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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 influence of external potassium Ko and tetraethylammonium on the cellular K content of hypoxic myocardium was investigated. Perfused rabbit hearts were submitted to 60 min hypoxia in medium containing 5 mM K throughout or either low (1.5 mM) or high (10 mM) K during the last 20 min of hypoxia. Paced electrical activity (2.5 Hz) was kept throughout the experiments. Tissue samples excised from the left ventricle were analyzed for total water, inulin space, and Na and K content. Lowering Ko to 1.5 mM increased both K loss and Na accumulation. Addition of 3.5 mM RbCl under these conditions reversed Na accumulation to levels found for hypoxia in normal medium but did not modify the cellular K loss. Tetraethylammonium (10 mM) did not alter Na accumulation but partly prevented the decrease in K content produced by hypoxia. A similar effect was observed by increasing Ko to 10 mM. At this high Ko prolongation of hypoxia did not enhance K loss. Abolition of electrical activity by TTX in a high K solution prevented K loss and reduced the sodium content. These results are consistent with the view that voltage-dependent channels are implicated in the K loss induced by hypoxia or ischemia. Furthermore, they indicate that the K loss may be modulated by external K because of the influence of the electrochemical gradient on passive K efflux and thus provide an explanation for the existence of a plateau in the early extracellular K accumulation observed 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.

The resting potential and the intracellular Na and K concentrations (Nai, Ki) were determined at several extracellular K concentrations (Ko) between 0.5 and 18 mM and after inhibition of the sodium pump with 0.5 microM ouabain. Exposure to low Ko (0.5 mM) produced a transient hyperpolarization (from -80 to -100 mV) followed by a depolarization that led to a stable potential of -60 mV within 25 min. Similar potential levels were observed in the presence of ouabain regardless of the Ko/Ki ratio. Intracellular sodium increased at Ko < 5 mM, whereas Ki rose at Ko less than or equal to 1.0 mM. Because of the large decrease of Ki at Ko = 0.5 mM, Ko/Ki was the same at 0.5 and 1 mM. However, the resting potentials at the steady state differed by 50 mV at these concentrations. A PNa/PK of 0.032 for the control conditions was obtained with the Mullins-Noda equation using 2.5 as the Na-K coupling ratio. This PNa/PK value yielded a Goldman potential of -69 mV; so we estimated that electrogenic sodium extrusion contributed -10 mV to the resting potential. The size of the electrogenic potential increased as Ko was lowered from 5 to 1 mM. This finding suggests that the control of the Na-K coupling ratio may be independent of the mechanism that controls the pumping rate.

Spontaneously beating explanted neonatal rat ventricle cells stop beating and show a steady potential (the mean resting potential, −46.2 mV at 6.0 mM Ko) when exposed to 10 mM Cao or 4 mM Mn. When Ko was increased, resting potential changed only slightly between 3 and 15 mM, but the resting potential versus Ko characteristically approached the slope of a K electrode at high Ko Elimination of Cl from the medium did not alter the K dependence of the resting potential. However, a hyperpolarization of 9 mV per 10-fold change was observed when Nao was decreased from 50 to 4 mM. Ouabain (10−4 M) depolarized the membrane within 2 min to a stable level of about −30 mV in spontaneously beating cells and in those treated with Ca channel blockers. This potential was considered as the diffusion component of the membrane potential, Vdiff. Consequently the difference between resting potential and Vdiff represents the ouabain-sensitive or the electrogenic component of the resting potential. Using linearized versions of the Mullins and Noda as well as the Goldman – Hodgkin – Katz equations, we calculated that a PNa/PK between 0.25 and 0.35, a Na/K exchange ratio of 2.0, and a Ki of 160 mM adequately described the K dependence of the resting potential. We demonstrated the contribution of electrogenic Na extrusion to the resting potential of mammalian cardiac cells in culture. Therefore the existence of a composite resting potential precludes the direct comparison of potential measurements obtained under conditions liable to independently modify either the diffusion or the electrogenic component.

In the perfused rabbit heart,the upstroke of the transmembrane action potential of fibers of the atrioventricular (AV) node presents two distinct components. The first depends strongly on extracellular sdoium concentration, but the degree to which it is activated is influenced by extracellular calcium, as indicated by the correlation between its Vmax and [Ca2+]0. The second component depends on calcium and sodium concentrations and is blocked by Mn ions. An analysis comparing action potentials from atrial (A), atrionodal (AN), and nodal (N) fibers shows that the second component of the upstroke of the action potential contributes 12%, 27%, and 34% to the total depolarization. The results suggest that the upstroke of the nodal action potential results from the activation of two inward currents, as in ordinary cardiac fibers. We postulate that (1) the degree of steady state inactivation of gNa is larger in N than in A fibers because of the low resting potential of the former, and (2) the contribution of the second channel to the upstroke depends on the time course of the previous depolarization and the potential level at which this component is activated.

