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(Circulation Research. 1995;76:802-811.)
© 1995 American Heart Association, Inc.

Articles

Spatial Distribution of Connexin43, the Major Cardiac Gap Junction Protein, Visualizes the Cellular Network for Impulse Propagation From Sinoatrial Node to Atrium

I. ten Velde, B. de Jonge, E.E. Verheijck, M.J.A. van Kempen, L. Analbers, D. Gros, H.J. Jongsma

From the Department of Physiology (I. ten V., B. de J., E.E.V.), University of Amsterdam (Netherlands); Laboratoire de Génétique et Physiologie du Développement (D.G.), Faculté des Sciences de Luminy, Université d'Aix-Marseille II (France); and the Department of Medical Physiology and Sports Medicine (M.J.A. van K., L.A., H.J.J.), University of Utrecht (Netherlands).

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Myocytes are electrically coupled by gap junctions, which are composed of low-resistance intercellular channels. The major cardiac gap junction protein is connexin43 (Cx43). The distribution of Cx43 has been studied by immunofluorescence to visualize the electrical coupling between atrial tissue and sinoatrial node. From modeling studies, this coupling was inferred to be gradual in order to shield the sinoatrial node from the atrial hyperpolarizing influence. The actual Cx43 labeling pattern did not show the expected gradient but instead a rather black and white staining in a striking pattern of strands of cells. We used an immunohistochemical marker (anti–-smooth muscle actin [SMA]) that specifically cross-reacts with guinea pig sinoatrial node cells together with Cx43 antibody to stain previously electrophysiologically mapped sinoatrial nodes. We found that in the guinea pig sinoatrial node the impulse originates in an SMA-positive, virtually Cx43-negative, region (primary pacemaker region). The impulse then travels obliquely upward to the crista terminalis through a region where layers of SMA-positive cells alternate with layers of Cx43-positive SMA-negative cells. The layers of Cx43-positive cells appear to become broader and thicker in the direction of the crista terminalis, whereas the layers of SMA-positive cells become thinner and narrower. Lateral contacts between Cx43- and SMA-positive cells were very sparse and only detected where the Cx43-positive strands ended (the region where SMA-positive cells fill the whole space between endocardium and epicardium, ie, the putative primary pacemaker region). From these results, we conclude that the primary pacemaker is shielded from the hyperpolarizing influence of the atrium by a gradient in coupling brought about by tissue geometric factors rather than by a gradient of gap junction density.

Key Words: gap junctions • connexin43 • sinoatrial node • immunofluorescence • impulse conduction

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The electrical impulse for the heartbeat is generated within the central part of the sinoatrial node and, from this primary pacemaker area, conducted through a transitional region to the working myocardium of the right atrium. The impulse is conducted slowly in the primary pacemaker area, with increasing speed through the transitional region, and even faster in the crista terminalis, which is the first activated thick atrial bundle.^{1 2} Conduction velocity is determined by the electrical membrane properties of the myocytes and their intercellular coupling: the size and location of gap junctions³ (arrays of low-resistance intercellular channels) in relation to the geometry of the cells. Gap junctions are scarce and small in the central part of the sinoatrial node, where their density is an order of magnitude less than in working myocardium, as determined by quantitative electron microscopy.⁴ From qualitative ultrastructural observation, it appeared that gap junctions became more frequent and larger in size in the transitional region, from the primary pacemaker area to the atrium.¹ The nodal cells in the transitional region gradually change in ultrastructure, size, shape, and arrangement from interwoven typical nodal cells (with only a few random oriented myofibrils in small cells) to parallel atrial cells (with many myofibrils oriented longitudinally in larger elongated cells).^{1 5 6 7} Membrane electrical properties seem to change concomitantly in this transitional zone. A gradual shift occurs from purely nodal characteristics (low maximal diastolic potential [MDP], high diastolic depolarization rate [DDR], and low maximal rate of rise [V_max]) to atrium-like characteristics (high MDP, low DDR, and high V_max).¹ However, Kodama and Boyett⁸ and Opthof et al⁹ showed that these changes were not caused by intrinsic differences in membrane properties of cells in the transitional region, because when they recorded action potentials in small isolated pieces of this tissue, the action potentials had DDRs equal to or even higher than those in primary pacemaker cells. Kirchhof et al¹⁰ provided evidence that this difference is caused by the electrotonic influence of adjacent atrial tissue, which exhibits a diastolic membrane potential hyperpolarized with respect to the MDP of primary pacemaker cells.

