Mitochondrial calcium content changes during each individual contraction cycle; a substantial amount of calcium is taken up during the systole and released during later systole and diastole.

SUMMARY1. At 36°C and 2 mm [Ca2+]0 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)

Cardiac muscle contraction is triggered by a small and brief Ca2+ entry across the t-tubular membranes, which is believed to be locally amplified by release of Ca2+ from the adjacent junctional sarcoplasmic reticulum (SR)

Electrically stimulated heart muscle preparations can be quickly frozen in undercooled propane at defined times of the mechanically controlled contraction cycle. The apparatus for triggered freezing of the muscle strips in undercooled propane is described in detail. Freeze substitution of some strips after freezing shows the degree of ice crystal formation without the potential interference of artifacts introduced later by cryosectioning and freeze drying. Ultrathin longitudinal and transversal cryosedions are cut with a LKB cryoultramicrotome at temperatures of -130 to -140°C, freeze-dried at lop6 Torr vacuum and carbon-coated before analysis. The freeze-dried cryosections are analyzed in a Siemens Elmiskop 102 electron microscope equipped with a Kevex energy dispersive system, and the elemental concentrations (in mMoVkg d.w.1 of Na, Mg, P,S,Cl,K, and Ca are determined in subcellular compartments of muscle frozen in different functional states. The methodology of quantitation, i.e, determination of elemental net peak and continuum, correction of continuum, preparation of standards, and deconvolution of overlapping peaks are described. The minimum detectable elemental concentration using the reported methods is in the range of a few mMol/kg d.w. This also applies to Ca, which can be accumulated in heart muscle in readily detectable amounts in intracellularly located stores as well as structures connected with the cell membrane. The present report shows that cryotechniques and x-ray microanalysis can be successfully applied to heart physiology.

Using x-ray microanalysis and cryoultramicrotomy, calcium and other diffusible elements were localized in heart muscle strips which had been shock frozen under different, defined conditions of electromechanical coupling. Guinea pig papillary muscles were shock frozen: 1) 1 1/2 seconds after paired stimulation, 2) 5 minutes after rest in normal bath medium and 3) 5 minutes after rest in bath medium to which noradrenaline was added. In 1) high calcium concentrations of 11.5 mmol/kg d.w. were regularly detected in sites at the level of Z-lines, which probably corre- spond to the Z rete of SR. In 2) in which the mechanogram of the first contraction after rest normally showed a small and retarded peak, the cell stores seemed to be nearly empty, with exception of a few regions between the mitochondria which revealed calcium accumulations of 77 mmol/kg d.w. These regions included JSR and/or T-tubuli. In 3) in which the mechanogram of the first contraction after rest normally showed a retarded peak with high tension, calcium was found in several cell structures. The highest amount, 25 mmol/kg d.w., was detected over the cell membrane. Measurable amounts were also detected over Z-lines and sarcomeres. In the present experiments, the respective rate of rise of tension, and time to peak tension, were extremely different. Possible correlations between different contraction patterns and different calcium stores involved in the various experiments have been discussed.