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Keywords:

  • human heart;
  • cardiac innervation;
  • immunohistochemistry;
  • cardiac conduction system;
  • infancy;
  • childhood;
  • adulthood;
  • senility

Abstract

  1. Top of page
  2. Abstract
  3. RESULTS
  4. DISCUSSION
  5. CONCLUSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES

In order to study the changes in the pattern of autonomic innervation of the human cardiac conduction system in relation to age, the innervation of the conduction system of 24 human hearts (the age of the individuals ranged from newborn to 80 years), freshly obtained at autopsy, was evaluated by a combination of immunofluorescence and histochemical techniques. The pattern of distribution and density of nerves exhibiting immunoreactivity against protein gene product 9.5 (PGP), a general neural marker, dopamine β-hydroxylase (DBH) and tyrosine hydroxylase (TH), indicators for presumptive sympathetic neural tissue, and those demonstrating positive acetylcholinesterase (AChE) activity, were studied. All these nerves showed a similar pattern of distribution and developmental changes. The density of innervation, assessed semiquantitatively, was highest in the sinus node, and exhibited a decreasing gradient through the atrioventricular node, penetrating and branching bundle, to the bundle branches. Other than a paucity of those showing AChE activity, nerves were present in substantial quantities in infancy. They then increased in density to a maximum in childhood, at which time the adult pattern was achieved and then gradually decreased in density in the elders to a level similar to or slightly less than that in infancy. In contrast, only scattered AChE-positive nerves were found in the sinus and atrioventricular nodes, but were absent from the bundle branches of the infant heart, whereas these conduction tissues themselves possessing a substantial amount of pseudocholinesterase. During maturation into adulthood, however, the conduction tissues gradually lost their content of pseudocholinesterase but acquired a rich supply of AChE-positive nerves, comparable in density to those of DBH and TH nerves. The decline in density of AChE-positive nerves in the conduction tissues in the elders was also similar to those of DBH and TH nerves. Our findings of initial sympathetic dominance in the neural supply to the human cardiac conduction system in infancy, and its gradual transition into a sympathetic and parasympathetic codominance in adulthood, correlate well with the physiologic alterations known to occur in cardiac rate during postnatal development. The finding of reduction in density of innervation of the conduction tissue with ageing is also in agreement with clinical and electrophysiological findings such as age-associated reduction in cardiac response to parasympathetic stimulation. Finally, our findings also support the hypothesis that, in addition to the para-arterial route, the parafascicular route of extension along the conduction tissue constitutes another pathway for the innervation of the conduction system of the human heart during development. Anat Rec 264:169–182, 2001. © 2001 Wiley-Liss, Inc.

The cardiac conduction system, consisting of the sinus node, the specialised atrioventricular junctional area and the ventricular conduction tissues, is responsible for the generation and coordination of transmission of electric impulse in the heart, resulting in its rhythmic and synchronized contraction. The morphology of the cardiac conduction system itself has been studied in detail in many animal species (Anderson, 1972a,b; Bleeker et al., 1980; Roberts et al., 1989; Forsgren et al., 1983; Op'thof et al., 1987), including the human being (Anderson et al., 1975; Smith et al., 1977; Davies et al., 1983a).

Interest in the analysis of cardiac innervation has recently been stimulated by the availability of sensitive immunohistochemical techniques, which have allowed better visualization and determination of the pattern of distribution of the various nerve subtypes (Weihe et al., 1984; Sternini and Brecha, 1985; Dalsgaard et al., 1986). Indeed, such techniques have been applied to the detailed analysis of cardiac innervation, including that of the conduction system, of several mammalian species (Roberts et al., 1989; Ursell et al., 1990, 1991a, 1991b; Choate et al., 1993; Slavikova et al., 1993; Zhang et al., 1993; Crick et al., 1996). Similar studies have also been performed on the human heart (Rechardt et al., 1986; Wharton et al., 1988, 1990; Chow et al., 1993, 1995; Gordon et al., 1993; Crick et al., 1994; Marron et al., 1994, 1995), but those with emphasis on the conduction system have been few (Chow et al., 1993; Crick et al., 1994). Moreover, while the innervation of the neonatal and adult human cardiac conduction system have been analyzed (Chow et al. 1993; Crick et al. 1994), information concerning its postnatal maturation during infancy and childhood, and the changes occurring during adolescence, adulthood and senility, is lacking.

On the other hand, it is well known that the cardiac rate of newborn infants is rapid, and subject to wide fluctuations (Behrman et al., 1992). The average rate ranges from 120 to 140 beats per minute, and may increase to 170 or more during crying and activity, or drop to 70–90 during sleep. As the child grows older, the average cardiac rate becomes slower, as low as 40 per minute in athletic adolescents. This variation may well be related in part to the functional development of the cardiac conduction system, and it is not unreasonable to speculate that changes in the pattern of innervation of the conduction tissues, for which detailed analysis has not been performed, may be one of the anatomical changes contributing to such an alteration.

