The Atrioventricular Node and Bundle in the Ferret Heart: A Light and Quantitative Electron Microscopic Study THOMAS A. MARINO Department of Anatomy, Temple Unwersity School of Medicine, Philadelphia, Pennsylvania 19140

ABSTRACT The cells of the atrioventricular (AV) junction in the ferret heart were examined using light microscopy, a wax-model reconstruction and quantitative electron microscopy to determine their organization and characteristics. A series of subdivisions of the specialized tissues of the AV junction was apparent at both the light and electron microscopic levels. A transitional zone was observed interposed between the atrial muscle cells and the AV node. The AV node consisted of a coronary sinus portion, a superficial portion and a deep portion. The AV bundle had a segment above the anulus fibrosus, a segment which penetrated the right fibrous trigone, a non-branching segment below the anulus fibrosus and a branched segment. At the ultrastructural level the AV junctional conduction tissues had fewer irregularly oriented myofibrils than did working atrial myocardial cells. T-tubules, present in atrial muscle cells, were not observed in the modified muscle cells of the AV node and bundle. Conventional intercalated discs also were not observed between the cells of the AV node or the AV bundle. Atrial myocardial cells had the highest percentage of the plasma membrane occupied by desmosomes, fasciae adherentes and gap junctions. The AV bundle cells had the highest percentage of appositional surface membrane and a relatively large fraction of plasma membrane occupied by gap junctions. Cells of the superficial portion of the AV node had the smallest percentage of the plasma membrane composed of gap junctions, desmosomes or fasciae adherentes, as well as the smallest fraction of the cell membrane apposed to adjacent cells. The stereological data indicate that the most useful distinguishing characteristic between atrial muscle cells and conduction cells was that a smaller percentage of the conduction cell sarcoplasm was occupied by mitochondria and myofibrils. The most useful characteristics that could be used t o differentiate between the regions of the AV junctional conduction tissues were the amounts and types of surface membrane specializations in the respective cell types. In the mammalian heart the atrioventricular (AV) node and bundle constitute the only normal pathway through which electrical impulses from the atria are transmitted to the ventricles, and this arrangement of AV junctional tissues permits coordinated contractions of the heart chambers. Due to the important location of the AV node and bundle, the complex cellular interrelationships both within and between these structures have been the focus of numerous light microscopic studies. Many of these comparative light microscopic AM. 3. ANAT. (19791 154: 365-392.

studies have been reviewed by Lev ('601, Robb ('65) and Truex and Smythe ('65).In a recent light microscopic study of the ferret heart, Truex (Truex e t al., '74; Truex, '74) described the AV node and bundle and suggested several advantages for using this animal for cardiovascular research. Truex pointed out that this mammal would be good for electrophysiologReceived June 20, '78. Accepted Oct. 5, '78. 'This work was supported in part by N.I.H. grants HL07047, HL19425, HL19606, CA08231. Present address: Department of Anatomy, University of Kansas Medical Center, Kansas City, Kansas 66103.

365

366

THOMAS A. MARINO

ical and ultrastructural studies and supported this contention by indicating that the ferret is a sturdy animal which has a small heart, a well differentiated AV node, and a n AV bundle which penetrates the right fibrous trigone. Truex (Truex et al., '74) divided the ferret AV node into a coronary sinus portion, a main portion and a cap-like portion above the proximal AV bundle. The AV bundle consists of a segment above the anulus fibrosus, a segment which penetrates the right fibrous trigone, a non-branched segment below the anulus and a branched segment (Truex et al., '74). For a greater understanding of the interrelationships between the AV node and bundle a t the light microscopic level, several investigators have utilized serial sections of the AV junction to reconstruct this area in three dimensions (Baird and Robb, '50; Rodbard, '58; Truex and Smythe, '67; Lev and Thaemert, '73; Anderson et al., '74, '75). Truex (Truex and Smythe, '67) and Anderson (Anderson et al., '75) have both pointed out the value of reconstructed models to visualize the structure of the AV node and bundle. This information also can be used to indicate the location of cells to be examined a t the fine structural level, as Thaemert ('70, '73) has done in the mouse heart. In addition to Thaemert's ('70, '73) meticulous ultrastructural studies of the mouse AV node, there have been numerous other fine structural studies of the AV junction in a variety of mammals. In early electron microscopic studies, the AV junctional cells were divided into one AV nodal cell type, and Purkinje cells within the AV bundle (Maekawa et al., '67; Kim and Baba, '71). The nodal cells are small with a paucity of myofibrils, while the Purkinje cells are larger, also having few myofibrils (Maekawa et al., '67). James and Sherf ('68a,b, '71) examined the human AV junction at the ultrastructural level and found that there are four cell types in the AV node and in the AV bundle. Most of the cells of the AV node are transitional cells, which are similar to the nodal cells described by Maekawa et al. ('67). "P" cells, which are larger than transitional cells with even fewer myofibrils, are interspersed within the node. Purkinje cells and working myocardial cells also were found in t h e human AV node. These latter two cell types were not seen as frequently in the AV node as were the transitional cells and "P" cells, however. The AV bundle also consists of the four cell types. In the AV

bundle, the Purkinje cells are the predominant cell type. Recently, DeFelice and Challice ('691, Challice ('711, Viragh and Challice ('73) and Tranum-Jensen ('761 have divided the rabbit AV junction into several regions. A transitional zone between atrial musculature and the AV node, two regions within the AV node, and the AV bundle have been described a t both the light and electron microscopic levels. These authors observed that each region of the AV junction consisted of a morphologically unique cell type. While muscle cells from other regions of the heart have been examined stereologically a t the fine structural level (Page et al., '71; Spira, '71; Matter, '73; Page and McCallister, '73a,b; Frank et al., '75; Phillips and Bove, '761, there has been only one quantitative electron microscopic study of the AV bundle (Arluk and Rhodin, '74). In this study the surface specializations of the AV bundle cells in the calf heart were analyzed. However, there have not been any quantitative ultrastructural studies of t h e AV nodal cells. Therefore, quantitative fine structural comparisons of the AV nodal cells to the AV bundle cells, and of these cells to the working myocardial cells in the same mammalian species, have never been made. The present study included a wax reconstruction of the AV node and bundle from 10-Fm-thickserial sections. This was followed by examination of these structures at the fine structural level. Finally, a systematic morphometric analysis of the cells of the AV junction was done a t the ultrastructural level on atrial muscle. cells, transitional cells, superficial AV nodal cells, deep AV nodal cells, and proximal and distal AV bundle cells. MATERIALS AND METHODS

A total of 11 adult ferrets were used in this study, with four male and three female hearts studied at the light microscopic level, and four female hearts examined at the electron microscopic level. Differences between male and female ferret hearts were not observed a t the light microscopic level. All the animals were anesthetized intraperitoneally with sodium pentabarbitol (35 mg/kg body weight), placed on a respirator, and then perfused through the inferior vena cava with 10 ml of a saturated solution of KC1, to stop t h e heart in diastole. This procedure was followed by a brief wash with either Ringer's or Locke's solution. Ferret hearts used for t h e light microscopic study were fixed with a retrograde perfusion

