Fine structural identification of individual cells subjected to microelectrode recording in perfused cardiac preparations

3ournal of Molecular

and Cellular

Cardiology

Fine Structural Microelectrode

14, 233-247

Identification Recording Jergen

tDepartment

(1982)

of Individual Cells Subjected in Perfused Cardiac Preparations

Tranum-Jensen

and

Michiel

J. Janset

*Anatomy Department C, University of Copenhagen Denmark of Cardiology and Clinical Physiology, University of Amsterdam, The Interuniversity Cardiology Institute, The Netherlands

(Received 26 September 1981, accepted in revised form

to

19 February

and

1982)

J. TRANUM-JENSEN AND M. J. JANSE. Fine Structural Identification of Individual Cells Subjected to Microelectrode Recordings in Perfused Cardiac Preparations. Journal of Molecular and Cellular CardioloQ (1982) 14, 233-247. We recorded transmembrane potentials from atrial, AV nodal and ventricular cells in perfused canine and rabbit heart preparations, and from Purkinje cells in superfused canine false tendons. Recording was maintained during perfusion with an aldehyde fixative. Action potential duration was greatly prolonged, and finally repolarization failed when the cells became fixed. After fixation the microelectrodes were withdrawn. Appropriate tissue blocks were embedded in Epon and serially sectioned at 4 pm. In 24 out of 34 attempts, the very cell recorded from was identified under the light microscope. After remounting and resectioning of the 4 pm sections for electron microscopy, the terminal electrode track was found in 12 instances, the very end of the track in six cases. Two main types of atria1 action potentials were recorded: with and without a plateau phase. After retrieval of the individual cells we found no basis for a morphological distinction between different cell types. In the AV node, electrotonic contact between two cells was established for distances up to 320 pm by delivering current pulses to one of two microelectrodes which were simultaneously in an intracellular position. Both “N” and “NH” types of action potentials could originate in the proximal atrioventricular bundle. Possibly the N-type in this location was induced by hypoxia. Impalements of Purkinje fibres and ventricular myocardial cells extended the observations on details of microelectrode penetration and impalement damage. The most conspicuous disorder observed in all cells impaled by a microelectrode was a local or universal hypercontraction of myofibrils. In all cases where stable recordings were obtained, the microelectrode tip was found close to the cell membrane opposite to the site of entrance. KEY WORDS : Cardiac potential configuration;

cells ; Microelectrode Impalement damage.

recordings

; Ultrastructure;

Electrotonic

contact;

Action

Introduction Microscopical retrieval of individual cells subjected to microelectrode recordings is the only direct means to unravel the morphological substrates for different electrophysiological specializations in heterogeneous cardiac tissues. This applies particularly to the regions of the sinuatrial and atrioventricular nodes where transmembrane potential characteristics change over short distances, and morphological differences are detected between neighbouring cells. In the atria, recordings of a spectrum of action potential configurations together with conflicting reports on

morphological heterogeneity has nourished a long standing controversy whether the tissue between the two cardiac nodes and between the right and the left atrium is uniform, or contains either specialized tracts or single specialized cells scattered between “ordinary” atria1 cells [II]. The techniques applied to achieve correlations between electrophysiological findings and morphological characteristics have been largely indirect, at best identifying a small volume of tissue from which microelectrode recordings were made [I, 18, 291. In one study lanthanum was

Supported by the Wijnand Pon foundation. Address correspondence to: J. Tranum-Jensen, Anatomy Department Copenhagen, Blegdamsvej 3C, DK-2200 Copenhagen N, Denmark. 0022-2828/82/040233+

15 303,00/O

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0 1982 Academic

Panum

Institute,

Press Inc.

University

(London)

Limited

of

234

J. Tranum-Jensen

injected into cells of the sinuatrial node via a microelectrode in order to identify cells showing pacemaker activity [Xl, in another, fluorescein was injected into a variety of cardiac cells [ZZ]. However, the possibility that the injected marker substances and the manoeuvres of injection can induce structural artefacts hampers such approaches. In the present paper we describe a method

and M. J. Janse allowing a precise identification of the very cell from which microelectrode recordings were obtained in a cardiac preparation. Additionally, the method provides insight into the degree of damage produced by the microelectrode to the cell membrane and the interior of the cell impaled. A preliminary report on the method has been presented at the workshop on the sinus node held in Maastricht [15].