In addition, several groups of investigators, using experimental animal models and by virtue of clinical observations, have provided evidence linking the autonomic nervous system and sudden cardiac death (Corr et al., 1986; Schwartz and Stramba-Badiale, 1988; Schwartz and Priori, 1990). For example, there is now general consensus that in the setting of acute myocardial ischaemia, sympathetic hyperactivity facilitates the onset of malignant arrhythmias, whereas vagal activation can exert an antifibrillatory effect, and these findings have been used in the identification of individuals at high risk of, and in the development of therapeutic strategies for the prevention of sudden cardiac death (Schwartz et al., 1994). In this respect, it is of interest to note that abnormalities of the cardiac conduction system have also been suggested to be the underlying cause for cases of sudden infant death syndrome (Davies et al., 1983b). Even though the issue remains controversial, detailed analysis of the autonomic innervation of the heart (notably that of the conduction system) in cases of sudden infant death syndrome, which has not yet been performed, would be of value.

Furthermore, while the anatomic basis of certain types of conduction defects, such as isolated congenitally complete heart block, is well described (Chow et al., 1998a), no consistent structural abnormality is found in others, as exemplified by the conduction defects associated with various types of neurological and myopathic syndromes (Davies et al., 1983c). In Duchenne muscular dystrophy, for example, the pathological changes of the heart, consisting of dilatation, hypertrophy, and fibrosis of both atria and ventricles, are fairly nonspecific. While it has been suggested that selective fibrosis in the posterobasal region of the left ventricle was responsible for the electrocardiographic changes characteristic of this condition (Davies et al., 1983c), this finding is by no means universal and other concomitant abnormality, notably that of the conduction system, may also contribute to the pathogenesis of the conduction defect. In this latter group of conduction disorders, it would be of interest to analyse the innervation of the conduction system, especially in light of the recent evidence of the dominant role of the autonomic nervous system in the genesis and modulation of cardiac arrthymias (Corr et al., 1986; Schwartz and Stramba-Badiale, 1988; Schwartz and Priori, 1990; Schwartz et al., 1994). In this regard, the importance of defining the normal pattern of innervation of the conduction system in various age groups cannot be overemphasized.

In the present study, therefore, we attempted to fill the gap in our knowledge by studying the changes in the pattern of innervation of the human cardiac conduction system from infancy to senility, employing a combination of immunohistochemical and histochemical techniques.

RESULTS

  1. Top of page
  2. Abstract
  3. RESULTS
  4. DISCUSSION
  5. CONCLUSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES

In histological sections, the location and extent of the conduction tissues were recognised by their distinct morphology, which has been fully described (Davies et al. 1983a). Under the fluorescent microscope, immunoreactive nerves appeared as fine wavy, bright green fluorescent fibrils, sometimes demonstrating varicose enlargement along their lengths, and could readily be distinguished from the bright yellow or yellowish green autofluorescence of lipofuscin pigments, elastic and collagenous tissue.

PGP (Overall) Innervation (Table 1)

Table 1. Changes in the pattern of PGP, DBH, and TH innervation of the human cardiac conduction system from infancy to senility
RegionInfantsChildrenAdolescents and adultsElders
PGPaDBHTHPGPDBHTHPGPDBHTHPGPDBHTH
  1. a

    aPGP, protein gene product 9.5; DBH, dopamine β-hydroxylase; TH, tyrosine hydroxylase.

  2. b

    bRelative number of immunoreactive nerves graded: 0, no immunoreactivity detected; ±, immunoreactive nerves rarely detected; 1+, sparse and scattered individual nerve fibres; 2+, moderate number of fibres; 3+, abundant nerve fibres; 4+, numerous nerve fibres; 5+, very numerous nerve fibres.

Sinus node4+b2+1+5+3+2+5+3+3+3+1+1+
Atrioventricular node3+2+1+4+2+2+4+2+2+2+1+1+
Penetrating bundle2+1+±3+2+2+3+2+2+2+1+1+
Branching bundle2+1+03+2+1+3+2+2+1+1+1+
Bundle branches1+±02+2+1+2+2+2+±±±

In infants, abundant protein gene product 9.5 (PGP)-immunoreactive nerve fascicles and fibers were present in the sinus node, both in the perivascular regions and among the nodal cells (Fig. 1B). Many of the nerve fibers were seen close to the nodal cells (Fig. 1C). Similarly distributed PGP nerves were also seen in the atrioventricular node, and in the penetrating and branching bundles (Fig. 2A), albeit in a significantly less density than that in the sinus node. In the bundle branches, only occasional PGP nerves were seen in their proximal portions (Fig. 3A).

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Figure 1. PGP innervation of the sinus node of the human heart in infancy and adulthood. A: Section of the sinus node (SN) of a two-day-old infant heart (case 3) stained with Masson's trichrome technique, showing the network of nodal cells grouped around branches of the nodal artery (outlined with dotted lines). M, atrial myocardium. Bar = 1 mm. B: Immunofluorescence photomicrograph of an adjacent section of the sinus node. The immunofluorescent nerves appear as white wavy lines. There is numerous (4+) nerves displaying positive immuofluorescence for protein gene product 9.5 (PGP). Bar = 1 mm. C: High power magnification to show the close apposition of nerve fibres to nodal cells (4+). Bar = 0.5 mm. D: In the sinus node (SN) of a 28-year-old adult heart (case 13), very numerous (5+) nerve fibres showing positive immunofluorescence for PGP are seen. M, atrial myocardium. Bar = 1 mm.

