(40-lOOup.) than ordinary heart muscle fibres and which are only slightly

Transcription

1 227 J. Physiol. (I 95 I) I"5, EFFECT OF CURRENT FLOW ON THE MEMBRANE POTENTIAL OF CARDIAC MUSCLE BY SILVIO WEIDMANN* From the Physiological Laboratory, University of Cambridge (Received 4 June 1951) Microcapillaries with an external tip diameter of 0 5p. (Ling & Gerard, 1949) can be inserted into single fibres of the cardiac syncytium and may be used to record resting and action potentials. The technique has so far been applied to the frog ventricle (Woodbury, Hecht & Christopherson, 1951) and to 'false tendons' of the dog and kid heart (Draper & Weidmann, 1951). The conductive system of the kid contains typical Purkinje fibres which are larger in diameter (40-lOOup.) than ordinary heart muscle fibres and which are only slightly contractile. In this preparation it is possible to insert two microelectrodes into the same fibre and to leave them intracellular for many minutes. Polarizing current can be applied through one electrode and the change in membrane potential -resulting from current flow recorded through the other electrode. The present paper contains an account of experiments designed to investigate the cardiac action potential by applying suitable pulses of current at various stages in the cardiac cycle. METHOD The method was the same as that described in an earlier paper (Draper & Weidman, 1951). Fig. 1 shows the essential elements of the recording and polarizing circuit. RESULTS Impedance changes in the course of the cardiac cycle The relative magnitude of the membrane resistance was measured in the following way: square pulses of polarizing current were applied through a first pair of electrodes placed on opposite sides of the surface membrane (Fig. 1). Potential changes resulting from current flow were recorded by a second pair of electrodes in a similar position. No voltage changes were observed when the tip of the recording microelectrode was just extracellular. This indicates that the voltage changes recorded from the inside of a cell were localized at * Present address: Department of Physiology, University of Berne, Switzerland.

2 228 SILVIO WEIDMANN the fibre membrane. Their amplitude was used to estimate changes in membrane resistance. The action potential of Purkinje tissue of the kid consists of an initial spike followed by a plateau and a phase of repolarization (Draper & Weidmann, 1951). During the period of mechanical rest there is generally some slow depolarization. In the present series of experiments the upstroke of the action potential was used to trigger the time base so that successive traces could be superimposed. The initial spike then appeared extended over the flyback of the sweep cycle while the plateau of the action potential, the phase ofrepolarization and the changes during diastole could be seen on the time base in the usual manner V as5mf <, XLIt3~~~~~~~~ Ag-AgCl Rfl Agar-Ringer electrode D~Eclaycrc]i Square pulse D. generator Fig. 1. Diagram of experimental arrangement. PE =polarizing electrode. RE =recording electrode. DCA =push-pull cathode follower and differential d.c. amplifier. R =high speed relay (Carpenter 3G2). P=potentiometer with 10 steps of 10kQ. A typical experiment is illustrated in Fig. 2 which was made by exposing the film for twenty cycles of activity. During this period rectangular pulses of anodal current lasting 40 msec. were applied to the membrane at a frequency of about 12 cyc./sec. These pulses were not synchronized with the action potential and therefore occurred at many different times in the cardiac cycle. The upper edge of the superimposed traces corresponds to an action potential in the absence of applied current while the lower edge represents action potentials displaced by anodal current. During diastole the process of charging and discharging the membrane was half complete in about 20 msec. At the crest of activity the half time was shorter than 1 msec. The breadth of the band in Fig. 2 was used as a qualitative index of the membrane resistance. The interpretation of this experiment is as follows: (1) During diastole the slow depolarization is associated with an increase in membrane resistance. (2) At the height of the spike the membrane resistance is greatly decreased.

3 POLARIZATION OF CARDIAC MUSCLE 229 (3) The resistance recovers during the few milliseconds between the crest of the spike and the beginning of the plateau. (4) During the plateau the resistance is comparable to that in diastole and increases as the phase of repolarization is approached. (5) The shortening of the time constant suggests that cardiac muscle is similar to other excitable tissues in that the membrane capacity changes less than the membrane resistance (cf. Cole & Curtis, 1939). Fig. 2. Impedance changes in the course of the cardiac action potential. Voltage calibration in steps of 10 mv. Time marks for left-to-right movement of the cathode ray at intervals of 100 msec. Duration of flyback of sweep cycle approx. 12 msec. A rough estimate of the change in membrane resistance may be obtained -from the equations of cable theory. For this purpose a relation used by Hodgkin & Huxley (1947) was employed in the following form log0 [=~~ee log,, f +(f-)(1 _VB toie where VB = amplitude of the electrotonic potential I distance between leading-in and recording electrode A =,space constant of the fibre in diastole 2= ratio of membrane resi-stance in dia-stole and at crest of spike. In practice it was convenient to use the ratio of current strengths required to produce the same potential change instead of the ratio of potentials produced by current of the same strength. In the experiment illustrated by Fig. 3 the

