Effect of reduced Na gradient on electrical activity in isolated bovine and feline ventricular myocytes

We have studied changes in electrical activity resulting from abrupt alterations of the Na gradient, using ventricular myocytes isolated from feline and bovine hearts. Attempting to investigate the ionic current possibly generated by Na–Ca exchange, we studied the effects of the changes in [Na]o in the presence of 20 mM CsCl to inhibit K currents. To facilitate the effect of Cs, we also used a K-free solution and a patch electrode filled with 150 mM cesium glutamate. The application of 20 mM Nao resulted in hyperpolarization and the action potential duration was reduced. Under voltage clamp, 20 or 45 mM Nao generated an outward current at all membrane potentials investigated. The initial part (100–200 ms) of this current was only partially inhibited by 5 mM NiCl2 which is known to fully block the Ca inward current. However, the outward current generated by the reduced [Na]o was fully inhibited by 20 mM MnCl2 (which presumably inhibits Na–Ca exchange). Our observations extend the work on multicellular cardiac preparations indicating that the outward current elicited by a sudden decrease in Na gradient could be generated by Na–Ca exchange. Although the characteristics of this outward current support certain concepts of the Na–Ca exchange in cardiac muscle, we cannot at present exclude a contribution of other membrane current(s).

Ca Entry and Contraction as Studied in Isolated Bovine Ventricular Myocytes

Single bovine ventricular myocytes were superfused with Tyrode solution containing 1.8 mᴍ 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 [Ca]0 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 mᴍ Ni or to 2 μᴍ 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 intra­ cellular Ca concentration (Δ[Ca]iT) was estimated by referring Ca entry to the cell volume (50 pl). Within 100 ms Δ[Ca]iT came to 25 μᴍ at control conditions, to 55 μᴍ at [Ca]0 = 3.6 mᴍ and to 88 μᴍ when 0.1 μᴍ adrenaline were present. The Δ[Ca]iT 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.