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Figure 2. PGP innervation of the penetrating bundle of the human heart in infancy and childhood. A: In the penetrating bundle (arrowheads) of a two-day-old infant heart (case 3), moderate number (2+) of PGP immunoreactive nerves are seen. Bar = 1 mm. B: In a 12-year-old child (case 9), abundant (3+) PGP immunoreactive nerves are seen in the penetrating bundle (arrowheads). Bar = 2 mm.

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In children, adolescents and adults, the pattern of distribution of PGP innervation of the sinus and atrioventricular node, as well as the penetrating bundle, was similar to but more densely populated than that observed in infants (Figs. 1–3). In addition, significantly more PGP immunoreactive nerves were seen in the bundle branches, and these nerves were present not only in the proximal portion of the conduction axis, but also in the peripheral regions (Fig. 3B).

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Figure 3. PGP Innervation of the left bundle branch of the human heart in infancy and childhood. A: Scattered individual (1+) PGP immunoreactive nerves (arrows), in close apposition to the conduction tissue, are see in the initial portion of the left bundle branch (LBB) of a two-day-old infant heart (case 3). They are absent in the distal portion of the LBB. E, endocardium; M, ventricular myocardium, Bar = 0.5 mm. B: In the heart of a 12-year-old child (case 9), moderate number (2+) of PGP immunoreative nerves are seen in the distal portion of the left bundle branch (LBB) as well. E, endocardium; M, ventricular myocardium, Bar = 1 mm.

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In the elders, the densities of PGP innervation in all regions of the conduction system of the heart were slightly less than those in adults. They were present in moderate number in the sinus and atrioventricular nodes, and only scattered PGP nerves were seen in the penetrating and branching bundles, they were rarely detected in the bundles branches.

In all instances, the densities of PGP immunoreactive nerves in the sinus and atrioventricular nodes, as well as those in the penetrating and branching bundles, were higher than those in the adjacent atrial and ventricular myocardium respectively.

DBH Immunoreactivity (Presumptive Sympathetic Innervation) (Table 1)

In infants, moderate numbers of dopamine β-hydroxylase (DBH)-positive nerves were present in the sinus and atrioventricular nodes, mainly in the perivascular regions, with extensions among the adjacent nodal cells (Fig. 4A). In the penetrating and branching bundles, only scattered individual DBH-positive nerves were seen in the perivascular locations whereas they were rarely detected in the bundle branches.

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Figure 4. DBH innervation of the conduction system of the human heart in infancy and childhood. A: In the sinus node of a two-day-old infant heart (case 3), moderate number (2+) of nerve fibres showing positive immunostaining for dopamine β-hydroxylase (DBH) are seen in the sinus node. Bar = 0.5 mm. B: In a 4-year-old child (case 7), abundant (3+) DBH immunoreactive nerves, in close apposition to the nodal cells, are seen in the sinus node. Bar = 1.5 mm. C: In a 12-year-old child (case 9), moderate number (2+) of DBH immunoreactive nerves, with varicose enlargements along their lengths and in close apposition to the conduction tissue, are seen in the left bundle branch (LBB). E, endocardium; M, ventricular myocardium. Bar = 1 mm.

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In children, adolescents and adults, in contrast, abundant DBH-positive nerves were seen in the sinus node, both in the perivascular regions and in close apposition to the nodal cells (Fig. 4B). Moderate number of DBH nerves were also noted in the atrioventricular node, penetrating and branching bundles as well as in the bundle branches (Fig. 4C).

In the elders, the density of DBH nerves declined, they were present in moderate number in the sinus node, and only scattered individual nerve fibres were seen in the atrioventricular node, penetrating and branching bundles, and they were rarely detected in the bundle branches.

As in the case of PGP innervation, the densities of DBH immunoreactive nerves in the sinus and atrioventricular nodes, and in the penetrating bundle, were significantly higher than those in the adjacent atrial and ventricular myocardium respectively, and in all age groups. In the bundle branches, however, the density of DBH-positive nerves in adolescents and adults was approximately half that of PGP nerves, but similar to those of DBH and PGP nerves in the adjacent ventricular myocardium.

TH Immunoreactivity (Presumptive Sympathetic Innervation) (Table 1)

In infants, only scattered individual tyrosine hydroxylase (TH) nerve fibers were present in the sinus and atrioventricular nodes, they were rarely seen in the penetrating bundle and were not detected in the branching bundle and bundle branches. In children, moderate numbers of TH nerves were seen in the sinus node, atrioventricular node and penetrating bundle, whereas only scattered individual nerve fibers were noted in the branching bundle and bundle branches. In adolescents and adults, abundant TH nerves were present in the sinus node, and they were seen in moderate number in the other areas of the conduction tissue. In the elders, the density of TH innervation declined so that only scattered individual TH nerve fibers were seen in the conduction system apart from the bundle branches where they were rarely detected.

