Relationships between voltage and tension in sheep cardiac Purkinje fibers

The two-microelectrode technique of voltage clamping sheep cardiac Purkinje fibers was used to examine the changes in contraction which occur during trains of voltage clamps. (A "train" is defined as a series of voltage clamps delivered at a particular rate, beginning after a rest long enough that the effects of previous stimulation have died away.) Contractions showed striking staircases, or progressive changes in peak isometric tension, during trains. Short clamps, clamps to voltages more negative than --20 or --30 mV, or holding potentials less negative than the resting potential favored negative staircases, while long clamps, clamps to positive voltages, and holding potentials near the resting potential each favored positive staircases. The staircase behavior appeared to be due to changes in the initial rate of recovery of the ability to contract. The changes in staircase behavior as a function of clamp voltage suggested that the relationship between peak tension and clamp voltage should depend on the experimental design. When the steady-state contraction was plotted as a function of clamp voltage, voltage-tension relations like those recently reported for working ventricle were obtained, with a threshold between --30 and - -40 mV and a steep relation between tension and voltage. When the first contraction after a rest was plotted, the threshold voltage was more negative, the curve was flatter, and the peak tensions at inside positive voltages were reduced.     

Slow inward current and contraction of sheep cardiac Purkinje fibers

A "slow" inward current (Is) has been identified in ventricular muscle and Purkinje fibers of several mammalian species. The two- microelectrode voltage clamp technique is used to examine some of the relationships between Is and contraction of the sheep cardiac Purkinje fiber. "Tails" of inward current occurring on repolarization and extrapolation of Is recovery each show that the Is system may not inactivate completely during prolonged depolarization. The rate of recovery of Is after a depolarization is slow, and when a train of 300- ms clamps (frequency 1 s-1) is begun after a rest, Is is larger for the first clamp than it is for succeedings clamps. For the first clamp after a rest, the thresholds for Is and tension are the same and there is a direct correlation between peak tension and peak Is for clamp voltages between threshold and minus 40 mV. After a clamp, however, the ability to contract recovers much more slowly than does Is. Therefore, since Is may occur under certain conditions without tension, the realtionship between Is and tension must be indirect. Calcium entering the cell via this current may replenish or augment an intracellular calcium pool.     

Effects of changes of intracellular pH on contraction in sheep cardiac Purkinje fibers

Intracellular pH (pHi) was measured with a pH-sensitive microelectrode in voltage-clamped sheep cardiac Purkinje fibers while tension was simultaneously measured. All solutions were nominally CO2/HCO3 free and were buffered with Tris. The addition of NH4Cl (5-20 mM) produced an initial intracellular alkalosis that was associated with an increase of twitch tension. At the same time, a component of voltage-dependent tonic tension developed. Prolonged exposure (greater than 5 min) to NH4Cl resulted in a slow recovery of pHi accompanied by a decrease of tension. Removal of NH4Cl produced a transient acidosis that was accompanied by a fall of force. In some experiments, there was then a transient recovery of force. If extracellular pH (pHo) was decreased, then pHi decreased slowly. Tension also fell slowly. An increase of pHo produced a corresponding increase of both force and pHi. The application of strophanthidin (10 microM) increased force and produced an intracellular acidosis. The addition of NH4Cl, to remove this acidosis partially, produced a significant increase of force. The above results show that contraction is sensitive to changes of intracellular but not extracellular pH. This pH dependence will therefore modify the contractile response to inotropic maneuvers that also affect pHi.     

Effect of diameter on membrane capacity and conductance of sheep cardiac Purkinje fibers

Membrane electrical properties were measured in sheep cardiac Purkinje fibers, having diameters ranging from 50 to 300 mum. Both membrane capacitance and conductance per unit area of apparent fiber surface varied fourfold over this range. Membrane time constant, and capacitance per unit apparent surface area calculated from the foot of the action potential were independent of fiber diameter, having average values of 18.8 +/- 0.7 ms, and 3.4 +/- 0.25 muF/cm2, respectively (mean +/- SEM). The conduction velocity and time constant of the foot of the action potential also appeared independent of diameter, having values of 3.0 +/- 0.1 m/s and 0.10 +/- 0.007 ms. These findings are consistent with earlier suggestions that in addition to membrane on the surface of the fiber, there exists a large fraction of membrane in continuity with the extracellular space but not directly on the surface of the fiber. Combining the electrical and morphological information, it was possible to predict a passive length constant for the internal membranes of about 100 mum and a time constant for chaning these membranes in a passive 100-mum fiber of 1.7 ms.     

