at the later time. 8. The slower development of the Fura-2 transient at + 30 mV than at -20 mV was mirrored in the time course of the inward Ca2+ current which was smaller but longer lasting at + 30 mV than at -20 mV. At the same time the initial delay of the Ca2+ transient was shorter at + 30 than at -20 mV. 9. The initial delay was briefer for Ca2+ transients (Fura-2) evoked by repolarization from + 100 to -40 mV than for Ca2+ transients evoked by depolarization from -40 to 0 mV. 10. The amplitude of the Fura-2 transient was reduced as the duration of a depolarizing clamp pulse (e.g. to 0 mV) was reduced to values below 10-20 ms. Similarly the development of the Ca2+ transient could be prematurely terminated by further depolarization to 100 mV. 11. We conclude that simultaneous measurements of both cell shortening and intracellular Ca2+ transients provide detailed data on excitation-contraction coupling in voltage clamped rat ventricular myocytes. Our results support the hypothesis that the release of Ca2+ from the SR is under direct, continous control of the Ca2+ influx through the Ca2+ channel.