Methods Isolated

herfused

and superfmed

Dogs and rabbits were anesthetized by intravenous injection of sodium pentobarbital. After intravenous administration of 5000 U.I. of heparin, the hearts were rapidly excised and placed in a large volume of oxygenated Tyrode solution at 37°C. Both the right and left coronary artery were then cannulated using a short length of polyethylene catheter of the largest possible diameter, and perfused at a hydrostatic pressure of 100 mm Hg with an oxygenated standard Tyrode solution (Na+ 156.5, Kf 4.7, Ca2+ 1.5, Mg2+ 0.7, H,PO,0.5, Cl- 137.0, HCO,- 28.0, glucose 20.0 mmol/l) to which 4% dextran of nominal mol. wt 70 000 (T 70 Pharmacia, Sweden) was added. The time interval between excision of the heart and beginning of coronary perfusion was about 3 min. The right atrium was opened according to reference [Zl] by cutting through the crista supraventricularis and superior vena cava. Additionally, the aorta was divided between the right and left coronary

Recording

artery. Opened this way, the crista terminalis is cut and atria1 activation is not completely normal. The majority of the ventricular myocardium was removed, and arteries in the cut surfaces of the interventricular septum and ventricular walls were clamped by small agraffes. The preparation was pinned to a ring of cork, over the central hole of which a nylon net was stretched, and transferred to a tissue bath, where, in addition to being perfused, it was also superfused with oxygenated Tyrode solution at 37°C. Further details of the preparation are given in reference [ 151. ‘Coronary flow for the rabbit preparation was approximately 10 ml/min, for the canine atria1 preparations it was in the order of 40 ml/min. From the canine hearts, strands of free running Purkinje fibres were removed and placed in the tissue bath where they were superfused with oxygenated Tyrode solution. The length of the strands varied from 5 to 8 mm, diameter varied from 0.7 to 1.1 mm.

and jixation

Microelectrodes were pulled from glass capillaries containing a small glass fibre in its lumen (Clark Electromedical Instruments, GC lOOF-4). They were filled with 3 M KC1 by the method of Tasaki et cl. [25]. Resistances were in the order of 20 MR. Because of the vigorous contractions of the perfused preparations, floating microelectrodes has to be used [30]. In most experiments two microelectrodes were simultaneously operated. Conventional, high input impedance pre-amplifiers and differential DC amplifiers were used. The outputs of the differential amplifiers were connected to an Ampex multichannel instrumentation tape recorder, running at a tape speed of 15 in/s. Signals were printed out on an Elema ink-writer or photographed from the screen of a memory oscilloscope (Tektronix 5103 N). The maximum upstroke velocity, dV/dtmay, was obtained by electronic differentiation of the action potentials, and displayed on the screen of the

preparations

techniques

oscilloscope. The frequency response of the recording system was determined by applying square wave pulses through a normal 20 Ma microelectrode and determining the upstroke velocity of the signal reproduced from the tape recorder. The r was 50 ys. When applying ramp pulses of different slopes it appeared that upstroke velocity of the reproduced signals were about 10% less than those of the input signals. The fact that of recorded action potentials was somed V/d&x times rather low cannot be due to the recording system. During recording of transmembrane potentials, the preparation was fixed by perfusion through the coronary arteries with a fixative (2.5% glutaraldehyde + 1% formaldehyde in sodium phosphate buffer 0.11 mol/l, pH 7.2) while the microelectrode was still recording intracellular action potentials. The preparation was fixed within seconds. Simultaneously with the coronary per-

Morphology

of Microelectrode

fusion of fixative, the superfusion was switched to fixative. Purkinje strands were fixed by superfusion only. Perfusion and superfusion with fixative was maintained for 20 min. Then, a second microelectrode filled with India ink (Pelikan C 11/1431a), and of which the very tip was broken, was positioned close to the recording microelectrode and by hydraulic pressure two tiny deposits of ink were placed subendocardially to mark the position of the recording microelectrode, which was then withdrawn from the preparation. The entire preparation was then kept in the fixative for 3 to 16 h at 4°C. A tissue block of 1 to 2 mm3 of precisely known orientation was cut at the position marked by the India ink dots. The block was washed for 1 h in the buffer of the fixative, and

Impaled

Cells

235

post-fixed in 2% 0~0, in the same buffer for 1 h. Following a 1 h wash in the buffer the block was dehydrated in a graded series of ethanol and embedded in Epon 812. The block was cut with glass knives into a near complete series of 4 pm sections which were mounted unstained in Epon. The section containing the microelectrode tip position was searched with phase contrast or interference contrast optics, and this section, together with its neighbouring sections were then remounted on pre-cast Epon blocks [31] and resectioned for electron microscopy. Usually, a series of about 50 useful ultrathin sections could be obtained from one 4 pm section. Sections were stained wit;1 uranyl acetate and lead citrate.