Fluctuations in membrane current driven by intracellular calcium in cardiac Purkinje fibers

Spontaneous oscillatory fluctuations in membrane potential are often observed in heart cells, but their basis remains controversial. Such activity is enhanced in cardiac Purkinje fibers by exposure to digitalis or K-free solutions. Under these conditions, we find that voltage noise is generated by current fluctuations that persist when membrane potential is voltage clamped. Power spectra of current signals are not made up of single time-constant components, as expected from gating of independent channels, but are dominated by resonant characteristics between 0.5 and 2 HZ. Our evidence suggests that the periodicity arises from oscillatory variations in intracellular free Ca that control ion movements across the surface membrane. The current fluctuations are strongly cross-correlated with oscillatory fluctuations in contractile force, and are inhibited by removing extracellular Ca or exposure to D600. Chelating intracellular Ca with injected EGTA also abolishes the current fluctuations. The oscillatory mechanism may involve cycles of Ca (or Sr) movement between sarcoplasmic reticulum and myoplasm, as previously suggested for skinned cardiac preparations. Our experiments in intact cells indicate that changes in surface membrane potential can modulate cytoplasmic Ca oscillations in frequency and perhaps amplitude as well. A two-way interaction between surface membrane potential and intracellular Ca stores may be a common feature of heart, neuron, and other cell types.     

4-Aminopyridine and the early outward current of sheep cardiac Purkinje fibers

We have studied the effects of the potassium-blocking agent 4-aminopyridine (4-AP) on the action potential and membrane currents of the sheep cardiac Purkinje fiber. 4-AP slowed the rate of phase 1 repolarization and shifted the plateau of the action potential to less negative potentials. In the presence of 4-AP, the substitution of sodium methylsulfate or methanesulfonate for the NaCl of Tyrode's solution further slowed the rate of phase 1 repolarization, even though chloride replacement has no effect on the untreated preparation. In voltage clamp experiments, 4-AP rapidly and reversibly reduced the early peak of outward current that is seen when the Purkinje fiber membrane is voltage-clamped to potentials positive to -20 mV. In addition, 4-AP reduced the steady outward current seen at the end of clamp steps positive to -40 mV. 4-AP did not appear to change the slow inward current observed over the range of -60 to -40 mV, nor did it greatly change the current tails that have been used as a measure of the slow inward conductance at more positive potentials. 4-AP did not block the inward rectifying potassium currents, IK1 and IK2. A phasic outward current component that was insensitive to 4-AP was reduced by chloride replacement. We conclude that the early outward current has two components: a chloride-sensitive component plus a 4-AP-sensitive component. Since a portion of the steady-state current was sensitive to 4-AP, the early outward current either does not fully inactivate or 4-AP blocks a component of time-independent background current.     

Effects of caffeine, tetracaine, and ryanodine on calcium-dependent oscillations in sheep cardiac Purkinje fibers

Membrane current and tension were measured in voltage-clamped sheep cardiac Purkinje fibers. Elevating the intracellular calcium concentration ([Ca2+]i) results in oscillations of membrane current and tension both at rest and during stimulation. During stimulation, an oscillatory transient inward current and an after contraction follow repolarization. We have examined the effects on the oscillations of changing the extracellular calcium concentration ([Ca2+]o) and of adding various drugs. In agreement with previous work, high concentrations of drugs that affect the sarcoplasmic reticulum, namely caffeine (10-20 mM), tetracaine (1 mM), and ryanodine (10 microM), abolish the oscillations. However, at lower concentrations, these three drugs have different effects on the oscillations. Caffeine (1-2 mM) decreases the oscillation amplitude but increases the frequency. Tetracaine (100-500 microM) has little effect on the magnitude of the oscillations but decreases their frequency. Ryanodine, at all concentrations used (0.1-10 microM), eventually abolishes the oscillations but, in doing so, decreases the magnitude, leaving the frequency unaffected. When [Ca2+]o was changed in order to vary [Ca2+]i, both the frequency and the magnitude of the oscillations always changed in the same direction. This suggests that these three drugs have effects in addition to just changing [Ca2+]i.     

