Large and rapid changes of myofibrillar total calcium during the cardiac cycle. Electron probe microanalysis of voltage-clamped guinea-pig ventricular myocytes

Summary

At 36° C and 2 mM [Ca²⁺]₀, single guinea-pig ventricular myocytes were voltage clamped with patch electrodes. 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).

The same paired-pulse protocol was applied to another group of myocytes from which contraction was recorded and [Ca²⁺]_i was estimated by microfluospectroscopy (50 µMNa-Indo-1). 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 ± SEM) and fell within 100 ms to about 450 nM.

ΣCa _myo,the total calcium concentration in the overlapping myofilaments (A-band), was measured by EPMA in 17 potentiated myocytes. During diastole, ΣCa _myo was 2.6±0.4 mmol/kg dry weight (dw), which can be converted to 0.65 mM (mmoles per liter myofibrillar space). Since [Ca²⁺]_i was 180 nM, we estimate that 99.97% of total calcium is bound.

A time-course for systolic ΣCa _myo was determined by shock-freezing 13 cells at different times after start of depolarization to +5 mV. ΣCa _myo was 5.5 ± 0.3 mmol/kg dw (1.4 mM) after 15–25 ms, 4.6±0.5 mmol/kg dw (1.1 mM) after 30–45 ms, and 3.1 mmol/kg dw (0.8 mM) after 60–120 ms. The fast time-course of ΣCa _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.

Supported by the Deutsche Forschungsgemeinschaft We 879/3-2 and Is 24/7-2