Results Attempts to retrieve the cellular origin of 34 microelectrode recordings were made. In 24 instances, the microelectrode tip position was identified under the light microscope: eight times in dog atrium, three times in dog Purkinje fibres, once in a dog ventricular myocardial cell, five

Atrial

times in rabbit atrium, six times in rabbit atrioventricular node and once in rabbit ventricle. In ultrathin sections the terminal electrode track was found in 12 cais and the very end of the track in six of these.

mycardium

Both in rabbit and dog atria, two main types’of transmembrane potentials were recorded: (a) action potentials which exhibited a pronounced plateau [Figures 1 (a) and 51, and (b) action potentials without a plateau, more or less triangular in shape [Figure 1 (b)]. As a third category, short (< 100 ms), spike like action potentials may be distinguished. In all 13 instances these potentials originated from atria1 myocardial cells in which no signs of specialization, mutually or relative to surrounding ordinary atria1 working myocardium, were detected by morphological criteria, such as cell diameter, myofibrillar content and depth below the endocardium. Figure 1 shows two simultaneously recorded action potentials from a perfused canine right atria1 preparation. The potentials shown in (a) were recorded from the lower part of the crisfa terminalis close to the coronary sinus, those in (b) from the upper part of the crista terminalis close to the sinuatrial ring bundle. The action potentials in (a) show a distinct plateau, those in (b) have a faster time course of repolarization. In figures 2 and 3 the tip position of the microelectrodes at sites (a) and (b), as identified in 4 pm serial sections are shown. Both tips were located in normal atria1 cells of the crista terminalis. (a) was 290 pm and (b) 120 pm below the endocardial surface. The approximate cell diameter at

(a) was 16 pm, at (b) 14 pm. In (b), an adjacent cell about twice as large had also been pierced. The coarsely granulated appearance of impaled

cA

_\,

l.,_L

_\_

1 50

mV

200ms‘ FIGURE 1. Transmembrane potentials of two cells, (a) and (b), simultaneously recorded from a perfused canine atria1 preparation. The potentials at (a), showing a distinct plateau, were recorded from the crisfa ~erminnlis close to the coronary sinus, those at (b), lacking a plateau. were from the upper c&n /ermirza/is close to the sinuatrial ring bundle. At each impalement site is shown the first (left) and the last, stable action potential recording just before the preparation was fixed. In between the electrode “left the cell” eight times at site (a) and nine times at site (b). By all readjustments the electrode was advanced into deeper layers. At both sites all action potentials recorded had basically the same configuration.

236

J. Tranum-Jensen

FIGURE 2. Low- and high power micrographs of the 4 ym section in which the tip of the microelectrode corresponding to the last recording in panel (a) of Figure 1 was retrieved (arrow). The electrode track is oriented at an angle of about 35” to the plane of sectioning, and had been followed from the endocardial surface (E) through a series of 51 sections. As often observed along the medial aspect of the crista terminalis, a population of small diameter cells (sd), related to the sinuatrial ring bundle, is found subendocardially. Upper (a) and lower (b) frame is photographed at different levels of focus.

FIGURE 3. Shows the identification of the cell from which the last recording in panel (b), Figure 1, originated. The full length of the electrode track is contained in the plane of sectioning. Cells pierced by the electrode all exhibit a coarsely granular appearance (asterisks) due to hypercontraction of myofibrils. Cap = capillaries.

and M. J. Janse

FIGURE 4. Electron micrograph of the track left by the tip of the microelectrode retrieved in the 4 pm section of Figure 3 (frame). The impression left by the tip indicates an outer tip diameter (t) of 0.4 pm. The tip’is at level with an intercalated disc (d). The crevice (c) in front of the track- is probably produced during retraction of the electrode from the fixed tissue. hf = Hypercontracted myofibrils, cap = collapsed capillary.

cells along the electrode track, seen in high power light micrographs (Figure 3), and including the very cell impaled, is produced by hypercontracted myofibrils. In both cases, the microelectrode tip was facing the inside of the membrane opposite the site of impalement. In Figure 4, an electron micrograph from the tip position at site (b) is shown. The channel left by the withdrawn microelectrode indicates an outer tip diameter of 0.4 pm, and the tip position was at level with an intercalated disc. During recording, the tip may have been pressed against the cell membrane. In the electron micrographs, cross sectioned hypercontracted myofibrils appear as near homogeneous electron dense material, and are seen only in cells containing the electrode track. The other cell contributing to the intercalated disc in Figure 4 is not hypercontracted. The impaled cells in sites (a) and (b) did mutually not differ with respect to diameter, myofibrillar or mitochondrial content, nor did they differ from the surrounding atria1 cells. In Figure 5 an atria1 action potential, displaying a clear plateau following a spike-like upstroke is shown during perfusion with a Tyrode solution containing 11 mM K+ (left) and just before fixation after return to standard perfusion with 4.7 rnM K+. Resting potential, and action potential upstroke showed comparatively little change during fixation, but action potential duration increased markedly.