THE AV NODE AND BUNDLE IN THE FERRET HEART

through the ascending aorta with 10%neutral buffered formalin after the Ringer's wash. Immersion fixation was continued overnight and the hearts were dehydrated, cleared and embedded in paraffin. They were sectioned serially in the frontal plane a t 10 pm and the sections were stained with either Masson's trichrome, hematoxylin - phloxine - saffranin (HPS), or the Holmes' silver technique. The serial sections of one adult heart were used for a wax reconstruction of the AV node and AV bundle. For this procedure the AV junction of every third 10 pm section was projected onto a sheet of paper, a t a magnification of 85 x , and traced. One-millimeter-thick sheets of different colored dental wax were cut to match the tracings of the different regions of the AV junction. The colored wax pieces of one tracing were fused together a s a composite wax plate. The series of wax plates was then joined together using the mitral and tricuspid valves as orienting landmarks, to reconstruct the AV node and bundle. The four adult female ferret hearts used for electron microscopic study were perfused with 1.25%glutaraldehyde in a sodium cacodylate buffer (pH, 7.3; 350 mOsm) following the Locke's solution wash (Tomanek and Karlsson, '73). After perfusion, the hearts were removed, immersed in cold fixative (4°C)for one to two hours, and then placed in sodium cacodylate buffer. One-millimeter slices of the AV junction were cut parallel to the frontal plane of the heart and post-fixed in 1%OsO, in sodium cacodylate buffer. The slices of the AV junction were dehydrated and embedded in Epon-Araldite (Molenhauer, '64). Each slice was embedded so t h a t it could be sectioned in the frontal plane of the heart. Both 1-pm sections and silver to gold thin sections were cut with glass knives on a Reichert Ultramicrotome OmUs. The 1-pm sections were stained with methylene blue and azure I1 (Richardson et al., '60). The thin sections were stained with lead citrate (Reynolds, '63) and uranyl acetate (Watson, '58). Thin sections were viewed and photographed on a Phillips EM-300 electron microscope at 60 kv. The ultrastructural stereology was based on 100 cell profiles from each of two female animals for every region examined, except for the atrial muscle cell profiles, where 50 profiles from each of two animals were used. The cells from each of the regions were chosen randomly by the following method. For each junction-

367

al region, a grid space was located which contained only cells from that particular region, and beginning with the upper left hand corner and moving to the right, every cell profile was photographed. Then the cells below t h a t in the next row were photographed. This was continued until 100 cell profiles were obtained from each animal for each junctional region. The electron micrographs were analyzed a t a final magnification of 35,500 x . Only whole cell profiles were examined and montages were constructed for those cells that were too large for one 16-by-20-inchprint. Morphometric analysis was done on a Thompson X, Y plotting table. This digitizer was interfaced to a DEC PDP-9 computer which in turn was linked to an off-line printer. The electron micrograph of each cell profile was placed on the Thompson table and the cell perimeter was traced. The computer then determined the perimeter length and the area of the cell profile. If a nucleus was present its perimeter was circumscribed and and the nuclear area was calculated. From this information the percentage of the cell volume occupied by the nucleus, and the sarcoplasmic volume were determined according to the stereological principles which permit the extrapolation of volume density from cross sectional area data (Weibel, '69). The sarcoplasm was defined in this study as that portion of the cell that did not contain the nucleus. Each mitochondrion within a cell profile was then circumscribed. This data provided several pieces of information. First, the total mitochondria1 area within a cell profile was determined. Also the number of mitochondria and the average area of a single sectioned mitochondrion were calculated. Finally, the percentage of the sarcoplasm occupied by mitochondria was obtained. The myofibrils also were traced and the total area of the myofibrils within a cell profile, as well as the percent volume of the sarcoplasm which consisted of myofibrils, was determined. Using the same procedures, similar data was obtained for the Golgi complex. The sarcoplasm devoid of mitochondria, myofibrils or the Golgi complex was designated as the clear area of a cell. The percentage of the sarcoplasmic volume composed of the clear area in each cell type was also calculated. Surface specializations of the cells in t h e six different regions of the AV junction were also analyzed. The length of the perimeter of a cell profile t h a t was apposed to another cell profile

368

THOMAS A. MARINO

was traced. The length of apposed membrane and the percentage of the sarcolemma which was apposed to another cell were calculated. Apposition was defined in this study as any two plasma membranes less than 30 nm apart without a basal lamina interposed between them. The desmosomes and fasciae adherentes were then traced and the length of these junctions per cell profile, as well as the percentage of the cell membrane that was occupied by desmosomes and fasciae adherentes, was calculated. Finally, gap junctions were traced, and the length of gap junctions per cell profile and the percentage of the sarcolemma occupied by gap junctions were obtained. The mean and the standard error of the mean were obtained for each item analyzed in each region. Then a statistical F test and a Newman-Keuls test were done with the aid of the PDP-9 computer to determine the significant cellular differences between the regions of the AV junction. RESULTS

General morphology of the A V junctional tissues In the light microscopic studies of ferret hearts which were cut in the frontal plane of the heart, it was apparent t,hat there were distinct differences in the cellular organization in various regions of the AV junction. One of the major differences was the variation in cell size in the different regions. It was also evident that the cells within each region were arranged differently and had a staining intensity unique to that particular region. Thus the ferret AV junction could be divided into several zones. Basically, the five major regions were the atrial muscle cells of the interatrial septum, the transitional zone interposed between the atrial muscle cells and the AV nodal cells, the AV node, the AV bundle and the interventricular muscle cells (fig. 1). It was also possible to distinguish three regions within the AV node. There was a superficial portion of the node which was composed of small, very diffusely located cells, a deeper portion which consisted of compact, multilaminated fascicles of cells (fig. l A ) , and a group of larger cells near the ostium of the coronary sinus. The AV bundle cells were histologically very similar throughout the bundle. However, the bundle could be subdivided on the basis of its relation to the anulus fibrosus and the right fibrous trigone. There was a proximal portion of the AV bundle located above the

anulus (fig. 1A); a segment which penetrated the right fibrous trigone (fig. 1B); a distal, non-branched segment below the anulus (fig. 1C); and, finally, a branched segment. Since the relationships between the various regions were complex, it was thought that a wax model reconstruction of one of the serially sectioned hearts would help to elucidate the basic organization of the ferret AV junction. Using the model, three relationships were observed. First it was observed that transitional cells were continuous with all three portions of the AV node. Secondly, the superficial portion of the node was located in a position between the coronary sinus portion and the deep portion of the AV node (fig. 2). Finally, the AV bundle cells were found to connect only to the cells of the deep portion of the AV node (fig. 2B). From the model it was also possible to determine that the superficial portion of the AV node was the largest region composed of nodal cells in the AV junction. These diffuse cells occupied a region at the base of the interatrial septum which was 1.3 mm in length, 1.0 mm in width and 0.6 mm a t its greatest height. The compact and multilaminated deep portion of the AV node was crescent-shaped and was arranged as a cap above the AV bundle (fig. 2A). This portion of the node was also 1.3 mm long, yet only 0.3 mm high and 1.2 mm wide. The length of the AV bundle from its junction with the AV node to the origin of the posterior fascicle of the left bundle branch was 1.3 mm in the young adult ferret heart. With these observations in mind, the cells of the AV junction were examined in greater detail with the light and electron microscope.