Electrogenic sodium extrusion in cardiac Purkinje fibers

Thin canine cardiac Purkinje fibers in a fast flow chamber were exposed to K-free fluid for 15 s to 6 min to initiate "sodium loading," then returned to K-containing fluid to stimulate the sodium pump. The electrophysiological effects of enhanced pump activity may result from extracellular K depletion caused by enhanced cellular uptake of K or from an increase in the current generated as a result of unequal pumped movements of Na and K, or from both. The effects of pump stimulation were therefore studied under three conditions in which lowering the external K concentration ([K]0) causes changes opposite to those expected from an increase in pump current. First, the resting potential of Purkinje fibers may have either a "high" value of a "low" (less negative) value: at the low level of potential, experimental reduction of [K]0 causes depolarization, whereas an increase in pump current should cause hyperpolarization. Second, in regularly stimulated Purkinje fibers, lowering [K]0 prolongs the action potential, whereas an increase in outward pump current should shorten it. Finally, lowering [K]0 enhances spontaneous "pacemaker" activity in Purkinje fibers, whereas an increase in outward pump current should reduce or abolish spontaneous activity. Under all three conditions, we find that the effects of temporary stimulation of the sodium pump are those expected from a transient increase in outward pump current, not those expected from K depletion.     

An upper limit for the electrogenic Na-K pump contribution to maximum diastolic potential in feline cardiac Purkinje fibers in steady state

We have estimated an upper limit for the electrogenic contribution of the Na-K pump to diastolic transmembrane potential. We simultaneously monitored the maximum diastolic potential and the extracellular space potassium activity during exposure to a very high concentration of ouabain. Exposure to ouabain caused a depolarization of approximately 3 mV (n = 33 experiments) over 34 +/- 3 s (mean +/- standard error) prior to any change in extracellular K activity. In four experiments, we monitored intracellular sodium activity and observed it to rise with approximately the same temporal lag (delay = 26 +/- 7 s). We also measured relative membrane conductance in one series of experiments and observed it to decrease to 91 +/- 2% of its control value by the time extracellular space K began to rise. Following the initial increase in extracellular space K activity the subsequent membrane depolarization is shown to be accurately predicted solely from the measured increase in extracellular space K activity as calculated from the Goldman equation. Limitations of the method and possible interpretations of the data are discussed. We interpret this ouabain-induced depolarization that occurs prior to the rise in external K to be an upper limit to the Na-K pump's electrogenic contribution to steady-state membrane potential.     

Strophanthidin inotropy in cardiac Purkinje fibers under conditions that vary cellular sodium or calcium

The role of sodium and calcium on strophanthidin inotropy was studied in canine cardiac Purkinje fibers perfused in vitro under conditions that vary cellular sodium and calcium. With high concentrations of strophanthidin (greater than or equal to 10(-7) M), force increases more in the presence of low [Ca]0 or high [Na]0 and less in the presence of a low sodium-calcium concentration solution than in Tyrode solution. In a solution with a low concentration of sodium-calcium containing strophanthidin, restoring [Na]0 to normal decreases and then re-increases force: when [Na]0 is decreased again, the force transiently overshoots. These effects of strophanthidin are exaggerated by metabolic inhibitors. In a low [Ca] solution, low concentrations of strophanthidin (3 X 10(-8) or 5 X 10(-8) M) re-increase force a little or not at all. On recovery, the transient force increase is not exaggerated by low strophanthidin and is absent after manganese exposure. The inotropy of low concentrations of strophanthidin is potentiated by norepinephrine, high [Ca]0 (4 mM), or by lowering [Na]0. Thus, the present results suggest that the inotropic action of high strophanthidin concentrations depends primarily on sodium and secondarily on calcium, and that the inotropic action of low concentrations of strophanthidin involves a modification of the cell response to calcium.     