As in the case of DBH innervation, the densities of TH immunoreactive nerves in the all regions of the conduction system, apart from the bundle branches, were significantly higher than those in the adjacent atrial and ventricular myocardium respectively, and in all age groups. In the bundle branches, however, the density of TH nerves in adolescents and adults was approximately half that of PGP nerves, but similar to those of TH and PGP nerves in the adjacent ventricular myocardium.

Cholinesterase Activity (Presumptive Cholinergic Innervation) (Table 2)

Table 2. Changes in the pattern of cholinesterase activity, uninhibited, pre-incubated with acetylcholinesterase inhibitor and pre-incubated with pseudocholinesterase inhibitor, of the human cardiac conduction system from infancy to senility
Region*InfantsChildrenAdolescents and adultsElders
ABCABCABCABC
  • *

    A = cholinesterase activity, uninhibited, B = cholinesterase activity, pre-incubated with acetylcholinesterase inhibitor, C = cholinesterase activity, pre-incubated with pseudocholinesterase inhibitor.

  • Relative density of enzyme-positive nerves graded: 0, no nerves; ±, stained nerves rarely detected; 1+, sparse and scattered individual nerve fibres; 2+, moderate number of stained nerve fibres; 3+, abundant stained nerve fibres; 4+, numerous stained nerve fibres; 5+, very numerous stained nerve fibres. Intensity of cholinesterase staining of the conduction tissue: 0, no staining; +, weak staining; ++, strong staining.

Sinus node
 Conduction tissue+++++000000000
 Nerves2+1+2+3+1+2+4+1+4+2+1+2+
Atrioventricular node
 Conduction tissue+++++000000000
 Nerves1+1+1+2+1+2+3+1+3+2+1+2+
Penetrating bundle
 Conduction tissue+++++000000000
 Nerves0002+1+2+2+1+2+1+1+1+
Branching bundle
 Conduction tissue+++++000000000
 Nerves0001+±1+2+1+2+1+±1+
Bundle branches
 Conduction tissue+++++000000000
 Nerves0001+±1+2+1+2+1+±1+

In infants, the specialised conduction cells comprising the sinus and atrioventricular nodes showed prominent pseudocholinesterase but weak acetylcholinesterase (AChE) activity. There were moderate number of nerve fibres showing positive pseudocholinesterase reaction, and relatively fewer AChE-positive nerves, among the nodal cells (Fig. 5A and B). A similar pattern of staining reaction, though weaker in intensity, was noted in the penetrating and branching bundles, as well as the bundle branches, but nerves showing positive cholinesterase reaction were not identified in these areas (Fig. 5C).

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Figure 5. Cholinesterase staining of the conduction system of the human heart in infancy. A: In a two-day-old infant heart (case 3), the nodal cells of the sinus node (outlined with dotted lines) show strong cholinesterase staining (++). Moderate number (2+) of nerve fascicles (arrow) with positive cholinesterase staining are seen among the nodal cells. Bar = 1 mm. B: High magnification to show the positive staining of the nerve fascicle and nodal cells. Bar = 0.5 mm. C: In a two-day-old infant heart (case 3), the branching bundle and initial portion of the left bundle branch show strong cholinesterase staining (++). However, nerves with positive cholinesterase staining are not identified in these regions. Bar = 1 mm.

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In children, adolescents, adults and elders, the specialised conduction tissues themselves showed negative cholinesterase reaction, whereas AChE-positive nerves were present in these areas. These nerves exhibited a decreasing density gradient from the sinus node (Fig. 6A and B), through the atrioventricular node, penetrating and branching bundle (Fig. 6C), to the bundle branches (Fig. 6D). In adolescents and adults, the density of AChE-positive nerves in the bundle branches was approximately half that of PGP-positive, and similar to that of DBH-positive nerves. Furthermore, the density of AChE innervation increased from infancy through childhood, reached its maximum in adolescence and adulthood, and gradually declined in the elders, to a level similar to that in infants.

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Figure 6. Cholinesterase staining of the conduction system of the human heart in adulthood. A: In the sinus node (outlined with dotted lines and on the right) of a 30-year-old adult heart (case 15), the conduction tissues are devoid of cholinesterase activity, however, there are abundant (3+) acetylcholinesterase (AChE) positive nerves (arrows). Bar = 1 mm. B:High magnification to show the abundant (3+) AChE-positive nerves (arrows) in the sinus node (SN), many of which are closely apposed to the nodal cells. M, atrial myocardium. Bar = 0.3 mm. C: In a 30-year-old adult heart (case 15), moderate number (2+) of AChE-positive nerves (arrows) are seen in the penetrating bundle. The conduction tissues are devoid of cholinesterase activity. Bar =1 mm. D: In the left bundle branch of a 30-year-old adult heart (case 15), moderate number (2+) of AChE-positive nerves (arrows), in close apposition to the conduction tissue, are identified. E, endocardium; M, ventricular myocardium. Bar = 0.5 mm.