The following question arises: How do cells in the center of the sinoatrial node maintain pacemaker activity in the face of the hyperpolarizing influence of the atrium? Joyner and van Capelle¹¹ addressed this question in a theoretical study. They coupled models of sinoatrial node cells with models of atrial cells to test which coupling resistance would allow pacing (impulse formation) and driving (propagation) of the preparation and thus check the atrial hyperpolarizing influence. Their functional model consisted of a circular sinoatrial node (center, 0.5-mm radius; arbitrary thickness) of weakly coupled sinoatrial node–model cells surrounded by a ring (radius, 1 mm) of sinoatrial node–model cells coupled with decreasing resistance of a factor 10 to the large (radius, 3 mm) outer ring of well-coupled atrial-model cells. The gradient of coupling was found to be essential in the model because without it only pacing and no driving occurred. In the heart, the sinoatrial node functions despite the atrial hyperpolarizing load; therefore, a gradient of coupling between cells in the transitional region may be part of the security mechanism for proper pacemaker functioning. However, whether the transitional area exhibits a gradual change in electrical coupling is questionable. Bouman et al¹² described an irregular pattern of electrotonic spread of current in the rabbit sinoatrial node instead of the more gradual pattern described by others.¹ This finding correlates with the immunocytochemical results of Oosthoek et al,¹³ who described an interdigitating pattern of sinoatrial node cells and atrial cells in the bovine right atrium. The latter authors could detect no gap junction protein between sinoatrial node cells and very little between sinoatrial node cells and atrial cells. The objective of the present investigation was to characterize the morphological substrate for the supposed gradient of coupling. In view of the result reported by Oosthoek et al, a clear distinction between atrial and sinoatrial node cells had to be made, which is difficult since gradual changes in morphology are described in the transitional zone.¹⁴ Therefore, the observation we made that a monoclonal antibody specific against the -actin isoform of smooth muscle (SMA) apparently specifically stains sinoatrial node cells of the guinea pig provided an important tool in delineation of the sinoatrial node.

To assess the distribution of gap junctions in the region of interest, we used site-directed antibodies against connexin43 (Cx43) and, for some experiments, against connexin40 (Cx40). Connexins, the proteins forming gap junctional channels, are encoded by a gene family.¹⁵ The transmembrane and extracellular domains of all connexins are largely homologous, whereas the cytoplasmic regions have unique amino acid sequences. Site-directed antibodies raised against unique peptide sequences of this cytoplasmic domain permit specific detection of a particular connexin. The major cardiac gap junction protein,¹⁶ Cx43 (named after its predicted molecular mass of 43 kD), has been demonstrated to be present in gap junctional membranes when immunocytochemical staining with a number of different anti-peptide antibodies is used.^{17 18 19 20 21 22}

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals and Tissue Preparation
Hearts of 20 young adult guinea pigs (HSD/CPB, Dunkin Hartley, Harlan, Zeist, The Netherlands) were excised after decapitation of the animals. Right atrial preparations containing the crista terminalis, the sinoatrial node, and a small edge of the intercaval region were made as described previously.²³ After electrophysiological mapping, 12 preparations were immersion-fixed 1 to 10 hours with either 2% freshly prepared paraformaldehyde in PBS (pH 7.4) or periodate-lysine-paraformaldehyde.²⁴ Neither procedure caused loss of antigenicity. After gradual infiltration to 20% sucrose/Tissue-Tek OCT compound (2:1) or 20% sucrose²⁵ in PBS, the preparations were frozen in liquid N₂ and serially cryosectioned (6 µm) perpendicular to the crista terminalis. Cryosections were collected on poly-L-lysine–coated slides, dried, and stored at -90°C.

Histology and Immunohistochemistry
Every tenth section was stained for histology according to the van Gieson method (Kiernan²⁶ ). Each subsequent section was stained for Cx43 by immunofluorescence. The antibody that we used was raised in the rabbit against a peptide corresponding to residues 314 to 322 of the COOH-terminus of rat Cx43 and affinity-purified against the peptide.²¹ It was previously characterized in the rat and shown to cross-react with many mammalian species, including guinea pig.²¹ The sections were rehydrated in PBS and incubated in 0.2% Triton X-100 in PBS for 1 hour, followed by 0.5 mol/L NH₄Cl in PBS for 15 minutes. The sections were preincubated in 1% bovine serum albumin in PBS for 30 minutes and incubated overnight with anti-peptide serum (60 µg/mL) diluted 1:10 in the preincubation buffer. The sections were rinsed three times for 10 minutes with PBS and again preincubated for 30 minutes. The secondary antibody, tetramethylrhodamine isothiocyanate (TRITC)–conjugated goat anti-rabbit IgG (Jackson), was diluted 1:10 and incubated for 30 minutes with 10% normal guinea pig serum to absorb cross-reactivity of the secondary antibodies to guinea pig tissue. After centrifugation (15 minutes at 15 000 rpm), the supernatant was used to incubate the sections for 1 hour. After the slides were rinsed three times for 10 minutes with PBS, they were mounted in PBS/glycerol (1:9 [vol/vol]) containing 2.3% (wt/vol) 1,4-diazabicyclo[2,2,2]octane (DABCO, Sigma Chemical Co). Negative controls consisted of either omission of the first antibody incubation or incubation with affinity-purified fractions of preimmune serum.²¹ In some experiments we tested for the presence of Cx40, for which we used an antibody (final concentration, 5 to 15 µg/mL) raised in rabbits against a peptide corresponding to residues 335 to 356 of rat Cx40, previously characterized and shown to cross-react with guinea pig heart.²⁷

SMA (Sigma) was used in a dilution of 1:1000 and detected with either fluorescein isothiocyanate (FITC)- or TRITC-conjugated donkey anti-mouse IgG (1:100, Jackson) in single- or double-staining experiments with Cx43 antibody. Sections were examined with an epifluorescence microscope (Nikon Diaphot) equipped with the appropriate filters.

