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In rat ventricular muscle, the action potential configuration and the ionic currents were studied as a function of frequency under current and voltage clamp conditions. Increase of stimulation frequency produced (a) a progressive loss of the plateau accompanied by a shortening of the action potential; (b) no significant change in the amplitude of the instantaneous outward current in [Ca 2+ ] o between 2.2 and 10.0 m m , and (c) an increase in the amplitude of the slow inward current, I si and a marked reduction of its inactivation time constant τ f up to frequency of about 75 stimuli/min. Above this frequency, the amplitude of I si decreased and τ f increased again.

The upstroke of the ventricular action potential in the rabbit consists of two depolarizing components with different rates of rise. The effects of hypoxia on the resting potential (RP); the upstroke phases (I and II) and the maximum rate of rise of phase I (V max) were studied at different external K concentrations (K0). Perfused hearts were submitted to N2-equilibrated media containing 1.5 to 10 mM K0. Exposure of oxygenated hearts to different K0 changed the regenerative response from a fast rising action potential at 1.5 mM K0 to a depressed fast response at 7.5 and 10 mM K0. Hypoxia decreased the action potential amplitude (APA) at all K concentrations. In K0 less than or equal to 5 mM the reduction of APA was due to a decrease in the amplitude of phase II of the upstroke but the maximum rate of rise (V max) did not change. In contrast, phase I of the upstroke was markedly depressed by hypoxia in high K0, but phase II was unmodified and its V max compared well with values reported for other normoxic cardiac cells. Hyperkalemia per se did not slow conduction during normoxia but increased conduction time in hypoxia. The resting potential of hypoxic cells was closer to the K equilibrium potential than in the control. The RP v. Ko/Ki relation suggested that electrogenic Na extrusion persists in hypoxia. The electrogenic fraction of the resting potential as determined from pump inhibition with 10(-4) M ouabain amounted to -6 mV. Our results did not indicate whether the differential effects of hypoxia on the upstroke components were potential dependent or were related to direct effects of K+ on the ionic currents that determine the action potential. The persistence of phase II during hypoxia in partly depolarized cells may assure the maintenance of propagated electrical activity under conditions that are likely to be encountered in vivo during cardiac ischemia.

The inhibitory effects of verapamil (84.4% inhibition) and D-600 (70.4% inhibition) on the conductance of the slow inward current were compared with that of Mn 2+ (99.7% inhibition). Hill coefficients of 2 for Mn 2+ and D-600 and one for verapamil were found. For racemic mixtures of verapamil and D-600 the apparent dissociation constants (K 0.5 ) were 1.4 and 3.6 μ m respectively, whereas the apparent dissociation constant of Mn 2+ was 625 μ m . With such high affinities, as compared to Mn 2+ , time constants of inhibition for D-600 and verapamil were larger and their offset rates were slower. It is concluded that Mn 2+ and the organic blockers have different mechanisms of action, that varapamil and D-600 have to pass through or enter into the membrane, and that the molecules of D-600 and verapamil possess one and two active groups respectively.

In cardiac muscle the steady membrane potential (Vm) behaves as a K-electrode at extracellular K concentrations (Ko) above 10 mm [3, 13]. At lower concentrations the Goldman equation describes Vm reasonably well [13]. Such a curve fitting assumes: (a) concentration-independent permeability coefficients, (b) a constant intracellular potassium concentration (Ki), and (c) a passive distribution of chloride. Considering that Ki varies with Ko in rabbit ventricle [11] we tried to fit the Vm v. Ko relation to the Goldman equation using assumptions (a) and (c) together with the measured variation of Ki. We will present data suggesting that the NaK exchange ratio is not rigidly linked to the activity of the sodium pump.

The effects of sodium and calcium concentration on the action potential of cell clusters derived from rat ventricle were studied. The maximum rate of rise (Vmax) decreased in low sodium (50 mm) medium, but the overshoot did not change. The Vmax did not vary with [Ca2+]o within the range of 0.4 to 5.0 mm, but the size of the overshoot increased with increasing [Ca2+]o. A change of 8.8 mV/decade of change of [Ca2+]o was observed. These results indicate that both calcium and sodium ions carry the inward current responsible for the upstroke. The insensitivity of Vmax to changes in [Ca2+]o can be explained if high calcium shifts the inactivation characteristic of the slow current (f∞) towards more negative potentials. The slope of the diastolic depolarization decreased in low sodium and in low calcium media, and increased in 5 mm [Ca2+]o. These findings, together with the different sensitivity of the upstroke and the diastolic depolarization to TTX [15] lead us to postulate that although calcium and sodium carry the current(s) involved in electrogenesis and automaticity, these phenomena may be mediated by two distinct types of ionic channels having different kinetics.

The effects of amphotericin B on contraction and electrical activity of isolated rabbit hearts were tested. The hearts were perfused with Krebs Henseleit solution at 33°C and constant coronary flow. Amphotericin B was used at concentrations of 1.25, 2.5, 5 and 10 μg/ml. Surface electrograms were displayed synchronously with atrial transmembrane potentials. In non-driven preparations, amphotericin caused progressive lengthening of PR interval and P wave. AV conduction block progressed towards complete atrioventricular dissociation. The effects were the same with all the concentrations used but the time course of the changes observed was related to the concentration. In driven preparations, the antibiotic produced a decrease in action potential amplitude. This was characterized by the progressive abolition of the slow phase of depolarization followed by a decrease in the amplitude of the fast phase of the upstroke. The maximum depolarization rate decreased. Progressive inactivation of atrial fibers occurred. Alternance of action potentials with electrotonic potentials was currently observed. Finally, the preparation became irreversibly unexcitable. It is proposed that amphotericin exerts its effects by progressive blocking of the ionic currents involved in excitation, and that it also interferes with the processes of activation and removal of inactivation.