Atrial muscle cells The atrial muscle cells, studied a t the light microscopic level, were the largest cells of the interatrial portion of the AV junction. These cells were found to stain darkly and were organized into distinct fascicles (fig. 3). The fascicles of atrial muscle cells were often noted to be composed of cells arranged in a different direction from the cells of adjacent fascicles. The atrial muscle cell nuclei were ovoid, with a very thin heterochromatic periphery. The nuclei were usually located centrally, deep within the atrial muscle cell sarcoplasm and occupied 3%of the total cell volume (table 1). The average atrial muscle cell profile was 36.4 pm' in area with a mean perimeter length of 25.4 pm (tables 1, 2). These cells

THE AV NODE AND BUNDLE IN THE FERRET HEART

were packed with ovoid mitochondria (fig. 4) which averaged 0.17 pm2 per mitochondria1 profile and which comprised 14%of the sarcoplasmic volume (fig. 22, table 1). The myofibrils, which were highly organized within the atrial muscle cells (fig. 51, occupied 54%of the sarcoplasmic volume (figs. 5, 22, table 1). The clear area of the atrial muscle cells was found to consist of 32% of the sarcoplasm (fig. 22, table 1). A small Golgi complex was noted in the atrial muscle cells, as were T-tubules, sarcoplasmic reticulum, rough endoplasmic reticulum and free ribosomes. Within some of the cells, atrial granules were observed (fig. 5). Most, but not all, of the cell-to-cell connections between the atrial muscle cells were through classical intercalated discs (fig. 5). It was determined morphometrically that 22%of the sarcolemma of a typical atrial muscle cell was apposed to adjacent atrial muscle cells (fig. 23). Analysis of this appositional membrane revealed that desmosomes and fasciae adherentes comprised 10%of the plasma membrane, while gap junctions were found along 2.6% of the atrial muscle cell sarcolemma (fig. 23). The remaining 9.3%of the cell membrane

369

apposed to other cells was non-specialized appositional membrane (fig. 23). The average length of the gap junctions per cell profile was 0.4 pm (table 1). Gap junctions were found in 41% of the atrial muscle cell profiles examined. Transitional cells The cells of the transitional zone were interposed between atrial muscle cells and the three portions of the AV node (figs. lA,B). At light and electron microscopic levels the transitional cells were found t o be smaller than atrial muscle cells and had an average profile area of 15.7 pm2 and a mean perimeter length of 15.1 pm (tables 1,2).In addition, the transitional cell sarcoplasm did not stain as intensely as the atrial muscle cell sarcoplasm (fig. 6). While the atrial muscle cells were arranged into distinct fascicles, the transitional cells did not appear t o be arranged in any such organized manner (fig. 6). The nuclei in transitional cells were ovoid and had a wider heterochromatic periphery than atrial muscle cell nuclei. The transitional cell nucleus occupied an average of 2% of the total cell volume (table 1).

Fig. 1 Three photomicrographs of frontal sections through the AV junction in the ferret heart. A A section through the atrioventricular node and atrioventricular bundle. The superficial and deep portions of the AV node are located above the AV bundle. The transitional zone is located between the atrial muscle cells of the interatrial septum and nodal cells. The AV bundle is above the anulus fibrosus (arrowheads). B A more anterior section where the AV bundle begins to penetrate the right fibrous trigone (arrowheads). The deep portion of the AV node is located above the AV bundle, with the transitional zone between the deep portion of the node and the interatrial septa1 muscle cells. C A more anterior section, with the AV bundle located below the anulus fibrosus (arrowheads) in the region where the posterior fascicle of the left bundle branch begins to arise.

370

THOMAS A. MARINO

The mitochondria within transitional cells occupied 12%of the sarcoplasmic volume, and the average sectioned area of a mitochondria was 0.07 p m 2 (fig. 22, table 1). The myofibrils, which were arranged irregularly within the transitional cells (fig. 7), did not have a uniform polarity. They accounted for 37%of the sarcoplasmic volume (fig. 22). The clear area of these cells occupied 51%of the sarcoplasm (fig. 22, table 1). As in the atrial muscle cells, a small Golgi complex was observed within the transitional cells, and sarcoplasmic reticulum, rough endoplasmic reticulum and free ribosomes were also seen. However, a transverse tubule system was seldom observed in the transitional cells. Since the myofibrils did not exhibit a uniform polarity within the transitional cells, t h e classical arrangement of junctions into conventional intercalated discs was not observed (fig. 8). It was determined that 36.4% of the

plasma membrane was apposed to adjacent cells (fig. 23, table 2). However, despite the increase in appositional membrane over the amount found in atrial muscle cells, only 8.1% of the plasma membrane was occupied by desmosomes and fasciae adherentes. A mere 0.7% of the sarcolemma consisted of gap junctions (fig. 23, table 2). The average length of gap junctions per cell profile was found to be 0.07 pm (table 2), and gap junctions were observed in 30% of the cell profiles that were taken from this region. Superficial AV nodal cells The cells of the superficial portion of the AV node were located between the transitional zone and the deep portion of the AV node (fig. l A ) , except posteriorly, where the superficial portion of the node lies deep to the coronary sinus portion of the AV node (fig. 2). The superficial AV nodal cells were small, with a

Fig. 2 Two views of the wax model reconstruction of the AV node and AV bundle. A Seen from the front, the AV bundle is located above the interventricular septa1 musculature and gives rise to the posterior fascicle of the left bundle branch. The AV bundle continues proximally to ascend to a position superior to the anulus fibrosus. Above the AV bundle is the deep portion of the AV node. A small coronary sinus portion of the AV node is located posterior to the superficial portion of the node. The transitional zone which surrounds the AV node is not represented in the model. B Seen from the left side, the AV bundle rises to join only the deep portion of the AV node. The superficial portion of the AV node lies between the coronary sinus portion of the AV node and the deep portion of the node.

371

THE AV NODE AND BUNDLE IN THE FERRET HEART

mean profile area of 17.2 pm2. The perimeter length was 16.4 l m (tables 1, 2). The superficial AV nodal cells were located diffusely within this portion of the AV node and were rather isolated from each other (fig. 9). The superficial AV nodal cells contained an ovoid nucleus which had a heterochromatic periphery (fig. 10). The nucleus occupied 4%of the total cell volume (table 1). The mitochondria of the superficial AV nodal cells were ovoid and scattered throughout the sarcoplasm (fig. 10).The mitochondria occupied 16%of the sarcoplasmic volume. The average cross-sectioned size of a single mitochondrion was 0.1 pm' (fig. 22, table 1).Myofi-

brils did not exhibit a uniform polarity within the superficial AV nodal cells (fig. 11) and comprised 37% of the sarcoplasmic volume (fig. 22, table 1).The clear area of the superficial AV nodal cells consisted of 47%of the sarcoplasm (fig. 22, table 1). A Golgi complex, sarcoplasmic reticulum, rough endoplasmic reticulum and free ribosomes were observed in the superficial AV nodal cells. A system of T-tubules, however, was never seen. Classical intercalated discs were not observed between the superficial AV nodal cells, even though most of the cell-to-cellcontact OCcurred a t the ends of these cells (fig. 11). Only 11.7%of the superficial AV nodal cell plasma

TABLE 1

Cellular characteristics ofsix regions ofthe AVjunction (mean Frontal section mean values for

Number of cell profiles Cross sectional area (pm2) Mitochondria within sarcoplasm (percent volume) Mitochondrion average size (rm2) Myofibrils within aarcoplasm (percent volume) Clear area within sarcoplasm (percent volume) Nuclear volume per cell volume (percent volume)

Atrial muscle cells

Superficial AV nodal cells

Transitional cells

(100)

(200)

2

S.E.)