Morphology

of Microelectrode

Impaled

237

Cells

200 ms

K+47 mM FIGURE 5. Transmembrane potentials from an atria1 cell in the middle of the crista terminalis of a perfused canine atria1 preparation. In the right panel the recording made just before fixation is shown. Note the spikelike upstroke followed by a plateau. In the left panel, it is shown how membrane potential, action potential amplitude and upstroke velocity are reduced during perfusion with 11 rnM K +. Horizontal line indicates zero potential.

The photomicrographs of Figure 6 demonstrate that the impaled cell was located in the fourth cell layer, 60 pm below the endocardial surface and had a diameter of 15 pm. A band of hypercontraction extends to the intercalated disc at one end

of the cell, but not to the intercalated disc at the other end. This is also clearly seen in the electron micrograph of Figure 7. In rabbit atria, similar results were obtained as in canine preparations. In particular, action potentials with a distinct plateau and action potentials with no plateau were found to originate from morphologically non-specialized atria1 fibres.

FIGURE

7. Electron

micrograph

resectioning the 4 pm section It can be seen, that the band

fibrils end

FIGURE retrieving 5 originated. by arrows

6. Shows the

three

consecutive

cell from which the Positions of intercalated in frames (b) and (c).

recordings discs

4 pm sections are

of Figure marked

state duces

(hf), of the

the

of ordinary a

sarcolemma The

does not reach cell,

terminal

rumed

the intercalated

remaining

contraction. cell

surface

at level of Z-lines. electrode

track

obtained

of Figure 6 (a) of hypercontracted myofibrils

after (frame). myo-

disc at one (cf)

being

in a

Hypercontraction (r),

due

to

probinding

id = Intercalated is marked

of

disc.

by an asterisk.

J. Tranum-Jensen

238 Rabbit

VetWiCk

Start

and M. J. Janse perfusion

with

fixative

5OmV

I

’

cl.5 s

FIGURE 8. Continuous recording of the transmembrane potential of a ventricular cell in a perfused rabbit heart preparation. Perfusion with fixative starts at the arrow, and after about 1 s action potential durations begins to lengthen. Note electrotonically mediated “humps” during the repolarization phase in the middle panel. In the lower panel, repolarization fails and the cell is fixed while its membrane potential remains in the plateau range.

Ventricular

myocardium

Figure 8 shows continuous recordings from a cell in the interventricular septum of a perfused rabbit heart preparation. As in atria1 fibres, fixation had little effect on resting membrane potential and action potential upstroke, but greatly prolonged action potential duration up to the point of complete failure of repolarization. The impaled cell was found in the 15th cell layer, 140 vrn from the endocardial surface [Figure 9(a)]. The electrode produced a deep invagination in the cell and the tip approached the cell membrane opposite the site of entrance [Figure 9(b) and (c)]. The tip was separated from the membrane by a thin (0.25 pm) layer of compressed myofibrils. The outer tip diameter has been 0.3 pm, and a small plug appears to have entered the lumen of the electrode, suggesting an internal diameter of about 0.15 pm [Figure 9(c)]. The full cross section of the impaled ccl1 shows hypercontraction. Another ventricular action potential was recorded from a canine ventricular trabecula. It appeared that a ventricular cell in the third cell layer was impaled, with F’urkinje cells overlying it (Figure 10). The electrode tip was deeply inserted into a hypercontracted cell, which had a mean diameter of 15 pm.

FIGURE 9. Light micrographs of the section in which the cell giving rise to the sequence of action potentials displayed in Figure 8 was retrieved. The characteristic funnel-shaped depression (d) of the endocardial surface at the site of entry of the microelectrode is very pronounced in this deep (140 pm) impalement. Cells along the track have been stretched, and those pierced by the electrode exhibit hyper-

contracted myofibrils. Figure 9(c) is an electron micrograph obtained after resectioning of the 4 pm section of Figure 9(b) (frame). The width of the tip opening has been in the order of 0.15 ym as indicated by the small plug (p) of compressed hypercontracted myofibrils (hf).

Morphology

of Microelectrode

Impaled

Cells

FIGURE 10. Light micrograph of the section in which the cell, from which a ventricular type of action potential had been recorded, was identified as a ventricular myocardial fibre located in the third cell layer of a trabecula in which Purkinje fibres (P) were located only subendocardially. The electrode produced a depression (d) of the endocardium. The termination of the electrode track is indicated by arrow.