4 230 SILVIO WEIDMANN ratio of current strengths was 22 and the electrode distance 0 33 mm. The space constant was not determined on this fibre but averaged about 2 mm. in other experiments (Weidmann, in preparation). With these figures equation (1) indicates a resistance ratio of 56: 1. This ratio is subject to the following sources of error: (a) The measurements were not made on a uniform fibre of infinite length but on one which fused into other fibres of the syncytium at a distance of one or Fig. 3. Potential changes due to square pulses of polarizing current applied in the middle of diastole. The record shows on the left the phase of repolarization, on the right the upstroke of the action potential. Seventeen traces are superimposed. Relative strength of polarizing current reading from bottom to top: 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, O, -1, -2, -3, -4, -5, -6. Time marks at intervals of 100 msec. two diastolic space constants. (b) Equation (1) applies to a fibre which is thin compared to its space constant. This approximation was entirely satisfactory in diastole but may have introduced some error at the crest of the spike since the space constant is then only about twice the fibre diameter. (e) Charging of the membrane capacity by the current pulses was only about 90% complete during diastole. These three sources of error tend to make the estimated ratio too low.

5 POLARIZATION OF CARDIAC MUSCLE Subthreshold potentials Fig. 3 shows a family of electrotonic potentials produced by square pulses of constant current which were timed to fall at a constant phase of the cycle. Successive traces were superimposed by synchronizing the sweep circuit with the upstroke of the action potential. It can be seen that anodal currents of increasing strength give changes in membrane potential smaller than those expected on the assumption of a constant membrane resistance. On the other hand the depolarization produced by a given increment of cathodal current increased continuously as the threshold was approached. The strongest cathodal current initiated an extrasystole. The cathodal part of the record looks strikingly similar to the curves obtained on invertebrate nerve (Hodgkin & Rushton, 1946) and skeletal muscle (Katz, 1948). One respect in which it differs is that the time course of the potential after the break of the current exhibits the phenomena of postcathodal depression and postanodal enhancement. Peculiarities of the pacemaker region The pacemaker was localized by recording simultaneously from two intracellular electrodes and by shifting the one nearest the point of origin of the spike until it gave the earliest upstroke. At this point the action potential was regularly found to have a slow upward curvature preceding the most abrupt part of the rising phase. This feature was absent or less marked in other parts of the fibre. It was also found that, in this region, application of a square pulse of subthreshold cathodal current increased the duration of diastole, presumably as a result of postcathodal depression, while the opposite effect was observed after the break of an anodal current. If strong enough the cathodal current evoked an extrasystole. These features are illustrated in Fig. 4. Propagated wave of repolarization In the experiment illustrated in Fig. 5 square pulses of anodal current were applied during the upstroke of the action potential and terminated at the beginning of the plateau. The current strength was increased in seven steps of equal increment. After the break of steps 1-4 the cathode-ray tube beam followed its usual course. With strength 5 a short spike preceded the return to the plateau. This overshoot became slightly larger after the break of strength 6 and appeared with a longer latency. An unexpected event appeared when a current of strength 7 was broken. The membrane potential did not return to the plateau but stayed near its resting level. The height of the short spike could be altered continuously by changing the current strength over a limited range. On the other hand, the reversal of the plateau seen in 7 was an all-ornothing event which had a definite threshold. This all-or-nothing repolarization was observed regularly in nine different kid hearts. The conditions for obtaining 231

6 232 SILVIO WEIDMANN Fig. 4. Superimposed traces from the pacemaker region showing changes in membrane potential produced by square pulses of cathodal current (relative strength 0, -1, -2, -3, -4). Time marks at intervals of 100 msec. Fig. 5. Potential changes produced by square pulses of anodal current applied at the beginning L of the action potential. Recording electrode close to the polarizing electrode (0-2 mm.). Relative strength of current from above downwards: 0, 1, 2, 3, 4, 5, 6, 7. Time marks at intervals of 200 msec.