Deep AV nodal cells

Proximal AV bundle cells

(200)

(200)

Distal AV bundle cells

(200)

(200)

36.42 22.67

f

15.7221.28

=

17.2420.91

f

11.8520.74

f

17.13k1.74

=

15.51?1.24

14.4 2 0 . 7

f

12.3 20.6

f

16.0 20.5

f

13.6 20.6

f

9.5 2 0 . 6

f

8.5 ?0.5

0.17k0.007

0.0720.003

0.1020.003

0.0750.42

0.0720.005

0.0520.004

53.9 21.7

f

36.5 21.5

= 36.6 21.0

f

32.2 21.2

f

26.9 21.2

f

32.9 2 1 . 4

31.6 22.2

f

51.1 21.8

f

47.3 21.2

f

53.9 21.5

f

63.4 k 1 . 5

f

58.5 21.7

2.9 20.7

2.0 20.6

3.8 20.6

3.5 20.8

1.0 20.3

1.8 20.4

=, no significant difference between adjacent figures.

t,significant difference a t the 95%level of confidence between adjacent figures.

TABLE 2

Surface specializations ofcells ofsix different regions ofthe AVjunction (mean Frontal section mean values for

Number of cell profiles Perimeter length (pm) Appositional membrane (percent of the cell membrane) Desmosomes (percent of thecellmembrane) Gap junctions (percent ofthecellmembrane) Gap junction length percellprofile (rm)

Atrial muscle cells

Superficial AV nodal cells

Transitional cells

Deep AV nodal cells

k

S.E.1

Proximal AV bundle cells

Distal AV bundle cells

(100) 25.35 21.28 f

(200) 15.1020.70

f

(200) 16.42k0.48

f

15.4620.69

f

17.65k1.05

=

(200) 17.1120.95

22.0 23.23

f

36.4 22.61

f

11.7 k1.72

f

21.0 22.26

f

52.7 k2.01

=

52.322.18

10.1 21.64

f

8.1 21.00

f

4.1 k0.74 f

6.8 20.87

=

8.5 21.03

=

8.0 20.95

2.6 T0.46

f

0.7k0.14

f

0.2 k0.05

f

0.5 20.13

f

1.4 20.15

f

2.0 20.21

0.4150.06

f

0.07k0.01

=

0.03&0.01

=

0.0520.01

f

0.20*0.02

f

0.29?0.04

= , no significant difference between adjacent figures. #,significant difference at the 95%level of confidence between adjacent tissues.

(200)

(200)

372

THOMAS A. MARINO

membrane was apposed to adjacent cells (fig. 23, table 2). Desmosomes and fasciae adherentes occupied a scant 4.1% of the plasma membrane, while the gap junctions comprised only 0.2% of the plasma membrane (fig. 23, table 2). The average length of gap junctions per cell profile was only 0.03 pm (table 2). Gap junctions were seen in only 7.5%of the profiles on which morphometry was done. The superficial AV nodal cells had the least amount of plasma membrane which consisted of surface specializations or appositional membrane, compared t o all the other cell types examined in this study.

cell membrane over the superficial AV nodal cell membrane which consisted of gap junctions. These gap junctions occupied 0.5%of the deep AV nodal cell membrane and their average length per cell profile was 0.05 pm (fig. 23, table 2). Gap junctions were observed in 16.5% of the deep AV nodal cell profiles that were examined morphometrically. Conventional intercalated discs were never observed between the deep AV nodal cells.

Proximal AV bundle cells The proximal AV bundle cells were located between the deep portion of the AV node and the anulus fibrosus (figs. lA,B). The cells of Deep A V nodal cells this segment of the bundle were organized The deep AV nodal cells were organized into into branching clusters (fig. 15). These cells multilaminated fascicles and were located be- had an average cell profile area of 17.1 pmZ tween the superficial portion of the AV node and a mean perimeter length of 17.7 pm and the AV bundle, posteriorly (fig. l A ) , and (tables 1,2). The nuclei of the AV bundle cells between the transitional cells and the bundle, contained scattered regions of heterochroanteriorly (fig. 1B). These fascicles were cres- matin and occupied 1%of the proximal AV cent-shaped and capped the AV bundle (fig. 2). bundle cell volume (table 1). At the electron microscopic level, the AV The deep AV nodal cells did not stain intensely (fig. 12). The average area of a deep AV bundle cells were observed to be irregularly nodal cell profile was 11.9 pm'. The mean pe- shaped (figs. 16, 17) and contained mitochonrimeter of these deep nodal cell profiles was dria which were scattered throughout the sar15.5 pm in length (tables 1, 2). The nuclei coplasm (fig. 16). The mitochondria made up within the deep AV nodal cells were ovoid and 10%of the sarcoplasmic volume. The average size of a sectioned mitochondrion was 0.07 occupied 4%of the total cell volume. The mitochondria within the deep AV nodal p m 2 (fig. 22, table 1). The irregularly arcells were found throughout the cell sarco- ranged myofibrils occupied 27% and the clear plasm (fig. 13). They occupied 14%of the sar- area comprised 63%of the proximal AV bundle coplasmic volume and the average profile of a cell sarcoplasmic volume (fig. 22). A Golgi mitochondrion was 0.07 pm2 (fig. 22, table 1). complex, sarcoplasmic reticulum, rough endoThe myofibrils were arranged irregularly with plasmic reticulum and free ribosomes were respect to their polarity within the deep AV seen in the proximal AV bundle cells, whereas nodal cells and comprised 32% of the sarco- T-tubules were never observed (fig. 17). As a result of the irregular arrangement of plasmic volume. Fifty-four percent of the sarcoplasm consisted of the clear area (fig. 22, myofibrils, classical intercalated discs were table 1).The deep AV nodal cells had a small not present between proximal AV bundle Golgi complex, sarcoplasmic reticulum, rough cells. However, the proximal AV bundle cells endoplasmic reticulum and free ribosomes. did have 52.7% of the plasma membrane apposed to adjacent cells (figs. 17, 23, table 2). However, a T-tubule system was never seen. The surface specializations between the Desmosomes and fasciae adherentes comdeep AV nodal cells were mostly a t the ends of prised 8.5% of the cell membrane area and gap these cells (fig. 14). There was an increase junctions occupied 1.4%of the plasma memin the percentage of surface specializations brane (fig. 23, table 2). The average length of along the deep AV nodal cell membrane, com- the gap junctions per cell profile was 0.2 pm pared to the percentage of the superficial AV (fig. 23, table 2). These junctions were obnodal cell membrane specializations. The ap- served in 54.5% of the cell profiles which were posed membrane comprised 21%of the plasma examined from this region. An unusual findmembrane, while desmosomes and fasciae ad- ing in this study was the occasional appearherentes were found along 6.8%of the plasma ance of a desmosome between two portions of membrane (fig. 23). There was a 2.5-fold in- the plasma membrane of the same cell (fig. crease in the percentage of the deep AV nodal 17). These desmosomes also were found in the

THE AV NODE AND BUNDLE IN THE FERRET HEART

non-branching segment of the AV bundle and in places the folded sarcolemma surrounded a collagen bundle (fig. 17). There was a small population of large cells interspersed throughout the proximal segment of the AV bundle (fig. 18) which, except for their size, resembled the other AV bundle cells. Typical junctions were seen between the large cells of the AV bundle (fig. 18). The large-cell to AV-bundle-cell contacts were also similar t o the cell-to-cell contacts that were seen between two AV bundle cells.