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In children, adolescents, adults and elders, the densities of AChE-positive nerves in the sinus and atrioventricular nodes, and in the penetrating and branching bundles, were significantly higher than those in the adjacent atrial and ventricular myocardium respectively. In the atrial myocardium, moderate number of AChE-positive nerve fibres were seen, whereas in the ventricular myocardium, only isolated and sparse AChE-positive nerve fibres were detected.

By comparing the staining pattern in the serial sections, it was noted that, in all cases and in all areas of the conduction tissue, nerves exhibiting positive cholinesterase reaction were distinct from those showing positive immunostaining with DBH and TH. Furthermore, nerves coexpressing positive cholinesterase reactivity and immunoreactivity for DBH or TH were not identified.

DISCUSSION

  1. Top of page
  2. Abstract
  3. RESULTS
  4. DISCUSSION
  5. CONCLUSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES

Background

Although detailed studies of cardiac innervation using sensitive immunohistochemical techniques have been performed on several mammalian species (Roberts et al., 1989; Ursell et al., 1990, 1991a, 1991b; Choate et al., 1993; Slavikova et al., 1993; Zhang et al., 1993; Crick et al., 1996), investigation of nerves associated with the conduction system of the human heart have been few (Chow et al., 1993; Crick et al., 1994). Moreover, since considerable variation exists in the density and distribution of subtypes of nerves in mammalian hearts (Davies et al., 1983a), it is potentially inaccurate to base concepts of maturation of human cardiac innervation on findings obtained from other species.

One of the main reasons for the paucity of published studies on the innervation of the human cardiac conduction system using immunohistochemical techniques (Chow et al., 1993; Crick et al., 1994) lies in the difficulty in obtaining tissue sufficiently fresh for such investigations. There is no reasonable alternative, apart from obtaining recipient heart at transplantation, other than to use postmortem tissue for such an investigation. The validity of employing relatively fresh autopsy tissue, nonetheless, has now been justified by our recent study (Chow et al. 1998b). In this study, we showed that accurate information concerning cardiac innervation could be achieved by studying the pattern of protein gene product 9.5 (PGP), dopamine β-hydroxylase (DBH), tyrosine hydroxylase (TH) and Neuropeptide Y (NPY) immunoreactive nerves in normal human hearts from autopsies performed within 6, 4, 2, and 3 days after death, respectively. Accordingly, all the hearts in the present study were obtained during autopsies performed within 18 hours after death. We believe that our current findings are valid, and give a genuine representation of the pattern of innervation of the human cardiac conduction system, as well as reflecting important changes occurring from infancy to adulthood.

Quantitation

In the assessment of the quantity of nerves in the conduction system during postnatal development, the application of automated quantitation seems logical as it is more objective (Crick et al., 1994). The equipment required for such analysis, however, is not available in our laboratory. On the other hand, automated quantitation is not entirely free of defects. From a functional perspective, it seems more reasonable to assume that the density of nerves per cell rather than unit area would serve as a better indicator for the degree of innervation. In automated quantitation, however, the density of innervation is measured in terms of nerves per unit area and other factors such as the age variation of the size of the myocardial cells, as well as the amount of interstitial fibroelastic tissue, are not taken into account.

It has been shown, for example, that the average volume occupied by the total sino-atrial nodal cells in adults is 2.4 times greater than that in infants, due mainly to the process of simple hypertrophy of the nodal cells (Shiraishi et al., 1992). Moreover, the volume of interstitial connective tissue of the sinus node in adults is 7.4 times that in infants (Shiraishi et al., 1992). Therefore, for the same density of innervation per nodal cell, the density calculated in terms of nerves per unit area will be higher in infants than adults.

Even though no perfect solution to this problem has been attained, we are of the opinion that a semiquantitative assessment of the density of innervation, as defined above, would serve the purpose of the present study. Furthermore, the additional advantage of this semiquantitative analysis are that it is simple and less time consuming, and can thus be incorporated in the routine service of most laboratories, where computer-assisted image analyser may not be readily available. More importantly, in our experience (Chow et al., 1993, 1995), as well as those of others (Ursell et al., 1990, 1991a,1991b; Wharton et al., 1990), valid and valuable results could be obtained by such semiquantitative analysis.

Overall (PGP) Innervation of Cardiac Conduction Tissue

PGP is a specific marker of nervous tissue (Thompson et al., 1983), and was therefore used in the present study to show the general pattern of innervation of the conduction system of the human heart during development. It revealed that, as in other species (Anderson, 1972a,b; Bleeker et al., 1980; Roberts et al., 1989), the various components of the human conduction system were more densely innervated than the adjacent atrial and ventricular myocardium. In addition, many nerves and their varicose segments were in close apposition to, and most probably making morphological contacts with the cells of the conduction tissue, indicating a likely direct neuroeffector relation. These findings are not unexpected in view of the significant role of the autonomic nervous system in the regulation of cardiac impulse. It is this intimate association of these nerves with the conduction tissues, which are themselves specialised myocardial cells, that provides the anatomic basis for such function.