A progressive conduction block leading to atrioventricular dissociation develops in perfused rabbit hearts within 20–30 min of exposure to Krebs containing 0.5 mM potassium (low K). A decrease in potassium permeability resulting in membrane depolarization (as seen in Purkinje fibers) could be responsible for the loss of excitability in nodal cells. We investigated the K dependence of the resting potential and the long-term effects of low K perfusion on the resting and action potentials of nodal cells in rabbit hearts. The resting potential of atrial, atrionodal, and nodal cells varied by 5 2, 41, and 34 mV per decade of change in Ko within the range of 5–50 mM K. Hyperpolarization of the resting membrane, a progressive decline in action potential amplitude, and a decrease in maximum rate of rise were observed in nodal fibers when exposed to low K. Loss of propagated activity occurred in the middle node within 20–30 min while the cells remained hyperpolarized. There was no evidence of electrogenic Na extrusion and it seems that the low nodal resting potential results from a high resting PNa/PK permeability ratio. The early decrease in rate of rise in low K probably reflects an increase in K-dependent outward currents, whereas the progressive deterioration and final loss of conducted electrical activity may result from an accumulation of internal Na and Ca overload produced by low K inhibition of the Na pump.

Intracellular chloride activity (aiCl) and membrane potential (Vm) were measured in rabbit papillary muscle under in vitro conditions. The cellular chloride concentration (Cli) was estimated from measurements of total water content, extracellular space, and total chloride concentration. The effects of therapeutic (10(-8) M) and toxic (10(-6) M) concentrations of ouabain on these parameters were tested. The chloride-sensitive microelectrodes were of the liquid-ion exchanger type. Selectivity for HCO-3 was taken into account in the calculation of aiCl. In 11 control experiments made with two different protocols aiCl was determined in subendocardial and in deeper cells. The mean membrane potentials were -78.7 and -78.0 mV and the mean cytoplasmic chloride activities were 17.5 and 17.7 mM, respectively. The chloride equilibrium potentials were -43.5 and -43.2 mV. These results indicated that chloride is not passively distributed in rabbit papillary muscle. Ouabain (10(-8) M) did not change Vm or aiCl. At a toxic concentration of ouabain, Vm fell to -68.0 mV in superficial cells and to -67.8 mV in deeper cells, but aiCl remained unchanged . These results suggested that under in vitro conditions intracellular chloride is distributed within more than one cellular compartment.

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 diffusion and electrogenic components of the resting potential of hypoxic ventricular muscle were separated by inhibition of the sodium pump with 10−4 M ouabain. The response to varying external K concentrations (Ko) was studied. Arteriaily perfused rabbit hearts were submitted to 60 min hypoxia in Krebs solution containing 5 mM K throughout or to different external K concentrations during the last 20 min of hypoxia. For K concentrations between 1.5 and 10 mM, hypoxia did not change the resting potential except for a slight hyperpolarization in 7.5 mM K. The diffusion component of the resting potential did not differ from the resting potential at Ko o values between 5 and 10 mM. The internal K concentration, Ki, was estimated from extrapolations to zero potential of the relation resting potential vs. Ko in normoxic and hypoxic hearts. These experiments revealed a decline of Ki of 16 mM with hypoxia. The variation of the diffusion potential with external K was fitted by a PNa:PK ratio five times lower than in normoxia. It has been concluded that an increase in K permeability and the persistence of electrogenic Na extrusion during hypoxia of rather short duration prevent membrane depolarization despite the myocardial K loss.

Insulin stimulates ionic transport by the sodium pump and induces hyperpolarization in skeletal and cardiac muscle among other cells. The insulin-induced hyperpolarization in most cases can be inhibited by exposure to cardiac glycosides or metabolic inhibition. However, extracellular accumulation of K ions leaking from hypoxic cells in superfused preparations may distort the effects of insulin on the resting potential. We used standard microelectrode techniques and perfused rabbit hearts submitted to hypoxia and substrate deprivation to reinvestigate the effects of insulin (6.4 nM) on the membrane potential. The membrane depolarized by about 6 mV and the action potential was reduced to a sharp spike without overshoot. Insulin restored the resting potential to control values but did not change the action potential configuration substantially. The insulin-induced repolarization was not due to reuptake of potassium as revealed by spectrophotometric determinations of myocardial K content. In addition, the diffusion component of the resting potential measured after inhibition of the sodium pump with 10−4 M ouabain was not modified by insulin. Our results suggest that an increase in the contribution of electrogenic Na extrusion to the resting potential underlies the repolarizing effect of insulin of hypoxic substrate-deprived myocardium.