Distal AV bundle cells The distal AV bundle cells were taken from the non-branching portion of the AV bundle which was located below the anulus fibrosus immediately proximal t o the branching AV bundle (fig. 1 0 . At the light microscopic level these cells closely resembled those of the proximal AV bundle (fig. 19). However, a fascicular arrangement of the clusters of AV bundle cells was more evident in this distal segment of the AV bundle. The nuclei of these AV bundle cells were irregular in shape and occupied 2%of the distal AV bundle cell volume. The large cells that were located in the proximal AV bundle were not seen in this distal AV bundle segment. The mitochondria of the distal AV bundle cells were located randomly throughout all regions of the cell and occupied 9%of the sarcoplasmic volume (figs. 20, 22, table 1). The average size of a mitochondria1 profile was 0.05 pm' (table 1). The myofibrils, which were not organized with a uniform polarity in the distal AV bundle cells, accounted for 33%of the sarcoplasmic volume, while the clear area occupied 58%of this volume (figs. 21, 22, table 1). The distal AV bundle cells did not have a T-tubule system but, a small Golgi complex, sarcoplasmic reticulum, rough endoplasmic reticulum and free ribosomes were seen in these cells. The surface membrane specializations of the distal AV bundle cells were essentially identical to those found in the proximal AV bundle cells (figs. 21,23, table 2). Classical intercalated discs were not seen between the distal AV bundle cells but occasionally desmosomes were seen between two portions of the plasma membrane of the same distal AV bundle cell. The only significant difference in membrane specializations between the two segments of the AV bundle was that there was a higher percentage of the plasma membrane

373

that consisted of gap junctions in the distal AV bundle cells than in the proximal AV bundle cells. In the distal AV bundle cells, 2.0%of the plasma membrane was occupied by gap junctions. The average length of gap junctions per cell profile was 0.29 pm (fig. 23, table 2). These junctions were found in 59.5 percent of the distal AV bundle cell profiles examined. While the difference in gap junctions existed between the two regions, the average length of the cell profile perimeter was not significantly different between the cells of the two segments of the AV bundle. DISCUSSION

In the present study, the AV node and AV bundle were examined at the light and electron microscopic levels. The light microscopic information was used to reconstruct the node and bundle in three dimensions to gain a further appreciation of the interrelationships between these structures. The present study has been the first t o characterize cells of the AV node and bundle quantitatively at the ultrastructural level, and t o compare directly the cells of these structures. This methodology also permitted the various AV junctional conduction cell types to be compared directly to working myocardial cells of the interatrial septum. One of the most distinguishing characteristics of the conduction cells in the ferret was that T-tubules were found in atrial muscle cells and occasionally in the transitional cells, but they never were seen in cells of the AV node or the AV bundle. Many authors also have reported that the AV nodal cells and bundle cells lack a transverse tubule system (Kawamura and James, '71; Kim and Baba, '71; Mochet et al., '75). In addition to the AV nodal and bundle cells not having T-tubules, Viragh and Porte ('73) did not find this system of tubules in the cells of the atrio-nodal junction of the monkey heart. Another clear difference between atrial muscle cells and conduction cells in the ferret was the decrease in myofibrillar volume in the conduction cells compared to atrial muscle cells. Also, the orientation of the myofibrils was less regular with regard to their polarity in the transitional cells, nodal cells and bundle cells compared to atrial muscle cells. Most studies have indicated that the AV nodal cells have fewer myofibrils than the atrial muscle cells (Maekawa e t al., '67; Kawamura and James, '71; Tranum-Jensen, '761. The observa-

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THOMAS A. MARINO

tion that there were fewer, less organized myofibrils within the AV bundle cells compared t o atrial muscle cells has also been reported in studies on other mammals (Maekawa et al., '67; Kawamura and James, '71; Challice, '71; Tranum-Jensen, '76). However, some studies have shown that the myofibrils are more organized in AV bundle cells than in the AV nodal cells (DeFelice and Challice, '69; Challice, '71; Viragh and Porte, '73). The present findings, in the ferret heart, are in agreement with the reports of Maekawa e t al. ('67) and Kawamura and James ('711, which indicated that there is a smaller fraction of the sarcoplasm comprised of irregularly oriented myofibrils in the AV bundle cells than in AV nodal cells. The decrease in myofibrillar volume in the conduction cells of the AV junction, compared to the atrial muscle cells, was accompanied by a higher fraction of clear area in the transitional cells, AV nodal cells and AV bundle cells in the ferret heart. The volume of sarcoplasm composed of the clear area is another criterion which could be used qualitatively to distinguish conduction cells from atrial muscle cells. The quantitative data indicated, however, that the clear area should not be used as a qualitative basis for differentiating transitional cells from AV nodal cells, and AV nodal cells from AV bundle cells, because differences between cell types, while statistically significant, were small and were not obvious upon visual examination. When considering the surface membrane specializations of the ferret AV junction conduction cells, the most distinctive morphological feature was that conventional intercalated discs were not observed in the transitional zone, in either region of the AV node or in the AV bundle. This lack of conventional intercalated discs is probably related to the nonuniform polarity of the myofibrils within these cells. The junctional components of the classical intercalated disc are oriented to the myofibrillar terminations on t h e plasma membrane as described by McNutt and Fawcett in atrial and ventricular muscle cells (McNutt and Fawcett, '69; Fawcett and McNutt, '69). The finding that classical intercalated discs were not present between conduction cells is in general agreement with the description of the AV node and bundle cell junctions as reported by Kawamura and James ('71). It should be added, however, that desmosomes, fasciae adherentes and gap junc-

tions often appeared together as a junctional complex in the cells of the cardiac conduction system of the ferret heart. The various surface specializations of the plasma membrane were also distinctive features of each cell type examined in the ferret AV junction. In the current study it has been demonstrated that AV bundle cells had the greatest percentage of the surface membrane apposed to adjacent cells. The percentage of appositional cell membrane decreased progressively in the transitional cells, atrial muscle cells, deep AV nodal cells and superficial AV nodal cells. This type of organization is very similar to that described for the monkey AV junctional conduction cells (Viragh and Porte, '73). In the rabbit, however, the AV nodal cells are closely apposed to each other, and there is less appositional membrane present between the cells of the atrial-nodal junctional zone (Challice, '71). Whether the cells of this junctional zone in the rabbit represent a cellular region similar to the transitional cells of the ferret heart is presently unknown. To substantiate this correlation there is a need for utilizing combined ultrastructural morphometry and electrophysiological techniques because the electrophysiology of the ferret AV junctional tissues is not known at the present time. I t has also been shown that the largest percentage of the plasma membrane which consisted of desmosomes and fasciae adherentes was in the atrial muscle cells of the ferret heart and progressively decreased in the AV bundle cells, transitional cells, deep AV nodal cells and superficial AV nodal cells. While Kim and Baba ('71) reported that desmosomes and fasciae adherentes were observed frequently between AV nodal cells, other investigators have found that these junctions did not occur as often between the cells of the AV node, compared to the working myocardial cells (Challice, '71; Viragh and Porte, '73). As noted previously, the atrial muscle cells had the greatest percentage of the sarcoplasm occupied by myofibrils, and the highest percentage of desmosomes and fasciae adherentes. This data was expected, considering the contractile function of working atrial muscle cells and the function of cellular adhesion ascribed to desmosomes and fasciae adherentes (McNutt and Weinstein, '73). It is interesting to note, however, that the decreased frequency of desmosomes and fasciae adherentes did not correlate with changes in the sarcoplasmic