In all age groups presently studied, the density of PGP nerves was highest in the sinus node, and showed a decreasing gradient from the sinus node, through the atrioventricular node to the bundle branches. This density gradient is similar to that reported in the sympathetic innervation of the conduction system of the canine heart (Ursell et al., 1990). It is dissimilar from that in the guinea pig (Crick et al., 1996), where all regions of the conduction system possess a similar density of PGP nerves. These findings illustrate the species variation that occurs with respect to cardiac innervation. Furthermore, the density of PGP nerves in all regions of the conduction system increased from infancy to childhood when the adult pattern was established, and declined in the elders to a level lower than that found in infants.

It is also of interest to compare the pattern of PGP innervation of the bundle branches in infants and adults. In infants, only scattered PGP nerves were found focally in the proximal portion of the bundle branches, and were extremely sparse in the distal portion. In contrast, not only did the density of PGP innervation in the proximal portion of the bundle branches in adults exceed that in infants, but moderate number of PGP nerves were also seen in the distal portion. The extension of PGP innervation from the proximal to the distal portion of the bundle branches with growth indicates that in addition to the para-arterial route as suggested by Forsgren (1987), this parafascicular route constitutes an alternative pathway for the innervation of the conduction system of the human heart during development.

Presumptive Sympathetic (DBH and TH) Innervation of Cardiac Conduction Tissue

The patterns of DBH- and TH-positive nerves supplying the human cardiac conduction system, as well as their changes with age, were basically similar. In some locations, a slightly higher density of DBH than TH nerves was observed. This minor variation in density may be related to the differences in structure, storage, and metabolism of these enzymes. For example, it has been shown that DBH, unlike TH, is partially particulate, is associated with the membrane of noradrenaline storage vesicles, and some of it is contained within the matrix of vesicles and released on exocytotic liberation of noradrenaline (Weinshilboum et al., 1971).

Similar to PGP reactive nerves, both DBH and TH nerves exhibited a decreasing density gradient from the sinus node to the bundle branches in all age groups. Their densities in all regions of the conduction system, apart from the bundle branches, were also higher than that in the adjacent atrial and ventricular myocardium. In addition, presumptive sympathetic nerves increased in density from infancy to childhood, when the adult pattern was established, and gradually declined in elders to a level similar to that in infants. These findings confirm our previous postulation that the patterns of developmental maturation of TH innervation of the conduction system of the human and canine heart are very similar (Ursell et al., 1990; Chow et al., 1993).

As DBH and TH are enzymes involved in the synthesis of catecholamines, they have been used as markers for locating presumptive sympathetic neural tissue (Molinoff and Axelrod, 1971). While this is valid in most instances, it is important to note the recent findings of Hardebo et al (1992). These workers showed that several neuropeptides, together with DBH and TH, coexist with vasoactive intestinal polypeptide and choline acetyltransferase in subpopulations of neurons in the cranial parasympathetic ganglia of rat. The presence of DBH and TH should thus be taken as supportive rather than conclusive evidence that tissue is noradrenergic in type. Nevertheless, it is interesting to note that, in the bundle branches, the density of DBH and TH nerves in adults was approximately half that of PGP, but similar to that of DBH, TH and PGP nerves in the adjacent ventricular myocardium. Thus, while the ventricular myocardium was predominantly innervated by these presumptive sympathetic fibers, which is in accordance with our previous finding in the neonatal heart (Chow et al., 1995), the bundle branches possess, in addition, a second population of nerves devoid of catecholamine-synthesising enzymes.

Presumptive Cholinergic Innervation (Cholinesterase Activity) of Cardiac Conduction Tissue

Corresponding to the findings of previous investigators using foetal hearts (Anderson and Taylor, 1972), we have observed moderate pseudocholinesterase activity in the conduction tissue of the human heart at birth. Only a few AChE-positive nerves were seen among the nodal cells of the sinus and atrioventricular nodes, but not in the bundle branches at this stage of development. In children, however, no cholinesterase activity was found in the conduction tissue. Furthermore, not only was there an increase in density of AChE-positive nerves in both the sinus and atrioventricular nodes, but they were also seen in the penetrating and branching bundles, as well as in the bundle branches. The density of innervation further increased in adults, but gradually declined in elders, to a level comparable to that in children. A decreasing density gradient from the sinus node to the bundle branches was observed in all ages.

Our present finding of AChE innervation in adults is exactly similar to that described by Kent et al. (1974). It also confirms our previous postulation (Chow et al., 1993) that, as the human cardiac conduction tissue matures, it gradually loses its content of pseudocholinesterase, but acquires a rich supply of AChE-positive nerves. In this respect, it is of interest to note that the conduction tissues in adult rabbit heart also display acetylcholinesterase activity (Anderson 1972c; Bojsen-Moller and Tranum-Jensen, 1972). Furthermore, Vitadello et al. (1990) found that neurofilament proteins were coexpressed with desmin in the conduction tissues of rabbit heart in all stages of development and persisted into adulthood. As the presence of acetylcholinesterase activity and expression of neurofilament proteins are characteristics of neural differentiation, the above findings, coupled with the expression of desmin, indicate that the conduction tissues of the heart function as a specialised neuromuscular system for the initiation and coordination of cardiac impulse.