7 POLARIZATION OF CARDIAC MUSCLE 233 it were that the current pulses should be of sufficient strength and that they should last for more than the first quarter of the action potential. In the experiment illustrated in Fig. 6 the distance separating the recording from the leading-in electrode was 4-2 mm. instead of 02 mm. as in Fig. 5. Square pulses of polarizing current were timed to coincide with the middle of the plateau. If these were less than a certain strength, they produced small and graded repolarizations, presumably by passive cable-like spread. However, above a certain strength a maximal repolarization was produced at a definite threshold. A comparison of records obtained from various distances Fig. 6. Effect of polarizing current on a region at some distance from the leading-in electrode (4-2 mm.). Square pulses of anodal current lasted from a to b. Ten traces are superimposed; relative strengths of current from above downwards: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9. Time marks at intervals of 100 msec. indicated that the subthreshold potentials spread with a decrement while the all-or-nothing repolarization was propagated without decrement as an 'offresponse'. Attempts to determine the velocity of this off-response were not entirely successful but indicated that it was much lower than that of the normal spike. It is therefore to be expectedl that the 'off-response' would disappear at a large conduction distance since it would be overtaken by the normal process of repolarization which occurs at a fixed time after the beginning of the spike. However, this point could not be verified since the total length of the fibres was insufficient for such an experiment. DISCUSSION The finding that the spike is associated with a profound decrease of the membrane resistance agrees with similar results obtained on the alga Nitella (Blin s, 1936; Cole & Curtis, 1938), the giant axon of the squid (Cole & Curtis,

8 234 SILVIO WEIDMANN 1939), skeletal muscle (Katz, 1941) and myelinated nerve (Tasaki & Mizuguchi, 1949). Previous impedance measurements on cardiac muscle have led to controversial results. Rapport & Ray (1927) recorded an impedance fall during the isometric contraction of a cannulated tortoise ventricle while Rosenblueth & del Pozo (1943) measured a systolic impedance rise in strips of turtle ventricle contracting isometrically. It is felt that the results obtained with hearts of cold-blooded animals should not be compared with the findings reported here for a mammalian preparation. For in Purkinje fibres of the kid the impedance fall is limited to the initial spike while there seems to be no such spike in the monophasic action potential of the turtle ventricle (Eyster & Meek, 1942), nor in that of the frog ventricle (Woodbury et al. 1951). Recent experiments with nerve and muscle indicate that the rising phase of the action potential is brought about by an increase in permeability which allows sodium ions to enter the fibre at a high rate (for references see Hodgkin, 1951; Huxley & Stampfii, 1951). It has also been suggested that repolarization during the falling phase occurs for two reasons (Hodgkin, Huxley & Katz, 1949; Hodgkin & Huxley, 1950). In the first place sodium entry is reduced by an inactivation process which decreases the permeability to this ion. In the second, the exit of potassium ions is accelerated by a rise in potassium permeability. Experiments described in a previous paper (Draper & Weidmann, 1951) suggest that the rising phase in cardiac muscle depends on an increase in sodium permeability and this conclusion is consistent with the present finding of a large increase in membrane conductance during the spike. The falling phase of the cardiac action potential is so different from that of nerve that it is doubtful if a hypothesis developed for the one tissue can be applied to the other. But it is interesting to consider briefly whether the general mechanism suggested for nerve is applicable to the present results. In order to explain the prolonged phase of depolarization one may suppose either that there is a long delay in the process which inactivates sodium permeability (PNa) or that the rise of potassium permeability (PK) is greatly retarded. It is also possible that one or other of these processes may be entirely absent, or that one or other may occur in two stages. During the plateau the membrane potential is not far from zero, indicating that PNa is approximately the same as PK. This situation might arise because PK has remained at its resting level whereas PNa has fallen from the high value which it attained at the crest of the spike. Or it might arise because PK has risen to the high value attained by PNa during the spike. The first suggestion would be consistent with a rapid but incomplete inactivation and a slow rise in PK, the second with a slow inactivation and a rapid rise in PK. It can be seen that only the first of these suggestions is compatible with the impedance measurements reported in this paper. For the membrane conductance has been shown to fall to a low level at the end of the spike and to remain at a low level during the plateau. This