THE AV NODE AND BUNDLE IN THE FERRET HEART

volume of myofibrils found in the respective conduction cell regions. The decreased frequency of desmosomes and fasciae adherentes could be correlated with the decreased percentage of the plasma membrane that consisted of gap junctions in the various examined cell regions. As pointed out previously, desmosomes, fasciae adherentes and gap junctions were often observed in the same region of the plasma membrane. It might be speculated that desmosomes and fasciae adherentes between cells of the conduction system might function primarily to maintain direct cellular apposition. This would allow the gap junctions to remain structurally intact and therefore permit intercellular communication via these gap junctions. It will be recalled that the percentage of gap junctions between atrial muscle cells was the highest for any region examined in the present study. The length and percentage of the plasma membrane occupied by these junctions decreased progressively in the transitional cells and superficial AV nodal cells, and then continuously increased in the deep AV nodal cells, proximal AV bundle cells and distal AV bundle cells. This pattern of gap junction distribution was also noted in the rabbit AV junction (DeFelice and Challice, '69; Challice, '71; Tranum-Jensen, '76). The paucity of gap junctions between AV nodal cells has also been described in a wide variety of other mammals (Maekawa et al., '67; Kawamura and James, '71; Kim and Baba, '71; Viragh and Porte, '73; Thaemert, '73). It has been generally accepted that the function of gap junctions is intercellular communication and adhesion (McNutt and Weinstein, '73). Weideman ('74, '76) stated that the gap junction plays a major role in the intercellular conduction of the cardiac electrical impulse. The internal resistance of cardiac muscle cells has been considered to be dependent on the gap junction resistance as well as the cytoplasmic resistance (Spira, '71). It can be assumed that the fewer the gap junctions per cell surface, the higher the internal resistance for a cell and the slower the conduction velocity of the electrical impulse from that cell to the next (Challice, '71). In other studies it has appeared likely that the infrequency of gap junctions in the vicinity of the AV node correlates well with the slow conduction velocities recorded electrophysiologically from this area (Viragh and Challice, '73; Viragh and Porte, '73). Since electrophysiological evidence was

375

not available for the ferret, only inferences may be made concerning the conduction velocities found in the AV junction of this animal. It might be postulated, however, that based on the gap junction data, the initial delay in conduction velocity occurs in the ferret transitional zone, as there was a substantial decrease in the percentage of gap junctions between these cells compared to atrial muscle cells. The slowest conduction of the cardiac electrical impulses would then occur in the superficial AV node, because this region has the smallest percentage of gap junctions between cells. From the stereological and morphological data presented here, it might also be anticipated that the conduction velocity would increase progressively in the deep AV node, the proximal AV bundle and the distal AV bundle. In the present study, it has been shown that several different regions can be distinguished within the AV junctional tissues of the ferret heart. The morphometric data presented in figures 22 and 23 demonstrate that there was a transitional zone in the ferret AV junction which contained a distinct cell type different from the atrial muscle cells and the AV nodal cells. A similar transitional zone has been described in other mammals by Anderson (Anderson and Lathum, '71; Anderson, '72; Anderson et al., '74, '75). This region also has been referred to as the "atrio-nodal (AN) region" by several other authors (Alanis et al., '59; Hoffman and Cranefield, '60; Paes de Carvalho, '61; DeFelice and Challice, '69; Challice, '71; Viragh and Challice, '73; Viragh and Porte, '73). The transitional zone may be comparable to the "approaches to the AV node" as described by Hecht et al. ('731, Titus ('73) and Truex ('74, as well. The division of the AV node into a coronary sinus portion, a superficial portion and a deep cap-like portion is in general agreement with the previous description of the ferret AV node that has been reported by Truex et al. ('74). While the superficial and deep portions of the AV node were composed of cells which were similar with regard to their organelles, changes in the surface membrane specializations were clearly evident upon morphometric inspection. This information adds further support for the subdivision of the AV node. After examination of the ferret AV bundle in the current study it was apparent that the results also are in agreement with the work of Truex et al. ('74). It will be recalled that the

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THOMAS A. MARINO

division of the AV bundle into four segments in the current study was based mainly on the relationships of this structure to the right fibrous trigone, the anulus fibrosus and the bundle branches. The morphometry also indicated that there were additional differences between the cells of the proximal and distal AV bundle. The division of the AV bundle into four segments is not unique to the ferret heart, as Hecht et al. ('73) have described similar segments in the human AV bundle. The subdivisions of the conduction cells of the ferret AV junction, as described above are comparable to the descriptions of this region in the rabbit and monkey heart (DeFelice and Challice, '69; Challice, '71; Viragh and Porte, '73; Tranum-Jensen, '76). The morphometric data indicate that the organization of the AV junction is more complex than the division of the AV junctional conduction cells into nodal cells and bundle cells, as was done in earlier studies (Maekawa et al., '67; Kim and Baba, '71). From the present study i t was also found that there were not four cell types in either the ferret AV node or bundle. Preliminary probit analysis of the present quantitative data based on the clear area and size of the cell profiles also indicated that "P' cells, as described by James and Sherf ('68b), do not exist in the ferret AV node (Marino, '77). A t present there is little consensus of opinion about the ultrastructural morphology of the AV junctional cells in the mammalian heart. In this study a methodology was employed to determine quantitatively how many different regions exist in the AV junction of the ferret heart. The morphometric data support a division of the AV node into a t least two regions. It also provides new evidence for a transitional zone in the AV junction. Finally, this data indicates that subtle changes do occur between cells of the proximal and distal segments of the AV bundle. The characteristics of these different cells from the AV junction of young adult ferrets also provide a needed baseline for future studies of the conduction system in embryonic, newborn and aged animals. Morphometric techniques not only allow for this type of quantitative comparison, but can be utilized further, with physiological, pharmacological and pathological studies, to provide new insights into the complex correlation of cell structure to its function in the different regions of the mammalian heart. ACKNOWLEDGMElNTS