Purkinje In canine false tendons, three Purkinje cell impalements were morphologically identified. Only one of the recordings could be considered normal. The action potential amplitude was 110 mV, resting membrane potential was -90 mV and dV/dtmsx was 210 V/s (Figure 11). This potential remained stable for several minutes before start of fixation. The electrode had advanced close to the membrane opposite the site of entry, and the tip was located in a small extension of a Purkinje cell which formed part of a bundle of two cells (Figure 12).

cells In ,another experiment action potentials of a cell displaying spontaneous diastolic depolarization were recorded [Figure 13(b)]. Spontaneous cycle length was 1010 ms. The.maximum diastolic potential was -68 mV, and action potential amplitude was subnormal at 72 mV. dV/dfmax was low, and there was no initial spike. As shown in Figure 13(a), the tip of the microelectrode had advanced to the middle of a Purkinje cell of the first cell layer, having a diameter of 25 pm. The interior of the cell showed very localized damage, only a local hypercontraction around the entrance

Dog Purkinje

FIGURE just before duration, M.C.C.

11. Transmembrane potentials and dV/dtmax of a Purkinje fixation (a) and during various stages of fixation (b), (c), and relative little change in dV/dtmax.

fibre in a superfused (d). Note prolongation

canine false tendon, of action potential

M

240

J. Tranum-Jensen

and M. J. Janse

site being present. Electron microscopy revealed that the electrode had passed the cell membrane without producing invagination, making a “clean cut” of 2 pm diameter. The track indicated an outer tip diameter of approximately 0.4 [*m (Figure 14). The action potentials displayed in Figure 15 are distinctly abnormal, and have a low upstroke velocity. The electrbde which recorded these potentials did apparently not penetrate the cell surface membrane, but produced only a dimple in the surface of a Purkinje cell of the first cell layer

(Figure 16). No hypercontraction was seen in this cell, whereas this was consistently seen in all other impalements. Unfortunately, no electron micrographs could be obtained from this cell.

FIGURE 12. Light micrographs of the section in which the Purkinje cell giving rise to the action potentials of Figure 11 was retrieved. The cell was located in the second cell layer and formed part of a bundle of two cells. The tip position is marked by an arrow. The dark material around the electrode track is hypercontracted myofibrils.

FIGURE 13. Retrieval of the Purkinje cell of first cell layer giving rise to the low amplitude action potentials shown in frame (b). The tip has advanced to a position centrally in the fibre close to the nucleus (N) (inset). Hypercontracted myofibrils, marked by small arrows, are seen around the site of entrance. co11 = major ct = Endocardial connective tissue, bundle of collagen.

FIGURE 14. Electron micrographs showing the site of entrance (a) and the terminal the membrane smoothly corresponding to the frames in Figure 13(a). The electrode “cuts” lesion at’its entrance (E). The outer tip diameter (t) has been approximately 0.4 pm, located cell nucleus (N). hf = Hypercontracted myofibrils, m = mitochondria.

track of the tip (bj, to produce a 2 pm in the vicinity of the

Morphology

of Microelectrode

Impaled

241

Cells

Dog Purkinje

,200s Just before

fixotidn

During

fixation

FIGURE 15. Action potentials recorded from a canine false tendon. These potentials shape and amplitude are abnormal and there is a fast spontaneous rhythm (cycle length

Atrior?entricular In three perfused rabbit heart preparations, simultaneous recordings were obtained from two atrioventricular nodal cells. Small current pulses of 100 nA were passed through one of the electrodes to establish whether the two impaled cells were electrotonically coupled. In Figure 17, two action potentials are shown during a Wenckebach phenomenon produced by rapid driving of the atrium. Extracellular electrograms from the c&n terminalis (upper trace) and His bundle (lower trace) were simultaneously recorded. The configuration of the AV nodal action potentials during this Wenckebach phenomenon are characteristic for so called N cells [l4] and 1201. In panel 2, current pulses are applied to microelectrode (a) and electrotonic potentials are observed in the transmembrane potential recording of (b). In panel 4, microelectrode (b) is withdrawn into the extracellular space, and again current pulses are applied to microelectrode (a). Although artefacts caused by the current pulses can still be seen, no displacement of the extracellular potential in (b) occurs, indicating that current did flow through intercellular connections during the situation

are distinctly 75 ms).

abnormal:

node when both electrodes were intracellularly. .4fter identification of the electrode tips in the fixed preparations, it was found that the tips were located in the enclosed nodal portion at the beginning of the atrioventricular bundle and were separated by a distance of 220 pm. During the experiment, the distance between the entry sites of both microelectrodes was measured through the dissecting microscope to be 400 pm. In another experiment two AV nodal cells were simultaneously impaled (Figure 18). Again, the configuration of the potentials during a Wcnckebaths periodicity produced by rapid atria1 pacing warranted the classification of thrse cells as N cells. Electrotonic deflections were seen in AVNz when current pulses were applied to AVN,. The microscopical retrieval revealed that the tips were 90 pm apart, and that both cells were located at the lower edge of the node in a tissue representing the beginning of the atrioventricular bundle (Figure 19). Moreover, this part of the node was not well perfused and cells showed signs of being hypoxic. The very tips were not found in ultrathin sections although the terminal M ?

