Potentiation of Contraction as Related to Changes in Free and Total Intracellular Calcium

Summary

In voltage-clamped guinea-pig ventricular myocytes, we studied the potentiation of contraction in dependence on the concentration of intracellular calcium; ionized calcium [Ca²⁺]_c was measured by Indo-1 microfluospectroscopy and total calcium (∑Ca) by electronprobe microanalysis (EPMA). After a 15 min rest period, [Ca²⁺]_c was approx. 90 nM and ∑Ca was below the detection limit (80 µM) in myoplasm (∑Ca_myo), junctional sarcoplasmic reticulum (∑Ca_SR) and mitochondria (∑Ca_Mito). Post rest, repetitive clamp steps (1 Hz) potentiated extent and rate of shortening by 300%. In the literature, post-rest potentiation is attributed to the replenishment of SR with releasable calcium; by EPMA the postulated increase in ∑Ca_SR was measured directly. Post-rest, the peaks of systolic [Ca²⁺]_c transients increased, however only by 40%. In addition, a moderate increase of end-diastolic [Ca²⁺]_c was measured. In an other series of experiments, contraction was potentiated by 800% increase by means of paired voltage-clamp pulses (1 Hz, 36 °C, 2 mM [Ca²⁺]₀). In the potentiated state, end-diastolic [Ca²⁺]_c was 180 nM and ∑Ca_myo was 0.65 mM. During systole, [Ca²⁺]_c peaked within 20 ms to 950 nM. ∑Ca_myo rose within 20 ms to 1.4 mM and fell within 40 ms to 1.1 and within 90 ms to 0.8 mM. In contrast, the time course of contraction was slow and peaked at a time (130 ms) when the [Ca²⁺]_c and ∑Ca_myo transients were finished. We suggest that Ca²⁺ bound to troponin C (TnC) controls only the onset but not the time course of myofilament interaction. From [Ca²⁺]_c and ∑Ca_myo we estimated a Ca²⁺ buffering capacitance of 1.5 mmol ∑Ca_myo per pCa change, only a fraction of which can be attributed to Ca²⁺ binding sites on TnC. A model explaining the results requires the assumption of 0.6 mM additional slow, high affinity Ca²⁺ sites and 2 mM fast, low affinity Ca²⁺ sites. We discuss that end-diastolic Ca²⁺ binding to these sites contributes to the potentiation of contraction. Junctional SR. At the end of diastole ∑Ca_SR was 2.4 mM which is 4 times larger than ∑Ca_myo. This difference disappeared 20 ms after depolarization (∑Ca_SR 1.1 mM), within another 20 ms it largely recovered (∑Ca_SR 2.0 mM). These properties suggest that the junctional SR is a compartment suitable not only for Ca²⁺ release but also for rapid Ca²⁺ reuptake. Mitochondria. Paired-pulse potentiation increased end-diastolic ∑Ca_Mito significantly (0.4 mM). During diastolic ∑Ca_Mito followed the changes in ∑Ca_myo with some delay (peak ∑Ca_Mito of 1.2 mM after 40 ms). According to biochemical literature, we interpret the changes in ∑Ca_Mito to stimulate the activity of dehydrogenases and to adapt the rate of ATP synthesis to the demands of muscle mechanics.