Our present finding of the loss of cholinesterase activity and the lack of expression of neurofilament proteins in the conduction tissues of adult human heart (unpublished observations) would therefore suggest that the neural characteristics of the conduction tissues of the adult human heart are less overtly expressed than those of the rabbit heart. The underlying explanation of this phenomenon remains obscure but may be related to the gradual acquisition of the rich supply of autonomic nerves in the conduction tissues of the adult human heart as the conduction tissues mature.

Functional Correlation

The density of AChE-positive nerves in the bundle branches of adults was approximately half that observed using PGP, and was similar to that of DBH nerves. These findings are intriguing in the context of the nonadrenergic component of innervation of the bundle branches alluded to previously. Approximately half of the PGP reactive nerves in the bundle branches contain DBH, while the remainder corresponds quantitatively with the density of AChE-positive nerves demonstrated in the same region. In addition, it is particularly important to note that, by comparing the staining pattern in the serial sections, nerves exhibiting positive cholinesterase reaction were distinct from those showing positive immunostaining with DBH and TH. Thus, it can be concluded that the AChE-positive nerves represent the majority of the population of nonadrenergic nerves in the bundle branches of the adult human heart, which are presumably cholinergic in type and vagal in origin. This hypothesis is in keeping with the electrophysiologic finding of the codominant effects of adrenergic and cholinergic transmitters on the cardiac conduction system (Corr, 1992).

Furthermore, it is of particular relevance to compare and contrast the maturational changes in the pattern of innervation of the human cardiac conduction system by presumptive sympathetic and parasympathetic nerves. In infants, significantly more DBH- and TH-reactive nerves were present in all regions of the conduction system as compared with AChE-positive nerves, suggestive of a sympathetic dominance in the innervation of the conduction tissue. In children and adults, however, the proportion of AChE-positive, presumptively parasympathetic, nerves progressively increased to the extent that, in adulthood, the density was comparable with that of nerves reactive to DBH and TH. This finding of initial sympathetic dominance in the neural supply of the conduction system in infancy, and the gradual transition to sympathetic and parasympathetic codominance in adulthood, correlates well with the clinical observation of rapid cardiac rate, with wide fluctuation, in infancy. The changes noted are also in keeping with the relatively slow, and more stable, heart rate observed in adulthood (Behrman et al. 1992). Indeed, it appears entirely reasonable to assume that this maturational change in the pattern of innervation may constitute one of the anatomical bases for such physiologic alteration.

The speculation is further supported by the finding that enhanced ventricular electrical stability produced by vagal stimulation is mediated by cholinergic fibers supplying the ventricular conduction system (Kent et al. 1974). The paucity of parasympathetic nerves supplying the conduction tissues of the infant heart might thus render the ventricle less electrically stable, thereby exaggerating the cardiac response during various physiological activities. Thus, it is well known that the cardiac rate of newborn infants may increase from an average of 120–140 beats per minute to 170 or more during crying, and falls to 70–90 or even exhibits a bradycardia during asleep (Behrman et al. 1992).

Finally, our finding of reduction in the density of innervation of the cardiac conduction system with ageing is also in keeping with clinical and electrophysiologic findings. One such examples is provided by the reduced prevalence of sinus bradycardia and arrhythmia on resting electrocardiogram with ageing, which is a reflection of the age-associated reduction in parasympathetic function (Pfeifer et al., 1983; Schwartz et al. 1991). Since the conduction tissues and presumably its nerve supply are more resistant to ischaemia than ordinary working myocardium (Davies et al., 1983e), and the coronary arteries of all the hearts employed in the present study were widely patent and the hearts showed no evidence of ischaemia, we are of the opinion that the reduction in the density of innervation of the human cardiac conduction system in the elders is largely if not merely a result of ageing.

CONCLUSION

  1. Top of page
  2. Abstract
  3. RESULTS
  4. DISCUSSION
  5. CONCLUSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES

In summary, our present study has shown that PGP, DBH, TH, and AChE innervation of the conduction system of the human heart show a similar pattern of distribution and developmental changes. The density of innervation is highest in the sinus node, and exhibits a decreasing gradient through the atrioventricular node, penetrating and branching bundles, to the bundle branches. Apart from AChE-positive nerves, the other types are already present in substantial quantities in infancy, but do increase in density in childhood, when the adult pattern is achieved. In contrast, only scattered AChE-positive nerves are found in the sinus and atrioventricular nodes, but not in the bundle branches of the infant heart, whereas these conduction tissues themselves possess a significant amount of pseudocholinesterase. With maturation into adulthood, however, the conduction tissues gradually lose their cholinesterase content but acquire a rich supply of AChE-positive nerves, comparable in density to those of DBH and TH nerves.