242

J. Tranum-Jensen

and

M.

J. Janse

FIGURE 17. Two simultaneously recorded action potentials (a) and (b) from the atrioventricular node of an isolated perfused rabbit heart preparation. Upper traces: atria1 electrogram, lower trace: His bundle electrogram. In panel 1, rapid atria1 pacing results in a 3 : 2 Wenckebach block. In panel 2 current pulses are applied through the &croelectrode in cell (a). In panel 4, the microelectrode in cell (b) is withdrawn and now records the extracellular potential.

Atrium

(al

,.-

AVN2

I

w-1

-An A

His A

(b)

,

t

H

V

AHV

1

-;.A AHV

1

A

-7. AH

7

7

Apply current through this micraelectrode

1

-

FIGURE 18. Two “N” potentials recorded from a perfused rabbit heart preparation (AVN, and AVN,). In the His bundle electrogram [lowest trace (a)] A indicates atria1 activity and H His bundle activity. In (a) a Wenckebach phenomenon is induced by atria1 pacing; In (b) current pulses arc applied through the intracellular electrode in AVN,, resulting in electrotonic potential displacements in AVN, [in the lowest trace the applied current pulses are displayed].

Morphology

of Microelectrode

microelectrode tracks were identified. Cells very close to the tip showed many nexus junctions and signs of hypoxia, such as edema, swollen mitochondria and marginal condensation of chromatin in the nuclei. Again, there was a large discrepancy between the tip distance and the distance between the entrance sites, which in this case was 170 pm.

Impaled

Cells

243

In a third experiment, two typical NH potentials were recorded [14, 201 with fast upstrokes which occurred just a few milliseconds before the extracellular spike in the His bundle electrogram. Electrotonic contact was established between these cells. The microelectrode tip positions were found in the atrioventricular bundle 320 pm apart.

FIGURE 19. Retrieval of the AVN,-cell [frames (a) and (b)] and the AVN,-cell [frames (c) and (d)] of Figure 18, shown in low and high magnification with interference contrast optics. The electrode track leading to AVN, is oblique to the plane of sectioning; that of AVN, is contained in the section of frames (c) and (d). Tip positions are indicated by arrows in frames (b) and (d). The perfusion of the tissue around the electrodes has been incomplete as indicated by a paucity of open capillaries, compared to surrounding tissue [marked c on frame (d)]. la = Left atria1 cavity, ivs = intraventricular septum, f = fat cells, blackened by 0~0,.

Discussion Our results show that it is possible in the majority of cases to identify the very cell from which microelectrode recordings were obtained; in our study in 24 out of 34 attempts. Since contractions in the perfused preparations were vigorous and floating microelectrodes had to be used, stable microelectrode recordings from superficially located cells were extremely difficult to obtain, and in fact we succeeded only once (Figures 5 and 6). In the majority of cases, stable recordings originated from cells located more than 100 urn, and sometimes more than 200 pm, below the surface. In superfused preparations, which are almost universally used for m,icroelectrode recordings, it is far easier to obtain stable recordings, and usually they can be obtained from superficial cells as in our false tendon preparations. However, superfused preparations have the disadvantage that

hypoxic cell damage is present already at depths between 100 and 150 pm below the surface of the preparation [15]. Cranefield and Greenspan [5] calculated that for cylindrical preparations, such as for example a papillary muscle, the rate of diffusion of oxygen and small molecules such as glucose becomes restrictive when the diameter of the preparation is more than 600 urn, and that the core of a thicker bundle will become severely hypoxic. In a plane sheet of tissue, such as an atria1 superfused preparation, the critical thickness is even less, because for a thickness of a sheet equal to the diameter of a cylinder, the surface area per unit volume across the sheet is half of that of a unit volume along the cylinder. In rabbit atria1 preparations, which morphologically appeared not well perfused, as could be judged from a small number of open capillaries,