Electrophysiology and Correlation of Impulse Propagation With Sinoatrial Node Morphology
Correlation of electrophysiology with histology of the sinoatrial node preparation was performed as described previously by Bleeker et al.¹ Guinea pig right atrial preparations, including the sinoatrial node and the crista terminalis but excluding the atrioventricular node, were mounted on a perforated silicon rubber block with the endocardial side up and superfused (5 mL/min) with a salt solution containing (mmol/L) NaCl 130.6, KCl 5.6, CaCl₂ 2.2, MgCl₂ 0.6, NaHCO₃ 24.2, glucose 11.1, and sucrose 13.2. The solution was saturated with 95% O₂/5% CO₂ and kept at 37±0.3°C. A bipolar surface electrode positioned on the thick part of the crista terminalis, as indicated in Fig 4b (asterisk), was used to record an atrial surface electrogram, which served as a time reference. Transmembrane potentials were recorded with conventional glass microelectrodes filled with 2.7 mol/L KCl and 2 mmol/L potassium citrate, which had a resistance of 20 to 25 M. They were mounted on a micromanipulator with Vernier scales, on which the coordinates of the impalements could be read with an accuracy of 10 µm. Each activation map was made with a single electrode, which was impaled successively at 60 to 80 different places in the preparation. The distance between impalements was 200 µm in the primary pacemaker area and 400 µm in peripheral regions. The activation moment was defined as the time at which the upstroke of the action potential was halfway between the maximal diastolic potential and the top of the action potential. Sinoatrial conduction time was calculated as the time between this activation moment and the first fast deflection of the atrial surface electrogram. Isochrones as depicted in Fig 4b were drawn by connecting impalement sites with the same sinoatrial conduction time. To align the electrical and morphologic map, the electrode was backfilled with 1% Alcian blue in 0.5 mmol/L sodium acetate (pH 4.0) at the end of the mapping procedure. The tip of the electrode was broken to lower resistance, and the tissue was marked with 10- to 50-µm-diameter Alcian blue dots iontophoretically by applying rectangular 0.5-mA pulses (500 Hz; duration, 30 to 300 microseconds) for 10 to 15 seconds (Lee et al²⁸ ). Dots were placed at the site of the dominant pacemaker, at the corners of a 200-µm–sided square around it, and at 400-µm interdot distances along lines parallel and perpendicular to the crista terminalis. In total, 11 to 15 dots were placed. After localization of the dots in the sections, the three-dimensional reconstruction of the Cx43 and SMA labeling patterns in the sinoatrial node region (see below) was projected onto the grid of the activation map, resulting in a two-dimensional reconstruction of the labeling patterns.

View larger version (18K): [in this window] [in a new window]   Figure 4. Correlation of connexin43 (Cx43) and -smooth muscle actin (SMA) distribution with impulse propagation in a guinea pig sinoatrial node preparation. a, Spatial distribution of Cx43 and SMA from cranial (I) to caudal (V) in sections perpendicular to the crista terminalis and the endocardium. Exclusively SMA-staining regions are green, and exclusively Cx43-staining regions are red. Regions where both Cx43 and SMA are detected, also demonstrated in Figs 2 and 3, are yellow. Connective tissue as identified in van Gieson–stained sections is blue. Five composite drawings at 900-µm distances through the sinoatrial node are shown. See "Materials and Methods" for details. b, Activation map of the preparation. Isochrones are projected on the endocardial surface together with a projection of the SMA and Cx43 staining patterns, performed as detailed in "Materials and Methods." Letters indicate impalement sites where action potentials depicted in panel C were obtained. The conduction time of each isochrone is indicated in milliseconds relative to the activation moment of the primary pacemaker located at impalement site g. CT indicates crista terminalis; IAS, interatrial septum; VCS, vena cava superior; and VCI, vena cava inferior. The asterisk indicates the recording site of the extracellular atrial electrogram. The projection of the Cx43 and SMA labeling pattern consists of four regions: green, region where only SMA staining is encountered between epicardium and endocardium; gray, region where endocardially located Cx43-positive cells overlay exclusively SMA-positive cells; hatched, region where exclusively SMA-positive cells overlay epicardially located Cx43-positive myocytes; and red, region where only Cx43-stained cells are encountered between endocardium and epicardium. For simplicity, the connective tissue is not indicated. c, Action potentials recorded at different sites in the preparation as indicated by letters in panel B. The asterisk indicates the action potential recorded from the earliest firing cell. The dashed line indicates the time reference of the atrial surface electrogram, depicted in the upper tracing of the three panels in c. Activation times are given in milliseconds.

Light Microscopic Reconstruction of Cx43 Labeling in Sinoatrial Node Region
Morphological observations were performed in 6-µm-thick sections perpendicular to the endocardial surface and the crista terminalis. The tissue was serially sectioned from the superior vena cava to the inferior vena cava (4 to 5 mm).