9 POLARIZATION OF CARDIAC MUSCLE 235 could not occur if repolarization from spike to plateau depended on an increase in PK but is quite consistent with its being brought about by a decrease in PNa. Beyond this point it seems unwise to speculate further. It is perhaps worth adding that Purkinje fibres in a sodium-free saccharose medium showed no sign of any 'delayed rectification' (Hodgkin et al., 1949) such as might be expected if there were a rise in potassium permeability. However, these experiments were not conclusive since the unsteadiness of the electrotonic potentials indicated that the application of relatively strong currents to a preparation in saccharose solution may have damaged the membrane in some way. The ability of cardiac muscle to propagate an all-or-nothing wave of repolarization is a new but not entirely unexpected finding. In the ordinary way an excitable fibre starts with a high membrane potential and propagates a negative wave because local circuit currents depolarize the membrane to a threshold potential beyond which it tends to attain a new active level. Granted a suitable relation between threshold and amplitude of the propagated wave there seems to be no reason why an excitable fibre should not start with a low membrane potential and propagate a wave of repolarization by local circuit action. The velocity of propagation would not be the same as that for the wave of depolarization, one of the reasons being that the relation between threshold and amplitude would be different in the two cases. SUMMARY 1. Polarizing current was caused to flow through the surface membrane of cardiac muscle by means of a microcapillary inserted into the myoplasm of a single Purkinje fibre of the kid. The membrane potential was recorded by means of a similar electrode inserted into the same fibre. 2. The amplitude of the electrotonic potential produced by a relatively weak square pulse of current was used to estimate the electrical resistance of the surface membrane. Impedance changes were recorded simultaneously with the action potential. During the greater part of systole (plateau of the action potential) the membrane resistance had values comparable to those found in diastole. But a profound fall in resistance was observed during the short spike at the onset of activity. 3. 'Graded activity' could be demonstrated by applying cathodal current pulses of variable strength during diastole. The action potential of the pacemaker region was found to be characterized by a slow upward curvature preceding the steep part of the rising phase. 4. The action potential could be 'switched off' by applying a relatively strong anodal current pulse. The off-response resembled the familiar on-response in that there was a definite threshold and that the response was propagated without decrement away from the stimulating electrode. PH. CXV. 16

10 236 SILVIO WEIDMANN 5. The findings are discussed on the hypothesis that the rhythmical changes in membrane potential result from a succession of permeability changes to ions, particularly sodium. This work was greatly helped by advice and criticism from Mr A. L. Hodgkin and Mr A. F. Huxley. To Dr R. S. Comline and collaborators I am indebted for kid hearts. The costs of the investigation were defrayed by the Rockefeller Foundation and the work was done during the tenure of a scholarship awarded by the Stiftung fur Biologisch-Medizinische Stipendien, Basel. REFERENCES Blinks, L. R. (1936). J. gen. Phy8il. 20, 229. Cole, K. S. & Curtis, H. J. (1938). J. gen. Phy8o. 22, 37. Cole, K. S. & Curtis, H. J. (1939). J. gen. Physio. 22, 649. Draper, M. H. & Weidmann, S. (1951). J. Phy8io. 115, 74. Eyster, J. A. E. & Meek, W. J. (1942). Amer. J. Phy8io. 138, 166. Hodgkin, A. L. (1951). Biol. Rev. (in the press). Hodgkin, A. L. & Huxley, A. F. (1947). J. Phy8i. 106, 341. Hodgkin, A. L. & Huxley, A. F. (1950). Abstr. XVIII int. physio. Congr. p. 36. Hodgkin, A. L., Huxley, A. F. & Katz, B. (1949). Arch. Sci. physiol. 3, 129. Hodgkin, A. L. & Rushton, W. A. H. (1946). Proc. Roy. Soc. B, 138, 444. Huxley, A. F. & Stimpffi, R. (1951). J. Physiol. 112, 496. Katz, B. (1941). J. Neurophysio. 6, 169. Katz, B. (1948). Proc. Roy. Soc. B, 135, 506. Ling, G. & Gerard, R. W. (1949). J. ceu. comp. Physiol. 34, 383. Rapport, D. & Ray, G. B. (1927). Amer. J. Phy8il. 80, 126. Rosenblueth, A. & del Pozo, E. C. (1943). Amer. J. Physiol. 139, 514. Tasaki, I. & Mizuguchi, K. (1949). Biochim. biophys. Acta, 3, 484. Woodbury, L. A., Hecht, H. H. & Christopherson, A. R. (1951). Amer. J. Physiol. 164, 307.