24-l

J. Tranum-Jensen

we found occasionally action potentials of very short duration that had a sharp spike like configuration. In two cases such action potentials (60 and 100 ms duration) were retrieved. They were recorded at a depth of 100 and 105 pm, respectively, and the cells, although displaying a relatively normal appearance were located within distances of 50 and 70 pm to a central zone of necrotic cells. The short duration, and the spike like configuration could have been due to the fact that deep cells in a not well perfused preparation, despite it being superfused as well, are hypoxic. Also, ions leaking from the deeper layer of necrotic cells may influence action potential characteristics. Even superficial cells which are well supplied with oxygen and substrate can be in electrotonic contact with deeper hypoxic cells, and the configuration of their action potentials may be influenced by those of the hypoxic cells. As a general statement, it can be said that action potentials recorded from superfused sheet like preparations of more than a few hundred micrometres thickness, and in which the depth of impalement is not defined, must be interpreted with great caution. Still, our results show that in well perfused atria1 myocardium, in which no signs of hypoxic damage is present throughout the whole atria1 wall, action potentials of different configuration can be recorded. In no case could we find evidence that atria1 potentials of different contiguration were generated by cells which exhibited morphological differences. Specifically, no signs of specialization were found in atria1 fibres from which action potentials displaying a pronounced plateau, and which might even be called “Purkinje-like”, were recorded. The reason why some atria1 cells produce “plateau” type action potentials, and others not, is not clear, but the extent of coupling to other cells may be a factor. When a particular cell in a multicellular preparation is in electrotonic contact with other cells, current flow provided by these cells when they are depolarized may prolong the action potential, and when such electrotonic contact is less developed, repolarization may begin earlier [19]. It is in this respect noteworthy that Horibe found that in atria1 tissue “the thicker the musculature, the larger the action potential and the more remarkable the plateau” [13]. In our experiments in well perfused tissue, fixed by coronary perfusion, only two morphological types of atrial, non-nodal cells were distinguished: (1) small cells, 3 to 6 pm diameter, close to the endocardial surface on the medial aspect of the crista terminalis, including the sinuatria1 ring bundle [21, 281. (2) In deeper layers on the crista terminalis and the interatrial septum, the atria1 cells were uniform with diameters from

and M. J. Janse 12 to 15 pm. No large diameter, pale looking cells with few myofibrils resembling Purkinje fibres were found in either dog or rabbit atria. It has recently been claimed that the deeper layers of the crista terminalis are composed of large-diameter, pale cells with widely separated myofibrils and with many T-tubules [2]. The micrographs published in support of this view indicate to us cell swelling resulting from hypoxic conditions, as usually observed in deeper layers of the crista in only superfused preparations. Atria1 cells are known to be very heterogeneous with respect to development of T-tubules [9, 241. Our present studies do not permit statements about the relative frequency of T-tubules among the cells retrieved, because nearly all cells were studied in cross sections which are not suited for a survey of T-tubules, in particular in the absence of an extracellular tracer. We attempted several times to record transmembrane potentials from the small diameter cells in the sinuatrial ring bundle, which was clearly visible through the dissecting microscope as a thin bundle medial to the crista terminalis. Even when during the experiments we felt convinced that we were recording from this bundle, subsequent identification of the microelectrode tip position showed that the whole sinuatrial ring bundle had been pierced by the electrode and that the tip was located in an underlying normal atria1 cell. The diameter of the cells in the sinuatrial ring bundle is so small [28] that microelectroderecordings from those cells in a contracting heart seem impossible. In the AV nodal area, four morphologically different cell types are present: atrial, transitional, midnodal and lower nodal cells [I, 271. Correlation between action potential configuration and cell type in this highly inhomogeneous area has been made in 20 pm cryostat sections, after iontophoretical injection of cobalt through the recording microelectrode [I]. Although this allowed action potentials of different configuration to be correlated with nodal architecture, identification at a cellular level was not possible. The results presented here show that identification at a cellular level in the AV node is possible. It is of interest to note that action potentials classified as “N” cells could be obtained from cells in the beginning of an atrioventricular bundle which showed hypoxic damage. Our experiments showed that electrotonic contact between cells, which morphologically are classified as belonging to the atrioventricular bundle, is “demonstrable” over distances of at least 320 pm. Unfortunately, we did not obtain clear data about other parts of the AV node, but de Mello found that in the proximal node, the “AN” zone, the space constant was on the average

