Ca Entry and Contraction as Studied in Isolated Bovine Ventricular Myocytes

Single bovine ventricular myocytes were superfused with Tyrode solution containing 1.8 mM CaCl2. The cells did not bear external load and contracted isotonically. Contraction and relaxation were characterized by the shortening and relengthening of the sarcomeres which resembled in their time course the isometric twitches of bovine papillary muscles. Resemblance was also found in regard to positive inotropic interventions as increase in the stimulation frequency, exposure to elevated Ca0 or to adrenaline. A two-microelectrode voltage-clamp technique was applied to the single myocyte. The transmembrane Ca inward current ICa was defined as difference current sensitive to 5 mM Ni or to 2 microM D600. During a voltage step from -45 to +5 mV, ICa peaked within 3 ms to -6 nA, afterwards it decayed to 15% of peak amplitude (incomplete inactivation with a 2 exponential time course). Experiments in Na-free media suggested that Na entry does not significantly contaminate ICa. Therefore, Ca entry could be calculated from ICa. The increment in total intracellular Ca concentration (deltaCaTi) was estimated by referring Ca entry to the cell volume (50 pl). Within 100 ms deltaCaTi came to 25 microM at control conditions, to 55 microM at Ca0 = 3.6 mM and to 88 microM when 0.1 microM adrenaline were present. The deltaCaTi values were sufficient to activate contraction without the necessity of Ca-release from SR. Despite the new data, the relationship between Ca entry and activation of contraction was complex: during the "positive Herztreppe" ICa slightly attenuated but contractility doubled. Therefore, the old EC-model (M. Morad and Y. Goldman, Progr. Biophys. Mol. Biol. 27, 257 (1973)) was adapted. The Ca-entry's capability to load and to overload the intracellular Ca store (SR) is discussed.