To obtain a two-dimensional morphological representation of the preparation coinciding with the electrophysiological maps, the following steps were taken: (1) Section 1 was stained for Cx43; section 5, according to the van Gieson method; and section 10, for SMA. The contour of the van Gieson–stained section was projected on drawing paper and outlined, and the Cx43 and SMA staining patterns were sketched into it. This procedure was repeated every 30 sections, ie, at every 180 µm. Five of the thus-obtained reconstructions are shown in Fig 4a. (2) These reconstructions were aligned to each other and to the activation map by eye by using the Alcian blue dots visible in the van Gieson–stained sections and taking into account the distance between the reconstructions. The location of each reconstruction was represented by a line drawn onto the activation map. (3) Each line was colored according to the staining encountered in the reconstruction it represented: red where only (from endocardium to epicardium) Cx43 staining was present, green where only SMA staining was present, gray where endocardially located Cx43-positive cells overlayed exclusively SMA-positive cells, and hatched where exclusively SMA-positive cells overlayed epicardially located Cx43-positive cells. The end points of the colored line segments of each of the lines spaced 180 µm apart were connected. In this way, a two-dimensional morphological map was obtained of which Fig 4b shows an example.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cx43 Staining Pattern in Histologically Characterized Sinoatrial Node
To obtain an overview of the Cx43 distribution in the sinoatrial region, three guinea pig right atrial preparations were serially sectioned perpendicular to the crista terminalis, including the presumed central part of the node. Based on the histological appearance on van Gieson–stained sections, a sinoatrial node region characterized by pale staining, high nuclear density, and many collagen fibers could be identified. The small nodal myocytes loosely packed in connective tissue were arranged in a central region where the nodal cells form an interweaving network and a peripheral region where they are arranged more in parallel.²³ In the central region, nodal cells fill all space between endocardium and epicardium. The central region extends toward the crista terminalis at the epicardial side of the sinoatrial node bordering a region of connective tissue. The region of peripheral nodal cells is found between the endocardial side of the central region and the crista terminalis²³ (Fig 1A). A section through the central region of each preparation was selected and labeled with anti-Cx43 for immunofluorescence detection. Cx43 was abundantly present between atrial cells but could barely be detected in the histologically defined central region (Fig 1B), with the exception of strands of labeled cells running along the endocardial side of the preparation.

View larger version (0K): [in this window] [in a new window]   Figure 1. A, Composite photograph of a van Gieson–stained section through the presumed central part of the guinea pig sinoatrial node. The central part of the sinoatrial node consisting of small interwoven cells is outlined by arrows. B, Immunofluorescent image of connexin43 distribution in a section 30 µm distant from the one shown in panel A. Arrow points to end point of endocardially located strands of connexin43-positive cells. All sections in this and following figures are displayed in the same orientation, unless otherwise stated: the crista terminalis is at the left; the interatrial septum, at the right; the top of the image is the endocardium; the bottom, the epicardium. Bar=100 µm.

In double-stained sections, it appeared that the endocardial layer consisted of several strands of strongly Cx43-labeled cells interdigitating with strands of Cx43-negative cells (Fig 2A and 2B). In each of the preparations, roughly the same staining pattern was observed. Nevertheless, there were clear differences due to (1) individual variability in location and size of the sinoatrial node, (2) deviation from the axis of sectioning, and/or (3) whether the node had been sectioned more caudally or more cranially.

View larger version (0K): [in this window] [in a new window]   Figure 2. Photomicrographs showing sections stained for connexin43 (Cx43, secondary antibody, tetramethylrhodamine isothiocyanate labeled, red) and for -smooth muscle actin (SMA, secondary antibody, fluorescein isothiocyanate labeled, green). In both panels, the endocardium is at the bottom of the figure and the epicardium is at the top. A, Section showing staining patterns that are virtually complementary. Strands of cells with abundant Cx43 labeling are located not only at the endocardial side of this section but also toward the epicardial side. Bar=100 µm. B, Higher magnification of an adjacent section taken more toward the septal side. This picture demonstrates again the complementary staining for SMA and for Cx43. Note the clear cross striations in some of the SMA-positive cells. Bar=50 µm.

SMA Staining of the Histologically Characterized Sinoatrial Node
Precise delineation of the sinoatrial node within the atrial tissue mass is difficult, since gradual changes in morphology are observed.¹⁴ Our observation that a monoclonal antibody against SMA cross-reacts with nodal cells provided an important tool in delineation of the histologically defined sinoatrial node. SMA antibody stained both the cytoplasm of nodal cells and of vascular smooth muscle cells homogeneously (Fig 3A), whereas atrial myocytes exhibited virtually no staining (see Fig 2B and compare lower left of Fig 3A and 3B). Since in some SMA-labeled cells cross striations due to the presence of myofibrils were observed (Fig 2B and small arrows in Fig 3A), it is clear that SMA labels a special class of myocytes, presumably nodal ones. This suggestion is strengthened by the fact that the Cx43 and SMA staining patterns were virtually complementary in double-stained sections (Fig 2A and 2B). For our purpose, it seems safe to designate Cx43-positive cells as atrial and SMA-positive cells as nodal.