Morphology

of Microelectrode

430 pm [8]. Utilizing a suction electrode technique Bukauskas & Veitikis [4] performed detailed measurements of length constants in longitudinal and transverse directions in the rabbit node. Average values in the longitudinal direction were 210 and 286 pm for two different areas of the AN zone; 170 pm in the N-zone, 416 pm in the NH zone and 970 pm in the His bundle. In all zones, except the N zone, length constants in the transverse direction were smaller. Based on the lack of diffusion of fluorescein injected into cells of the “N” zone of the AV node, Pollack [22] concluded that N-cells may not be sufficiently well coupled to permit impulse propagation by intercellular current flow alone. One might question how the “N” zone was defined, since no action potential recordings were shown, and also because the structures showing fluorescence had diameters in the order of 100 pm, whereas cells in the central part of the AV node, corresponding to the midnodal cell zone [I, 271 have diameters less than 10 pm. It is a very difficult undertaking to maintain two microelectrodes simultaneously in an intracellular position for a period long.enough to establish presence or absence of electrotonic contact in such small cells in a beating, perfused preparation. For that reason, we were unable to complete our original plan, namely to map in detail the distances over which electrotonic contact was demonstrable in the different areas of the AV node. The question whether in all regions of the AV node cells are coupled to such a degree that propagation by local current flow is permitted can, therefore, not be conclusively settled although this is likely in view of the findings of Bukauskas and Veitikis [4]. It must be emphasized that, especially in such an inhomogeneous area as the AV node, where superficial cells may be very different from deeper cells [6], identification of the very cell recorded from is required before safe statements about electrophysiological characteristics of the morphologically different, or “typical” AV nodal cells, can be made. Upon contact with fixative by vascular perfusion the response of cells, irrespective their location, followed a general pattern of increasing action potential duration till finally the cells failed to repolarize. Resting potentials atrd upstroke velocity were relatively little affected. These phenomena could partly be ascribed to the changed ionic composition of the perfusate, and partly to the action of the aldehydes in the fixative. In the experiments of Fozzard and Dominguez [IO], in which the action of low concentrations of glutaraldehyde and formaldehyde was studied, a marked prolongation of action potential duration was observed, as well as a-hyperpolarization, and an increase in action potential amplitude and upstroke velocity. In our experiments, applying

Impaled

Cells

2-15

much higher concentrations daldehydes, upstroke velocity on occasion remained constant, but as a rule decreased. An advantage of the method presented here over techniques in which substances such as lanthanum [.X1, cobalt [I], fluorescein [22] etc. are injected into cells which are subsequently identified, is that ultrastructural artefacts possibly induced by the marker substances need not be considered. Also, our method visualizes the distortion of the tissue around the microelectrode as well as the damage produced in the very cell impaled. The most conspicuous change in the interior of impaled cells was local or universal hypercontraction. This was most probably caused by the entry of calcium ions via the leak between microelectrode shaft and severed sarcolemma. A possible contribution by intracellular release of Ca2+ from the microelectrode glass may also be considered 131. It is of interest that this hypercontraction could be very localized (see Figure 7), indicating that the influx of calcium ions was not massive, and perhaps might occur only for a short period of time. Another conspicuous finding was that in those instances where stable recordings of higher amplitude were obtained, the microelectrode was always deeply inserted into the cell, its tip being close to the membrane opposite to the site of entrance. In those instances where the tip was found in the middle of a cell, the transmembrane potential was either of very low amplitude, or very unstable. Transmembrane potentials have been recorded from single, isolated cells, either from chick embryo cells in tissue culture [7], or from cells isolated from adult myocardium [23]. In the last study the membrane potential during the first millisecond after impalement was in the order of -70 mV, but quickly declined. Only in special conditions, such as an elevated extracellular calcium concentration, could stable resting membrane potentials in the order of -70 mV be maintained. De Haan and Gottlieb [7] could successfully impale isolated cells in tissue culture in 8.3O$ of the attempts, whereas impalement in conjoint cells adhering to and electrotonically interconnected with one or more neighbours, were successful in 75.3O Success was defined as a resting potential zf -40 mV or more, and a recording period of at least 10 s. In ascites tumour cells and red blood cells, it was found that membrane potential was high in the first millisecond after the membrane was pierced by a microelectrode, but quickly thereafter assumed low values [16, 171. Similar observations on a rapid degradation of membrane potentials by impalement with microelectrodes were made in the early studies of Hogg et al. on cultured embryonic rat heart cells [12]. It is reasonable to suppose, as was argued by de Haan

246

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and M. J. Janse transmembrane potentials, even though the microelectrode did not truly penetrate the cell. Such potentials might be called injury potentials and could be due to an increased current leak through part of the cell membrane, which was damaged by the pressure exerted by the tip of the microelectrode. Although, the method presented here is very time consuming it offers in our view the only way in which a precise correlation between electrophysiological and morphological characteristics can be obtained in those areas of the heart where cellular inhomogeneities exist.

and Gottlieb [7] that in the case of impalement of a single cell, substantial exchange of intracellular and extracellular material may occur before the opening between pierced membrane and microelectrode shaft becomes sealed off, and that this leads to a decrease in resting membrane potential. When the impaled cell is electrotonically coupled to other cells, those cells might serve as a source of current, which would tend to maintain the resting potential of the impaled cell. Finally, as shown in Figures 15 and 16, it is possible to obtain recordings which, although of abnormal configuration, have the appearance of

Acknowledgements We gratefully acknowledge the excellent technical Charles Belterman; and the skilful photographical

assistance of Lisette Hansen, work of Birgit Risto and Dirk

Wim ter Smitte van der Moot.

and

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