The author would like to express his appre-

ciation to Doctors R. C. Truex, J. R. Troyer, L. G. Paavola and A. D. Conger for their invaluable help and guidance during the course of this study. He is indebted to Doctor S. J. Phillips for providing the computer program which was used for this study. Special thanks to Doctor A. A. Bove and Mr. C. Hall for providing the computer equipment and programming assistance which permitted this project to be completed. He would also like to thank Mrs. V. Crissman, J. Rome and D. Marino for their assistance in the preparation of this manuscript. LITERATURE CITED Alanis, J., E. Lopez, J . J. Mandoki and G. Pilar 1959 Propagation of impulses through the atrioventricular node. Am. J. Physiol., 197: 1171-1174. Anderson, R. H. 1972 Histologic and histochemical evidence concerning the presence of morphologically distinct zones within the rabbit atrioventricular node. Anat. Rec., 173: 7-24. Anderson, R. H., A. E. Becker, C. Brechenmacher, M. J. Davis and L. Rossi 1975 The human atrioventricular junctional area. A morphological study of the AV node and bundle. Europ. J. Cardiol., 3: 11-25. Anderson, R. H., M. J. Janse, F. J. van Capelle, J. Billette, A. E. Becker and D. Durrer A combined morphological and electrophysiological study of the atrioventricular node of the rabbit heart. Circ. Res., 35: 909.922. Anderson, R. H., and R. A. Lathum 1971 The cellular architecture of the human atrioventricular node, with a note on its morphology in the presence of a left superior vena cava. J. Anat., 109: 433-455. Arluk, D. J., and J. A. G. Rhodin 1974 The ultrastructure of calf heart conducting fibers with special reference to nexuses and their distribution. J. Ultrastruct. Res., 49: 11-23. Baird, J. A,, and J. S. Robb 1950 Study, reconstruction and gross dissection of the atrioventricular conducting system of the dog heart. Anat. Rec., 108: 747-762. Challice, C. E. 1971 Functional morphology of the specialized tissues of the heart. Methods Archiev. Exp. Pathol., 5: 121-172. DeFelice, L. J., and C. E. Challice 1969 Anatomical and ultrastructural study of the electrophysiological atrioventricular node of the rabbit. Circ. Res., 24: 457-474. Fawcett, D. W., and N. S. McNutt 1969 The ultrastructure of cat myocardium. I. Ventricular papillary muscle. J. Cell Biol., 42: 1-45. Frank, M., I. Albrecht, W. W. Sleator and R. B. Robinson 1975 Stereological measurements of atrial ultrastructure in the guinea-pig. Experentia, 31: 578-580. Hecht, H. H., C. E. Kossman, R. W. Childers, R. Langendorf, M. Lev, K. M. Rosen, R. D. Pruitt, R. C. Truex, H. N. Uhley and T. B. Watt 1973 Atrioventricular and intraventricular conduction. Am. J. Cardiol., 31: 232-244. Hoffman, B. F., and P. F. Cranefield 1960 Electrophysiology of the Heart. McGraw Hill, New York. James, T. N., and L. Sherf 1968a Ultrastructure of the human atrioventricular node. Circulation, 37: 1049-1070. 1968b Ultrastructure of myocardial cells. Am. J. Cardiol., 22: 389-416. 1971 Fine structure of the His bundle. Circulation, 44: 9-28. Kawamura, K., and T. N. James 1971 Comparative ultrastructure of cellular junctions in working myocardium

THE AV NODE AND BUNDLE IN THE FERRET HEART and the conduction system under normal and pathologic conditions. J. Mol. Cell. Cardiol., 3: 31-60. Kim, S., and N. Baba 1971 Atrioventricular node and Purkinje fibers of the guinea pig heart. Am. J. Anat., 132: 339-354. Lev, M. 1960 The conducting system. In: Pathology of the Heart. S. E. Gould, ed. Charles Thomas, Springfield, pp. 132-165. Lev, M., and J. C. Thaemert 1973 The conduction system of the mouse heart. Acta Anat., 85: 342-352. Maekawa, M., Y. Nohara, K. Kawamura and K. Hayashi 1967 Electron microscope study of t h e conduction system in mammalian hearts. In: Electrophysiology and U1trastructure of the Heart. T. Sano, V. Mizuhira and K. Matsuda, eds. Grune and Stratton, New York, pp. 41-45. Marino, T. A. 1977 A morphometric study of the ferret AV node. Anat. Rec., 187: 645 (Abstract). Matter, A. 1973 A morphometric study on the nexus of r a t cardiac muscle. J. Cell Biol., 56: 690-696. McNutt, N. S., and D. W. Fawcett 1969 The ultrastructure of the cat myocardium. 11. Atrial muscle. J. Cell Biol., 42: 46-67. McNutt, N. S., and R. S. Weinstein 1976 Membrane ultrastructure a t mammalian intercellular junctions. Prog. Biophys. Mol. Biol., 26: 45-101. Mochet, M., J. Moravec, H. Guillemot and P. Y. Hatt 1975 The ultrastructure of rat conduction tissue: An electron microscope study of the atrioventricular node and bundle of His. J. Mol. Cell. Cardiol., 7: 879-889. Molenhauer, H. 1964 Plastic embedding mixtures for use in electron microscopy. Stain Technol., 39: 111-114. Paes de Carvalho, A. 1961 Cellular electrophysiology of the atrial specialized tissues. In: The Specialized Tissues of the Heart. A. Paes de Carvalho, W. C. de Mello and B. F. Hoffman, eds. Elsevier Pub. Co. Amsterdam, pp 115-130. Page, E., L. P. McCallister and B. Power 1971 Stereological measurements of cardiac ultrastructures implicated in excitation-contraction coupling. Proc. Nat. Acad. Sci. ( U S A . ) ,68: 1465-1466. Page, E.,and L. P. McCallister 1973a Quantitativeelectron microscopic description of heart muscle cells. Application to normal, hypertrophied and thyroxin-stimulated hearts. Am. J. Cardiol., 31: 172-181. 1973b Studies on the intercalated disc of rat left ventricular myocardial cells. J. Ultrastruct. Res., 43: 388-411. Phillips, S. J., and A. A. Bove 1976 The sinoatrial node of the ferret-initial fine structure morphometry. Anat. Rec., 184: 502 (Abstract). Reynolds, E. S. 1963 The use of lead citrate a t high pH a s an electron-opaquestain in electron microscopy. J. Cell Biol., 17: 208-212. Richardson, K. C., L. Jarret and E. H. Finke 1960 Embed-

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ding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol., 35: 313-323. Robb, J. S. 1965 Comparative Basic Cardiology. Grune and Stratton, New York. Rodbard, S. 1958 A reconstruction of serial sections of the atrioventricular node. Anat. Rec., 130: 462 (Abstract). Spira, A.W. 1971 The nexus in the intercalated disc of the canine heart: Quantitative data for an estimation of its resistance. J. Ultrastruct. Res., 34: 409-425. Thaemert, J. C. 1970 Atrioventricular node innervation in ultrastructural three dimensions. Am. J. Anat., 128: 239-264. 1973 Fine structure of the atrioventricular node as viewed in serial sections. Am. J. Anat., 136: 43-66. Titus, J. L. 1973 Normal anatomy of the human cardiac conduction system. Mayo Clin. Proc., 48: 24-30. Tomanek, R. J.,and W. L. Karlsson 1973 Myocardial ultrastructure of young and senescent rats. J. Ultrastruct. Res., 42: 201-220. Tranum-Jensen, J. 1976 The fine structure of the atrial and atrioventricular (AV) junctional specialized tissues of the rabbit heart. In: The Conduction System of the Heart. Structure, Function and Clinical Implications. H. J. J. Wellens, K. I. Lie and M. J. Janse, eds. H. E. Stenfert B.V.-Leiden, Netherlands, pp. 55-81. Truex, R. C. 1974 Structural basis of atrial and ventricular conduction. Cardiovascular Clin., 6: 2-24. Tmex, R. C., R. Belej, L. Ginsberg and R. L. Hartman 1974 Anatomy of the ferret heart: An animal model for cardiac research. Anat. Rec., 179: 411-422. Truex, R. C., and M. Q. Smythe 1965 Comparative morphology of the cardiac conduction tissues in animals. Ann. N.Y. Acad. Sci., 127: 19-23. 1967 Reconstruction of the human atrioventricular node. Anat. Rec., 158: 11-19. Viragh, S., and C. E. Challice 1973 The impulse generation and conduction system of the heart. In: Ultrastructure of the Mammalian Heart. C. E. Challice and S. Viragh, eds. Academic Press, New York, pp. 43-89. Viragh, S., and A. Porte 1973 On the impulse conducting system of the monkey heart (Macaca mulatfa). 11. The atrioventricular node and bundle. 2. Zellforsch., 145: 363-388. Watson, M. L. 1958 Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol., 4: 475-478. Weibel, E. R. 1969 Stereological principles for morphometry in electron microscopic cytology. Int. Rev. Cytol., 26: 235-303. Weideman, S. 1974 Generation and conduction of the cardiac impulse: Problems and recent results. Acta Med. Philippina, 10: 73-78. 1976 Cell coupling in cardiac muscle. Pontif. Acad. Sci. Scripta Varia, 9: 1-16.