View larger version (124K): [in this window] [in a new window]   Figure 3. Photomicrographs showing sections stained for -smooth muscle actin (SMA, A) and connexin43 (Cx43, B). In this figure, endocardium is at the bottom and epicardium is at the top. A, Anti-SMA stains smooth muscle cells of vessels (large arrows) strongly and nodal cells moderately. Small arrows indicate cross striations in nodal myocytes. Stars indicate corresponding points in panels A and B. B, Large arrows indicate blood vessels that are not stained by anti-Cx43; small arrows, low staining intensity for Cx43. Comparison with panel A shows that this decreased staining is located near SMA-positive cells. Bar=50 µm.

Cx43 and SMA Spatial Distribution Patterns in Electrophysiologically Mapped Sinoatrial Node
To estimate the extent of tissue unlabeled with Cx43 in relation to the light microscopic morphology and to the impulse propagation pattern, the Cx43 distribution pattern of three right atrial preparations was studied after electrophysiological mapping. In one of these, the SMA labeling pattern was also studied. As explained in "Materials and Methods," a composite drawing of the SMA and Cx43 labeling pattern was prepared every 180 µm. Five of these drawings at equal distances of 900 µm throughout the nodal region are shown in Fig 4a to provide a spatial impression of the shape of the different staining patterns. The isochrones obtained after electrophysiological mapping of the same preparation are depicted in Fig 4b together with a projection of the Cx43 and SMA staining pattern on the endocardial surface, performed as outlined in "Materials and Methods." The gray region in Fig 4b represents an area in the preparation where Cx43-positive cells, either exclusively so or intermingled with SMA-positive cells (red or yellow in Fig 4a), are located endocardially with respect to an exclusively SMA-positive region (green in Fig 4a). The hatched area in Fig 4b represents a comparable region located epicardially with respect to the exclusively SMA-stained region. The green area in Fig 4b delineates that part of the preparation where SMA-positive cells fill all space between the endocardium and epicardium.

The isochrones projected on the morphological map in Fig 4b show that activation starts in a small group of cells in the intercaval wall 1 mm septally from the crista terminalis and travels from this primary pacemaker preferentially in an oblique upward direction to the crista terminalis. In the septal direction, isochrones are closely spaced, indicating slow conduction. It seems that the septum is activated via a circuitous pathway around the inferior part of sinoatrial node, as has been reported previously.²³ The primary pacemaker is located in the SMA-positive region, whereas the preferential conduction pathway toward the crista terminalis is within a region where SMA-positive and Cx43-positive cells intermingle (compare Fig 4a, drawings II and III, with Fig 4b, gray area). Slow conduction in the septal direction is probably caused by the presence of connective tissue between the primary pacemaker region and the septal atrial tissue.

Fig 4c shows action potentials recorded at different sites in the preparation as indicated in Fig 4b. It is clear that action potentials recorded within the SMA-positive Cx43-negative region (c, e, g, and i) exhibit typical nodal characteristics, ie, low V_max and steep DDR, whereas those recorded in the region of intermingling show considerable higher V_max and slower (d and k) or virtually nonexistent (b and f) DDR. The latter action potentials have atrium-like characteristics (compare with action potential a). Because the recordings were obtained from superficial endocardial cells, it is probable that in these cases cells were impaled in a Cx43-positive SMA-negative region overlying the region of intermingling as seen in Fig 4a, drawings II and III.

To gain insight into the correlation between electrical activity and tissue architecture of the nodal region, we studied the immunocytochemically different regions more in detail.

SMA Distribution Pattern
As can be seen from Fig 5 the SMA-stained myocytes lie epicardially at the crista terminalis side and endocardially at the septal side of the sinoatrial node. At the crista terminalis side, they are separated from epicardially located atrial myocytes in the cranial part of the sinoatrial node by connective tissue (Fig 4a, drawings I through III). At the septal side of the node, they lie obliquely above a mass of atrial cells, especially in the cranial half of the sinoatrial node (Fig 4a, drawings I through III). The van Gieson–stained sections used for the drawings also showed this abrupt transition of bundles of different cell types, which in some more cranially located sections were separated by a thin sheet of connective tissue, causing the circuitous action potential propagation pathway mentioned before.

View larger version (51K): [in this window] [in a new window]   Figure 5. Composite photograph of -smooth muscle actin labeling pattern in a section from the central part of the sinoatrial node. Arrows indicate blood vessels. This is the same preparation as shown in Fig 4. Bar=100 µm.

Mosaic Distribution Pattern
Although at both the crista terminalis side and the septal side there are places where the SMA-positive region adjoins Cx43-positive regions directly, we also found an extensive region where both types of cells intermingle, primarily located endocardially at the crista terminalis side of the Cx43-negative region (Fig 6). In the cranial part of the sinoatrial node (Fig 4a, drawings I and II), this so-called mosaic region has a maximal thickness of 200 µm. From the cranial to the caudal part, it changes from a thick bundle of cells more or less oriented parallel to the crista terminalis to a thin sheet of cells oriented with their long axis perpendicular to the crista terminalis. Apart from this intermingling of Cx43-positive cells with SMA-positive cells, Cx43 labeling intensity seems to diminish at certain places within a narrow zone one to three cells wide (Fig 3).