In clusters of trypsinized ventricle cells from neonatal rats which exhibit slow response action potentials, streptomycin in concentrations from 0.17 to 5.5 mM significantly inhibits the beating rate. Microelectrode experiments performed at a concentration of 5.5 mM revealed a reduction in the slope of diastolic depolarization from 149 to 53 mV/s whereas the maximum diastolic potential depolarized from −42.4 to −33.6 mV which entailed a decrease in overshoot and maximum rate of rise of the action potential. We conclude that the decrease of the slope of diastolic depolarization mainly determines the slowing of the beating rate and that streptomycin interferes with the pacemaker mechanism usually associated with the slow response.

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.

Isolated rabbit hearts undergo ventricular depolarization when exposed to 0.5 mM K – Krebs during 30 to 45 min. A ventricular tachyarrhythmia develops when these hearts are allowed to repolarize in 1.5 mM K – Krebs. Ventricular fibrillation usually follows. These arrhythmias do not cease spontaneously. Initially, perfusion with 0.5 mM K hyperpolarized the membrane to −100 ± 1.2 mV [Formula: see text], decreased the plateau level, greatly increased the action potential duration, and slowed down intraventricular conduction, despite the level of membrane polarization and a high rate of rise of the upstroke. After a delay of 15 to 30 min, a progressive depolarization occurred and a stable potential level of −61 ± 1.9 mV was reached. The arrhythmia elicited by perfusion with 1.5 mM K was of ventricular origin but did not appear when the previous exposure to 0.5 mM K was reduced to 10 min. Addition of verapamil (1.0 μM) to the 1.5 mM K medium did not prevent the early appearance of the arrhythmia, but restored driven electrical activity after variable delays despite the persistence of alterations in the action potential configuration. Verapamil also abolished the slow depolarizing phase of the upstroke. It is proposed that the abnormal electrical activity arose in a partly depolarized Purkinje network and it may have been triggered by the electrical nonhomogeneity created by the repolarization of ventricular cells in an extracellular K+ concentration of 1.5 mM.

Voltage and current clamp experiments were performed on rat ventricular strips under anoxia. 1. Under the influence of anoxia the membrane depolarized by 5 to 10 mV and the action potential amplitude decreased by 15 mV. The plateau disappeared and the duration of the action potential was shortened. 2. The slow inward current was reduced by 50 to 80% and its reversal potential became more negative by about 31 mV. The conductance of the slow inward channel decreased by 26%. 3. The net outward current was slightly depressed.

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.

The ventricular membrane potential was measured in the perfused rabbit heart under control conditions and during total cardiac ischemia produced by an arrest of the coronary perfusion. The steady-state inactivation characteristic of the sodium system and the sodium dependence of the upstroke velocity were determined by measurements of the maximum rate of rise ([Formula: see text]) as a function of the resting potential (RP) and the [Na]o to [Na]i ratio. The intracellular concentrations of sodium and potassium ([Na]i, [K]i) were estimated from measurements of the cellular electrolyte content, the total water content, and the volume of the extracellular space. Ischemia produced a net sodium gain of 58 mmol/kg dry weight and a slight loss of potassium. As a consequence, [Na]i increased and [K]i decreased. Under ischemia the resting potential was closer to the potassium equilibrium potential than in the controls. The changes in action potential configuration and plateau level suggested that ischemia inhibited the slow inward current. The [Formula: see text] decreased within 10 min of ischemia. Neither membrane depolarization nor a reduction in the sodium gradient could entirely explain the low [Formula: see text] of ischemic cells. It is concluded that the electrophysiologic effects of ischemia result from changes in ionic gradients and membrane electrical properties.

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.

The electrophysiologic effects of a toxic concentration of ouabain (10−5 M) were studied in frog atrial trabeculae. The toxic concentration was determined by the appearance of a negative inotropic effect and an increase in basal tension. Current- and voltage-clamp measurements were performed. Ouabain did not alter the passive electrical properties of the preparation. Under current-clamp conditions the membrane depolarized and the action potential amplitude as well as its maximum rate of rise decreased. The current–voltage curve for the fast inward current was shifted toward more positive potentials and the maximum sodium current decreased. The maximum sodium conductance was also reduced. The process of reactivation of the fast inward current was accelerated. The slow inward current and the maximum slow conductance also decreased under ouabain. These effects could explain the negative inotropic action of high concentrations of glycosides, as well as the action potential changes observed by several investigators. They also help to understand the arrhythmogenic effects of high concentrations of digitalis.