The effect of amphotericin B (1.3, 2.6, and 5.2 μ m ) on the ionic currents determining the cardiac action potential was investigated in the frog atrial trabecula. The experiments were performed with the double sucrose gap technique. Under current clamp conditions, amphotericin B depolarized the membrane by about 15 mV, reduced the action potential amplitude, abolished the plateau, and slowed down the last phase of repolarization. At low concentrations, the antibiotic depressed the fast inward current by this effect was reversed by hyperpolarization of the preparation. At 5.2 μ m , the inhibition of the fast inward current was only partly reversed by hyperpolarization. At all concentrations, the antibiotic shifted the h ∞ curve towards more negative potentials, and at 5.2 μ m decreased its slope. A substantial decrease of the slow inward current was observed under amphotericin B. This effect was not reversed by hyperpolarization and the f ∞ curve was unaffected by the antibiotic. Amphotericin B did not significantly change the delayed outward current but substantially increased the leakage current.

The effects of lowering the temperature from 33° to 23°C on the electrical activity and intracellular Na and K concentrations of isolated rabbit hearts were studied. The preparations were at a steady state for experimental periods of up to 80 min insofar as the extracellular space and the ionic content is concerned. Low temperature produced a decrease in intracellular sodium in both ventricles and an increase in intracellular potassium in the left ventricle. These ionic shifts did not alter the resting potential. The action potential amplitude and the overshoot were higher at 23°C than at 33°C. The maximum depolarization rate decreased and the action potential duration alternated regularly at 23°C. The decrease in V max is explained by a shift of the h∞ vs V curve towards more negative potentials. It is postulated that the cyclic changes in duration are due to the temperature and frequency dependence of the outward currents IK2 and Ix1 respectively.

The effects of tetrodotoxin (TTX) and verapamil on atrial, atrionodal and nodal action potentials were studied in the perfused rabbit heart. Control recordings from atrionodal and nodal fibers showed that the upstroke was composed of a fast depolarization (phase I) followed by a slow depolarization (phase II). The amplitude of these phases and their maximum rate of rise ( V max ), as well as action potential amplitude and resting potential, were determined under control conditions and in the presence of TTX (0.3 μ m ) or verapamil (1.1 μ m . TTX selectively depressed phase I in the action potentials from all fiber types. Concomitantly, the amplitude of phase II increased, but its V max did not change. In contrast, the effects of verapamil were limited to phase II, its V max , and the plateau. The slow depolarization was greatly depressed or abolished in the nodal action potential under verapamil. These results support the hypothesis that the fast sodium channel is present in the membrane of nodal cells and that the upstroke of the nodal action potential results from the activation of two inward currents, but the fast inward current contributes less to depolarization in nodal than in atrial fibers because of the low resting potential of the former. It is estimated that the slow inward current contributes about 40% of the total depolarization in nodal fibers.

The effects of ouabain (0.5 × 10−6 m ) on the intracellular ionic concentrations and the transmembrane potential were studied. Isolated rabbit hearts were perfused at 33°C and constant coronary flow. Krebs solution containing [14C]inulin was used to measure the extracellular space (ECS). The cell Na and K content were determined after 10 and 60 min perfusion with ouabain. The membrane potential was measured throughout the experiment. Ouabain produced a decrease in ECS and cell potassium ([K]i) and an increase in intracellular sodium ([Na]i) and water. There was a time lag between the change in [Na]i and the change in [K]i. A positive inotropic effect occurred during the first 10 to 20 min of perfusion with ouabain. The resting potential (RP) and action potential amplitude (APA) decreased with a similar time course, reaching values of 63 and 55% of the control by the end of the 60 min period. The maximum rate of depolarization ( V max ) decreased after 10 min, levelling off at about 50% of the control at 30 min. An early lengthening of the plateau was followed by progressive shortening of the action potential. It is postulated that the depolarization is due to inhibition of an electrogenic ionic pump, that the changes in APA are due to ionic shifts and depolarization, and that the values of V max are largely determined by shifts in the h∞ curve.

Cell clusters derived from ventricles of newborn rats were found to have action potentials insensitive to TTX but which were abolished by 2 mM manganese. Phase plane trajectories indicated that there was only one current component responsible for the upstroke of the action potential. The mean values for the electrical parameters of these cells were: maximum diastolic potential: −41.6 mV; resting potential: −38.2 m V; firing level: −26.0 m V; action potential amplitude: 59.8 mV; overshoot: 18.5 mV; maximum rate of depolarization 6.56 Vs −1 ; time to peak: 16.2 ms. The maximum inward current was estimated as 15 μA/cm 2 . Circumstantial evidence suggests that in these cells pacemaker activity depends on a slow inward current sensitive to manganese. This preparation could serve as model to study the properties of ectopic pacemakers in ventricular cells.