X-ray microanalysis of single cardiac myocytes frozen under voltage-clamp conditions

By means of a patch pipette, an isolated ventricular myocyte was transferred into the taper of a silver holder covered by pioloform film. Once the cell was on the film, the cell was voltage clamped (pulses from -45 to +5 mV at 0.5 Hz). The amount of Ca entry was estimated from the Ca current. When contractility (cell shortening) was potentiated with either five pulses of 0.2 s or four pulses of 1 s, shock freezing was timed 116 or 816 ms after start of the clamp pulse. Electron micrographs from freeze-substituted cells revealed the good preservation of the intracellular compartments. The myocytes were cut at -150 degrees C, and the cryosections were freeze dried. In representative examples, the amount of Ca entry is compared with the subcellular Ca distribution as it is analyzed with energy dispersive X-ray microprobe analysis in cytoplasm, junctional sarcoplasmic reticulum (SR), mitochondria, and the subsarcolemmal space (sarcolemma, peripheral SR, fringe of cytosol).