The effects of acidosis and bicarbonate on action potential repolarization in canine cardiac Purkinje fibers

Studies were performed on canine cardiac Purkinje fibers to evaluate the effects of acidosis and bicarbonate (HCO3) on action potential repolarization. Extracellular pH (pHe) was reduced from 7.4 to 6.8 by increasing carbon dioxide (CO2) concentration from 4 to 15% in a HCO3-buffered solution or by NaOH titration in a Hepes-buffered solution. Both types of acidosis produced a slowing of the rate of terminal repolarization (i.e., period of repolarization starting at about -60 mV and ending at the maximum diastolic potential) with an attendant increase in action potential duration of 10--20 ms. This was accompanied by a reduction in the maximum diastolic potential of 2--8 mV. In contrast, if the same pH change was made by keeping CO2 concentration constant and lowering extracellular HCO3 from 23.7 to 6.0 mM, in addition to the slowing of terminal repolarization, the plateau was markedly prolonged resulting in an additional 50- to 80-ms increase in action potential duration. If pHe was held constant at 7.4 and HCO3 reduced from 23.7 mM to 0 (Hepes-buffered solution), the changes in repolarization were nearly identical to those seen in 6.0 mM HCO3 except that terminal repolarization was unchanged. This response was unaltered by doubling the concentration of Hepes. Reducing HCO3 to 12.0 mM produced changes in repolarization of about one-half the magnitude of those in 6.0 mM HCO3. These findings suggest that in Purkinje fibers, HCO3 either acts as a current that slows repolarization or modulates the ionic currents responsible for repolarization.     

Influence of chloride, potassium, and tetraethylammonium on the early outward current of sheep cardiac Purkinje fibers

In voltage clamp studies of cardiac Purkinje fibers, a large early outward current is consistently observed during depolarizations to voltages more positive than -20 mV. After the outward peak of the current, the total membrane current declines slowly. Dudel et al. (1967. Pfluegers Arch. Eur. J. Physiol. 294:197--212) reduced the extracellular chloride concentration and found that the outward peak and the decline of the current were abolished. They concluded that the total membrane current at these voltages was largely determined by a time- and voltage-dependent change in the membrane chloride conductance. We reinvestigated the chloride sensitivity of this current, taking care to minimize possible sources of error. When the extracellular chloride concentration was reduced to 8.6% of control, the principal effect was a 20% decrease in the peak amplitude of the outward current. This implies that the membrane chloride conductance is not the major determinant of the total current at these voltages. The reversal potential of current tails obtained after a short conditioning depolarization was not changed by alterations in the extracellular chloride or potassium concentrations. We suspect that the tail currents contain both inward and outward components, and that the apparent reversal potential of the net tail current largely reflects the kinetics of the outward component, so that this experiment does not rule out potassium as a possible charge carrier. The possibility that potassium carries much of the early outward current was further investigated using tetraethylammonium, which blocks potassium currents in nerve and skeletal muscle. This drug substantially reduced the early outward current, which suggests that much of the early outward current is carried by potassium ions.     

Intracellular pH and cell-to-cell transmission in sheep Purkinje fibers.

Intracellular pH (pHi) is a significant modifier of cell-to-cell communication in some tissues but its role is uncertain in heart tissue. The present studies examined the effect of cytosolic protons on electrotonic spread and conduction velocity in cardiac Purkinje fibers. Cable analysis provided values for internal longitudinal resistance (ri) and pH-selective microelectrodes monitored pHi during CO2 and HCO3- alterations. Resting fibers developed changes in ri that were proportional to intracellular free proton concentration ([H+]i) during CO2 changes at constant [HCO3-]. However, the effects on ri were small between pHi 6.9-7.8 and predicted only a 2.2% increase in ri per 10 nM increase in [H+]i. Other findings suggested that titration of cytosolic protons may not directly produce the changes in ri: (a) For an equal change in [H+]i, the effects on ri were roughly three times greater (6.8% increase per 10 nM rise in [H+]i) if bicarbonate was lost during CO2 changes. (b) pH-associated changes in ri were preceded by a time delay (1-5 min) producing hysteresis in the [H+]i-ri relation during successive perturbations. (c) The same CO2 variations modified the direction and magnitude of ri differently during pacing than at rest. The cumulative results suggest that the action of protons on ri in the heart may be subordinate to another regulator or mediated by another pH-dependent substance or reaction.     

