1. At 36-degrees-C and 2 mM [Ca2+]o single guinea-pig ventricular myocytes were voltage clamped with patch electrodes. With a paired-pulse protocol applied at 1 Hz, a first pulse to +5 mV was followed by a second pulse to +50 mV. When paired pulsing had potentiated the contraction to the maximum, the cells were shock-frozen for electron-probe microanalysis (EPMA). Shock-freezing was timed at the end of diastole (-80 mV) or at different times during systole (+5 mV). 2. The same paired-pulse protocol was applied to another group of myocytes from which contraction and [Ca2+]i was estimated by microfluospectroscopy (50-mu-M-Na5-Indo-1). Potentiation moderately reduced diastolic sarcomere length from 1.85 to 1.82-mu-m and increased diastolic [Ca2+]i from about 95 to 180 nM. In potentiated cells, during the first pulse, contraction peaked within 128 +/- 25 ms after start of depolarization. [Ca2+]i peaked within 25 ms to 890 +/- 220 nm (mean +/- S.E.M.) and fell within 100 ms to about 450 nM. 3. SIGMA-Ca(myo), the total calcium concentration in the overlapping myofilaments (A-band), was measured by EPMA in seventeen potentiated myocytes. During diastole, SIGMA-Ca(myo) was 2.6 +/- 0.4 mmol (kg dry weight (DW))-1 which can be converted to 0.65 mM (mmoles per litre myofibrillar space). Since [Ca2+]i was 180 nM, we estimate that 99.97% of total calcium is bound. 4. A time course for systolic SIGMA-Ca(myo) was determined by shock-freezing thirteen cells at different times after start of depolarization to +5 mV. SIGMA-Ca(myo) was 5.5 +/- 0.3 mmol (kg DW)-1 (1.4 mM) after 15-25 ms. 4.6 +/- 0.5 mmol (kg DW)-1 (1.1 mM) after 30-45 ms, and 3.1 mmol (kg DW)-1 (0.8 mM) after 60-120 ms. The fast time course of SIGMA-Ca(myo) suggests that calcium binds to and unbinds from troponin C at a fast rate. Hence, it is the slow kinetics of the cross-bridges that determines the 130 ms time-to-peak shortening. 5. Mitochondria of potentiated cells contained during diastole a total calcium concentration, SIGMA-Ca(mito), of 1.3 +/- 0.2 mmol (kg DW)-1 (0.4 mM). During the initial 15-25 ms of systole, SIGMA-Ca(mito) did not change, however, during 30-45 ms SIGMA-Ca(mito) rose to 3.7 +/- 0.5 mmol (kg DW)-1 (1.2 mM). The data suggest that SIGMA-Ca(mito) can follow SIGMA-Ca(myo) with some delay, thereby participating in both slow diastolic and fast systolic changes in total calcium (SIGMA-Ca), at least under the given conditions. 6. Junctional and corbular sarcoplasmic reticulum (SR) at the level of the Z-line had a diastolic total calcium concentration, SIGMA-Ca(SR), of 8.7 +/- 1.3 mmol (kg DW)-1 (2.4 mM), significantly higher than SIGMA-Ca(myo). Twenty milliseconds after the beginning of depolarization SIGMA-Ca(SR) had fallen to 4.1 +/- 0.4 mmol (kg DW)-1 (1.1 mM), during the following systole SIGMA-Ca(SR) recovered. The difference between SIGMA-Ca(SR) and SIGMA-Ca(myo) has the property expected of a 'Ca-release compartment'. 7. Our data indicate for the myofibrillar space a Ca2+-buffering capacitance of 1.5 mmol SIGMA-Ca(myo) per pCa unit between pCa 7 and 6. The concentration of troponin C is insufficient to account for this value. To present a model explaining the results requires the assumption of additional Ca2+-binding sites. The effect of potentiation on diastolic [Ca2+]i and SIGMA-Ca(myo) requires a postulation of 600-mu-M additional Ca2+, Mg2+ sites and the rapid systolic transients require the assumption of 2000-mu-M additional 'fast' Ca2+-binding sites.