Abbreuiations

Ag. Atrial granules AVB, Atrioventricular bundle BV, Blood vessels C, Collagen CSN, Coronary sinus portion of the AV node DN, Deep portion of the AV node F, Fascicles of cells IAS, Interatrial septum ID, Intercalated discs IVS, Interventricular septum JC, Junctional complex

LA, Left atrium LBB, Left bundle branch LV, Left ventricle M, Mitochondria Mf, Myofibrils MV, Mitral value NB, Nerve bundle RA, Right atrium RV, Right ventricle SN, Superficial portion of the AV node T, Transitional zone TV, Tricuspid valve

PLATE I EXPLANATION OF FIGURES

Figs. 3-5 Atrial cells from t he interatrial septum.

3 Photomicrograph of t he interatrial septal muscle cells. The cells are arranged in fascicles. Within each fascicle the cells are all oriented in the same direction. Note the numerous blood vessels between and within the fascicles. X 550. 4 Low-power electron micrograph of the muscle cells within a fascicle. The cells are tightly packed with mitochondria and well organized myofibrils. X 2,500. 5 The myofibrils and mitochondria, as well as the intercalated disc seen in this electron micrograph, typify t he organization of these structures within atrial muscle cells. Atrial granules can be found in some of the cells of the interatrial septal musculature. X 14,250.

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THE AV NODE AND BUNDLE IN THE FERRET HEART Thomas A. Marino

PLATE 1

379

PLATE 2 EXPLANATION OF FIGURES

Figs. 6-8 Cells of the transitional cell zone. 6

Light micrograph of the transitional zone, in which t h e cells no longer are arranged into fascicles. X 550.

7 Low-power electron micrograph of transitional cells which have a decreased size and a smaller percentage of sarcoplasm occupied by myofibrils and mitochondria compared to atrial muscle cells. X 2,500. 8

380

Electron micrograph of transitional cells. The myofibrils do not exhibit a uniform polarity and the mitochondria are smaller than those in t h e atrial cells. Although desmosomes (arrow) occur along the cell membrane, classic intercalated discs are not seen. X 14,250.

THE AV NODE AND BUNDLE IN THE FERRET HEART Thomas A. Marino

PLATE 2

381

PLATE 3 EXPLANATION OF FIGURES

Figs. 9-11 Cells of the superficial portion of the AV node.

9 Photomicrograph of cells of the superficial portion of the AV node. These cells are isolated from each other by a connective tissue matrix. Note the large nerve bundle and numerous blood vessels among the cells of the superficial AV nodal cells. X 550. 10 Low-power electron micrograph demonstrating a paucity of cell-to-cell contacts in this area, as well as the arrangement of the mitochondria and myofibrils in these cells. X 2,500. 11 Electron micrograph of superficial AV nodal cells. The major portion of the cell is isolated from other cells, while most of the cell contacts occur at the tapering ends of these cells, where gap junctions can be found (arrow). X 14,250.

THE AV NODE AND BUNDLE IN THE FERRET HEART Thomas A. Marino

PLATE 3

383

PLATE 4 EXPLANATION OF FIGURES

Figs. 12-14 Cells of the deep portion of t h e AV node.

12 Photomicrograph of deep AV nodal cells. These cells are arranged into a multilaminated cap above the AV bundle. x 550. 13 Low-power electron micrograph. Despite the compact arrangement of the deep AV nodal cells, adjacent cells are separated from each other and have few areas of cell contact. Note the irregular organization of the mitochondria and the non-uniform polarity of the myofibrils within the deep AV nodal cells. X 2,500. 14 Electron micrograph of the deep AV nodal cells in which the mitochondria and

myofibrils are irregularly organized. Note t h e regions of cell-to-cell contact at t h e ends of the deep AV nodal cells (arrows). X 14,250.

384

THE AV NODE AND BUNDLE IN THE FERRET HEART Thomas A. Marino

PLATE 4

385

PLATE 5 EXPLANATION OF FIGURES

Figs. 15-17 Cells of the AV bundle above the anulus fibrosus. 15 Photomicrograph of the AV bundle cells, showing their organization into branching clusters of cells. Nerve bundles can be seen within the AV bundle. x 550. 16 The cells of t h e AV bundle appear as highly apposed clusters of cells, as can be seen in this low-power electron micrograph. Note the irregularly arrangement of the mitochondria and myofibrils in these cells. x 2,500.

17 Electron micrograph of t h e AV bundle. The myofibrils and the mitochondria are sparse. These extensively apposed cells contact other AV bundle cells along all regions of t h e cell membrane. Some AV bundle cells have desmosomes between two portions of the plasma membrane of the same cell (arrow). X 14,250.

386

THE AV NODE AND BUNDLE IN THE FERRET HEART Thomas A. Marino

PLATE 5

387

THE AV NODE AND BUNDLE IN THE FERRET HEART Thomas A. Marina

EXPLANATION OF FIGURES

18 In this low power electron micrograph, a large cell is seen (single arrow) among the other cells of the AV bundle above the anulus fibrosus. Note t h e junctional complex (double arrow) between this large cell and another AV bundle cell. x 2,500.

Figs. 19-21 Cells of the non-branching AV bundle. 19 Photomicrograph of cells of the non-branching AV bundle. In this region the cells appear to be arranged into fascicles. X 550. 20 In this low-power electron micrograph the cells of AV bundle continue to be highly apposed to each other and the myofibrils do not have a uniform polarity. x 2,500. 21 The extensively apposed nature of the AV bundle cells is seen in this electron micrograph. The mitochondria are small and the myofihrils did not exhibit a uniform polarity. Note also the irregular pattern of the junctions x 14,250.

388

PLATE 6

THE AV NODE AND BUNDLE IN THE FERRET HEART Thomas A. Marino

PLATE 7

389

PLATE 8 EXPLANATION OF FIGURES

Figs. 22,23 Morphometric data for the cells of the AV junction. 22 Bar graph of t h e percentage of the sarcoplasm which was composed of mitochondria, myofibrils and the clear area for the different regions of the ferret AV junction. Numbers in the blocks are t h e mean 2 standard error. 23 Bar graph of the percentage of t h e surface membrane which consisted of non-specialized appositional membrane, desmosomes and fasciae adherentes, and gap junctions. Numbers immediately above the blocks are t h e mean for t h e non-specialized appositional membrane, the mean f the standard error for the percentage of desmosomes and fasciae adherentes, and the mean f t h e standard error for the percentage of gap junctions. The number above the bar for each cell type is the mean k the standard error for the total appositional membrane.

390

-

THE AV NODE AND BUNDLE IN THE FERRET HEART Thomaa A. Marino

PLATE 8

CLfAR AREA

MIOIIORILS

MITOCUONDRIA

n 3l.t k! 2.2

5l.l f 1.1

C ' I 41.3 f l2

63.4

MUSCLf CfUS

CELLS

SUPERFICIAL A V MODAL CfLLS

AV M O O N CELLS

f

1.5

A V BUNDLf CfLLS

A V WNDLE CELLS

@

A V JUNCTIONAL REGION

391