View larger version (88K): [in this window] [in a new window]   Figure 6. Crista terminalis side of the sinoatrial node. Photomicrographs show the distribution of connexin43 (Cx43) staining (A) and -smooth actin (SMA) staining (B) of two sections, 60 µm apart, used for composite drawing II of Fig 4a. At the upper (endocardial) region, a strand of Cx43-positive cells is seen (A), below which a Cx43-negative region is encountered, in which we find SMA-positive cells (B). Bar=50 µm.

In the middle of the nodal region, where the right atrial wall is very thin (Fig 4a, between drawings III and IV), all cells between the endocardium and epicardium are SMA positive (Fig 7B). At most sites, however, an endocardial layer of Cx43-positive cells covers the SMA-positive region (Fig 7A, arrow), which is at some sites interrupted with Cx43-negative SMA-positive cells. It spans roughly half the distance between the crista terminalis and septum. In the caudal part of the sinoatrial node, this layer seems to intertwine with SMA-positive cells (Fig 8). The mosaic pattern (yellow in Fig 4a) is in most sections found endocardially with respect to the exclusively SMA-positive region (green in Fig 4a). Also, in a number of caudal sections epicardial strands of Cx43-positive cells intertwine with SMA-positive cells, thereby forming an epicardial mosaic region (Fig 4a, drawing IV). Here, the exclusively SMA-positive region is sandwiched between mosaic regions containing atrial myocytes running mainly perpendicular to the crista terminalis.

View larger version (142K): [in this window] [in a new window]   Figure 7. Central thin part of the sinoatrial node region. Photomicrographs show the distribution of connexin43 (Cx43) staining (A) and -smooth muscle actin (SMA) staining (B) in two sections, 60 µm apart, used for composite drawing III of Fig 4a. This region contains mainly SMA-positive cells. One thin strand of Cx43-positive cells (arrow in A) can be discerned. Bar=50 µm.

View larger version (55K): [in this window] [in a new window]   Figure 8. Longitudinally cut cells in caudal part of the sinoatrial node in two sections 60 µm apart. Photomicrographs show that strands of connexin43 (Cx43)–positive cells (A) and -smooth muscle actin (SMA)–positive cells (B) are intertwined. Sections are displayed in composite drawing V of Fig 4a. Arrow in panel A points to very small dots of Cx43 labeling. Bar=50 µm.

Other Connexins
Recently, it was reported that the canine sinoatrial node region contains Cx40-positive cells.²⁹ Because we were unable to detect Cx43 between SMA-positive cells in the guinea pig sinoatrial node, we wanted to know whether Cx40 might possibly be present in this tissue as in canine sinoatrial node. Fig 9 shows the crista terminalis side of sections near drawing III in Fig 4a, ie, near the primary pacemaker area. In the left panel, a section stained with Cx43 antibody is shown in which an endocardial strand shows clear labeling, whereas the more epicardially located (presumably nodal) cells are devoid of staining. The right panel shows an adjacent section stained with Cx40 antibody. Here again the endocardial strand shows clear labeling, whereas the epicardially located cells are devoid of staining.

View larger version (113K): [in this window] [in a new window]   Figure 9. Crista terminalis side of sinoatrial node (endocardium at bottom). Photomicrographs show two sections 60 µm apart stained with anti–connexin43 antibody (A) and anti–connexin40 antibody (B). Labeling is positive for both connexins at the endocardial side, whereas virtually no staining is seen in the more epicardially located cells. The big dot in panel B is a staining artifact. Bar=50 µm.

Cx45 was also shown to be present in canine sinoatrial node.²⁹ In the present study, we could not assess whether this is also true for guinea pig sinoatrial node, because of lack of a well-characterized antibody.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we investigated the distribution of gap junctions in and around the guinea pig sinoatrial node by immunocytochemistry. Our aim was to substantiate the hypothesis of Joyner and van Capelle¹¹ that the center of the sinoatrial node, the primary pacemaker of the heart, is shielded from the hyperpolarizing influence of the surrounding atrium by a gradient of coupling. As a marker for gap junctions we used an antibody against Cx43, the major gap junction protein in the heart.^{16 21} Although several other gap junction proteins have been reported to be present in hearts of different mammalian species,^{29 30 31 32 33 34 35 36} no evidence exists for other gap junction proteins to be more ubiquitous in heart as Cx43.

SMA Antibody as a Marker for Guinea Pig Sinoatrial Node Cells
Comparison of SMA distribution patterns with activation maps (Fig 4b and 4c) shows that the primary pacemaker area exclusively contains SMA-positive cells. The antibody homogeneously stains cells in the central nodal area where virtually no Cx43 staining can be detected and in equally Cx43-empty regions between abundantly Cx43-stained strands of presumably atrial cells in the periphery of the node (Fig 2). Because SMA-stained cells show cross striations (Figs 2B and 3) and action potentials with nodal characteristics (steep DDR, low V_max; Fig 4c) were recorded from them, these cells are sinoatrial node cells. Markers for the conduction system in adult heart, including the sinoatrial node, have been described by others. Oosthoek et al¹³ found that a monoclonal antibody against the bovine conduction system (antibody 445-6E10, which recognizes a desmin-related protein) also stained the morphologically identified bovine sinoatrial node, but not rat or human sinoatrial node cells, whereas Gorza and colleagues^{37 38} reported specific marking of all sinoatrial node cells in rabbit by an anti-neurofilament antibody. Whether anti-SMA reacts with smooth muscle actin present in sinoatrial node cells of guinea pig or cross-reacts with a related protein is not known.