The effects of sodium pump activity on the slow inward current in sheep cardiac Purkinje fibres

The effects of Na pump activity on the slow inward current, Isi, magnitude and twitch tension were investigated in sheep cardiac Purkinje fibres. A two-microelectrode voltage-clamp method was used, tension being measured simultaneously. Na pump activity was lowered either by reducing the extracellular K concentration, [K]O, or by applying the cardiotonic steroid strophanthidin. Reduction of [K]O from 4 to 0 mM leads to time-dependent increases in Isi magnitude and twitch tension. The increases of Isi and tension could be reversed by adding Tl, Rb, Cs or NH4 ions to the K-free superfusate. The actions of these ions are attributed to the known ability of these cations to activate the external site of the Na pump. This conclusion is supported by the observation that such activator cations do not reverse the increases in Isi and tension produced by strophanthidin. We conclude that the effects of low [K]O on Isi are mediated by Na pump inhibition. Similarly the Na pump inhibition produced by strophanthidin increases Isi and tension, although, in this case, other mechanisms may also contribute. Measurements of the activity of the electrogenic Na pump show that elevated intracellular Na ion concentration secondary to Na pump inhibition and not the instantaneous Na pump turnover rate mediates the increase in Isi magnitude.     

Cell-to-Cell diffusion of Procion Yellow in sheep and calf Purkinje fibers

Summary Sheep and calf Purkinje fibers (false tendons) were cut near one end and exposed to a solution containing no calcium and the dye Procion Yellow (M4RS, molecular weight near 700). Fifteen minutes later the damaged end was sealed by applying calcium ions (Tyrode solution). Traces of Procion Yellow were detected within the intracellular compartment at a distance of 2.4 mm from the site of damage when the preparations had been washed in dye-free solution for 4 hr. This indicates that the dye had diffused through about 20 cells in succession. There was no detectable uptake of Procion Yellow through intact surface membranes. Visual curve fitting to quantitative data on concentrationvs. distance gives an apparent diffusion coefficient (cell junctions and myoplasm in series) of 3×10^–8 cm² sec^–1, as against 1×10^–6 cm² sec^–1 in an agar gel. It is concluded that specialized contact areas between neighboring cardiac cells represent a considerable yet not an absolute hindrance to the movement of this particle.     

Relation between intracellular Na ion activity and tension of sheep cardiac Purkinje fibers exposed to dihydro-ouabain.

The intracellular Na ion activity (aiNa) and the contractile tension (T) of sheep cardiac Purkinje fibers were simultaneously measured employing recessed-tip Na+-selective glass microelectrodes and a mechano-electric transducer. The aiNa of 6.4 +/- 1.6 mM (mean +/- SD, n = 56) was obtained in fibers perfused with normal Tyrode's solution. The changes in aiNa and T were measured during and after the exposure of fibers to a cardiac glycoside, dihydro-ouabain (DHO) in concentrations between 5 X 10(-8) M and 10(-5) M. The exposure time to DHO was 15 min. Both aiNa and T did not change in fibers exposed to 5 X 10(-8) M DHO, and the threshold concentration for the effect of DHO appeared to be around 10(-7) M. In DHO concentrations greater than the threshold, the increases in aiNa and T strongly correlated during the onset of DHO effects. The recoveries of aiNa and T were variable and slow, being dependent on the DHO concentration. In those fibers which recovered from the effects of DHO, the time-course of aiNa recovery was similar to that of T recovery. In fibers exposed to DHO of 5 X 10(-6) M or greater, the apparent toxic effects were observed in both action potential and contraction after an initial increase in T. The fibers manifesting the apparent toxic effects has a aiNa of approximately 30 mM or greater. The results of this study indicate that the increase in aiNa is associated with the positive inotropic action of the cardiac glycoside.