Cells staining both with anti-Cx43 and anti-SMA were extremely scarce (see Figs 3 and 4) and were found only in regions where atrial cell sheets ended in the SMA-positive network of cells. Anti-SMA antibody staining apparently only allows distinction of two types of cells in the nodal region: atrial cells and nodal cells. Transitional cells as described by others on morphological grounds^{5 6 7 14} were not seen. There is some variation in the intensity of SMA staining (see Figs 2 and 5), but this is neither correlated with localization in the node nor with variation in Cx43 staining.

Is Cx43 Expressed in Sinoatrial Node?
In the central part of the sinoatrial node of all species investigated, gap junctions are scarce but definitely present, as judged by electron microscopy.^{4 23 39} Anti-Cx43 antibody staining, however, virtually fails to detect them^{13 40 41} in rat, bovine, and human sinoatrial nodes or, as confirmed in the present study, in guinea pig sinoatrial node (Fig 1B). The study of Trabka-Janik et al⁴² reporting that hamster sinoatrial node contains a considerable amount of Cx43 will be discussed below. The failure to detect Cx43 can be caused either by a low expression level of Cx43 and insufficient sensitivity of the techniques used to detect it or by the presence of other connexins.

Recently Cx40 and Cx45 have been reported to be present in canine sinoatrial node gap junctions.^{29 43} Although Cx40 was abundantly present in guinea pig atrial tissue,^{27 36} we were not able to detect it in the sinoatrial node (Fig 9B). Because we could not assay for the presence of Cx45, we cannot exclude the possibility that guinea pig sinoatrial node cells are coupled by Cx45-containing gap junctions. Based on the experiments reported in the present study, we conclude that between guinea pig sinoatrial node cells, Cx40 and Cx43 at least are equally difficult to detect.

To assess the limit of detection of connexins in small gap junctions by immunofluorescence, we perused literature data. In pancreatic ß cells, minute and scarce gap junctions containing Cx43 are present.^{44 45 46} From studies by Meda et al,⁴⁶ we calculated that gap junctions containing 40 channels can just be detected by immunofluorescence, whereas from the data of Gros and colleagues,^{47 48 49} the lower limit of detection turned out to be 75 channels per gap junction. Since the mean number of channels in rabbit sinoatrial node gap junctions, and presumably also in guinea pig sinoatrial gap junctions, is reported to be 90 channels,⁴ they will be marginally detected with the fluorescence microscope. Therefore, we conclude that gap junctions between guinea pig sinoatrial node cells whether they contain Cx40, Cx43, or Cx45 are barely detectable with immunocytochemical techniques, because of the small number of antigenic sites, as has been reported previously, for rat sinoatrial node.^{40 50}

The quite substantial labeling with Cx43 antibodies that we find in the sinoatrial region of the guinea pig (Fig 2) is not due to gap junctions connecting nodal cells proper but between atrial cells intercalated between nodal cells. This is in contrast to Trabka-Janik et al,⁴² who showed clear staining of hamster sinoatrial region with Cx43 antibodies, indicating in their view that in this species gap junctional coupling between nodal cells is considerable. Because Trabka-Janik et al did not discern between nodal cells and atrial cells within the node as we did (compare with our Fig 2), the possibility remains that also in the hamster, strands of atrial cells are intercalated with nodal cells. Anumonwo et al⁵¹ described Cx43-containing gap junctions in the rabbit sinoatrial node, but here again no distinction was made between nodal cells and intranodal atrial cells as described by Masson-Pévet et al.⁶ Recently Davis et al²⁹ demonstrated the presence of Cx40 and Cx45 in the canine sinoatrial node region. Cx43 was not detected in this region. All three connexins were found to be present abundantly in the surrounding atrium. In the latter study, no electrophysiological identification of the primary pacemaker area was performed, and no specific marker for nodal cells was used. Therefore, it is difficult to exclude the possibility that in the case of the canine sinoatrial node the connexin-positive cells were of atrial origin.

Propagation of Activation From Sinoatrial Node to Atrium
As can be seen from Fig 4b, the impulse originates in a region where SMA-positive cells fill the space between endocardium and epicardium. The impulse preferentially travels in an oblique cranial direction toward the crista terminalis in a mosaic region overlaying an SMA-positive region (combining Figs 4a and 4b). When the recording site is near the mass of atrial cells in the crista terminalis, the chance of recording atrial-like action potentials from a mosaic region is greater than when it is far from it (compare action potentials b and f with k). As can be inferred from Figs 2 and 3, the proportion of atrial cells increases in the crista direction (ie, in the direction of sites b and f) and is low at recording site k.

As discussed above, it is difficult to assess the number of gap junctions in the nodal region. The few Cx43-stained spots localized between SMA-positive cells and atrial cells were always in a region with few atrial cells and a predominance of SMA-positive cells.

If our observations are correct, atrial cells and sinoatrial node cells should be found directly adjacent not only by use of immunofluorescence and activation maps but also by use of other techniques. From ultrastructural studies, it has been reported both for rabbit⁶ and guinea pig²³ sinoatrial node that atrial cells exhibiting organized myofibrils are present directly adjacent to cells that by morphological criteria are described as purely nodal (empty cytoplasm, many glycogen granules, extensive caveolae, and few poorly organized myofibrils). In experiments aimed at measuring the space constant of rabbit sinoatrial node, Bouman et al¹² found that sometimes externally applied membrane voltage displacements at the site of earliest activation did not decay exponentially with distance, as expected in their experimental setup, but instead increased. This phenomenon, which occurred mainly perpendicular to the crista terminalis, can be understood if it is assumed that sinoatrial cells were impaled in the neighborhood of the current electrode and that when the electrode was moved in the direction of the crista terminalis, cells in atrial strands intercalated in between sinoatrial node cells were impaled. The occurrence of different cell types at epicardium and endocardium only 0.2 mm apart from the primary pacemaker to the crista terminalis side has previously been shown by combined electrophysiology and electron microscopy.²³ Taken together, all these observations imply that strong electrotonic interaction between atrial and nodal cells intermingled in mosaic regions is absent. Gap junctions are scarce, and a clear-cut gradient of gap junction density is not observed. This is in agreement with Oosthoek et al,¹³ who described a comparable architecture in the bovine sinoatrial node.

How then is activation of the atrium by nodal cells brought about without the atrium silencing nodal activity? From our data and literature, the following possibility can be surmised: SMA-positive nodal cells are weakly coupled to each other, thereby forming a network of synchronously firing pacemaker cells. As pointed out by several authors,^{52 53 54} synchronization of sinoatrial pacemaker cells requires only few gap junction channels. Estimates range from three 50-pS channels⁵⁴ for synchronization of the firing rate to 100 of these channels⁵² between two cells for complete synchronization (ie, simultaneous firing without measurable latency). Atrial cells are well coupled by Cx43-containing gap junctions. Direct coupling between the mass of atrial cells and nodal cells is supposed to be weak or absent, because gap junctions can barely be detected by immunocytochemistry and the impulse does not propagate preferentially across this border, as inferred from electrophysiological measurements.²³ Impulse transmission from sinoatrial node cells to atrial cells occurs at the end of thin strands of atrial cells invaginating into the mass of sinoatrial cells, where few gap junctions between atrial and nodal (SMA-stained) cells are detected (Figs 2B and 3). Several synchronously firing sinoatrial node cells are coupled to only few atrial cells and are able to depolarize them to threshold. The impulse travels in the atrial strands toward the crista terminalis, at first slowly, as can be seen from the activation map of Fig 4b, because the strands of cells are thin, but then with increasing speed as the number of interconnected atrial cells increases. The same configuration ensures that sinoatrial pacemaker cells are shielded from the hyperpolarizing influence of the atrium: in the region of contact between nodal and atrial cells, the input resistance of the coupled nodal cells is relatively low, and the input resistance of the thin end of the atrial strand of cells is relatively high. Hyperpolarizing current from the atrial cells thus has little influence on the membrane potential of coupled nodal cells, whereas depolarizing current from these nodal cells, small as it may be, is sufficient to depolarize the atrial cells to threshold because of their high input resistance.

It should be emphasized that this hypothesis is based on morphological and electrophysiological data, which have a different spatial resolution. Whereas the morphology has a resolution of tens of micrometers, the electrophysiological resolution is on the order of hundreds of micrometers. To narrow this gap, optical measurements of activation spread using voltage-sensitive dyes are planned. Nevertheless, it seems safe to conclude that the absence of extensive coupling between atrial tissue and sinoatrial node cells and the presence of mosaic regions in which atrial and nodal cells intermingle is important for the small sinoatrial node to drive the much larger atrium. Model simulations to gain insight in this issue are well under way.

	Acknowledgments

 
This study was supported by the Netherlands Organization for Scientific Research (NWO grant 900-516-093 to Drs ten Velde, Jongsma, and Verheijck), the Institut National de la Santé et de la Recherche Médicale (INSERM grant 92-0409 to Dr Gros), the Direction des Recherches et Etudes Techniques (DRET grant 92-159 to Dr Gros), the Association Français contre les Myopathies (to Dr Gros), and the Netherlands Heart Foundation (NHS grant 91059 to Drs van Kempen, Analbers, and Jongsma).

	Footnotes

 
Reprint requests to Dr H.J. Jongsma, Department of Medical Physiology and Sports Medicine, Utrecht University, PO Box 80043, 3508 TA Utrecht, Netherlands.

Previously published as preliminary results in abstract form (Histochem J. 1992;24:575).

Received July 12, 1994; accepted January 10, 1995.

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