Our findings of initial sympathetic dominance in the neural supply of the human cardiac conduction system in infancy, and its gradual transition into a sympathetic and parasympathetic codominance in adulthood, correlate well with, and may constitute one of the anatomical bases for the physiologic alterations in cardiac rate known to occur during development. The findings are important clinically because they show that, in addition to marked differences in innervation of the conduction tissues among species, equally important changes occur with development in the pattern of innervation in the same species, namely, human.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. RESULTS
  4. DISCUSSION
  5. CONCLUSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES

We obtained 24 human hearts at necropsy from individuals without congenital cardiac anomalies, and dying from causes unrelated to the heart. Their age ranged from newborn to 80 years. The relevant clinical information is summarised in Table 3. In the present study, hearts were assigned into four major groups, namely, infants (age less than 1 year, cases 1–5, n = 5), children (ages from 1 to 15 years; cases 6–9, n = 4), adolescents and adults (ages from 16 to 50 years; cases 10–19, n = 10), and elders (age greater than 50 years; case 20–24, n = 5).

Table 3. Clinical information of the cases analysed
CaseSexAgeDelay timea (hr)Cause of death
  1. a

    aDelay between death and necropsy.

  2. b

    bThe gestation periods of these cases range from 38 to 39 weeks.

1FStillbirthb15Cord around neck twice
2MDay 1b12Tracheal agenesis
3MDay 2b18Short-limbed dwarfism (thanatophoric dysplasia)
4FDay 10b6Severe asphxia neonatorum
5M5 months18Bronchopneumonia
6M3 years11Multiple injuries due to accidental fall
7M4 years9Multiple injuries due to traffic accident
8F11 years13Asthma
9M12 years9Suicide (jump from height)
10F18 years6Suicide (jump from height)
11F21 years15Suicide (hanging)
12F23 years18Suicide (hanging)
13M28 years16Suicide (hanging)
14M30 years9Miliary tuberculosis
15M30 years13Suicide (hanging)
16M37 years12Septicaemia (intravenous drug addict)
17F43 years8Suicide (hanging)
18F48 years10Suicide (hanging)
19M48 years11Suicide (hanging)
20M53 years8Spontaneous intracerebral haemorrhage
21M65 years15Bronchopneumonia
22M70 years11Suicide (cut throat)
23F80 years9Aortic dissection
24M80 years15Septicaemia secondary to pyelonephritis

Preparation of Tissue

The hearts were collected within 18 hours of death, following the ethical standards of the institutions from which they were obtained. Tissue blocks containing the sinus node, atrioventricular node, penetrating bundle, branching bundle and bundle branches were prepared by the method described by Davies et al. (1983d). In brief, the ring of tissue from the sinuatrial junction, including portions of the superior caval vein and right atrial appendage, was removed, divided into 3 to 4 smaller strips, immediately frozen in isopentane previously cooled in liquid nitrogen, and then serially sectioned in a cryostat at a thickness of 10 μm at right angles and perpendicular to the terminal groove, which contains the entirety of the sinus node. The block containing the atrioventricular junctional area extended from the orifice of the coronary sinus to the supraventricular crest, including about 1 cm of the atrial and 1.5 cm of the ventricular tissues on either side of the atrioventricular junction. After taking a transverse block across the lower portion of the ventricular septum from this block, it was divided into 3 to 4 smaller blocks by parallel cuts at right angles and perpendicular to the atrioventricular junction. Each block was again frozen in isopentane previously cooled in liquid nitrogen, and then serially sectioned in a cryostat at 10μm thickness.

On the average, for each heart, this resulted in approximately 250 sections from the sinus node, 200 sections from the transitional and compact regions of the atrioventricular node, 100 sections from the penetrating bundle, and 300 sections from the branching bundle and bundle branches. We also took 30 serial sections from the transverse block of the muscular ventricular septum to demonstrate the left bundle branch in cross section. The tissue sections were mounted on gelatin-subbed glass slides, and stored at −70°C. Initially, every twentieth section was stained with Masson Trichrome technique (Fig. 1A), and these were used to evaluate and reconstruct the three dimensional morphology of the specialised conduction tissue of the heart. The intermediate sections were then studied by enzyme and immunohistochemical techniques as deemed appropriate.

Immunohistochemical and Enzyme Histochemical Analysis

In the present study, we used protein gene product 9.5 (PGP), a sensitive and specific marker for neural tissue (Thompson et al. 1983), to demonstrate the general pattern of innervation of the cardiac conduction system, and restricted ourselves to the investigation of nerves containing the classical neurotransmitters, namely, noradrenaline and acetylcholine. The former was studied by using immunohistochemical staining against dopamine β-hydroxylase (DBH) and tyrosine hydroxylase (TH), both of which are enzymes involved in the synthesis of catecholamine (Molinoff and Axelrod, 1971), while staining for acetylcholinesterase activity was used for the demonstration of presumptive cholinergic fibres. While we recognised that monoclonal antibodies towards choline acetyltransferase offer a more specific method for the identification of cholinergic neural tissue (Eckenstein and Thoenen, 1982), we have been unsuccessful in the application of this method in the postmortem tissue employed for the present study, presumably due to the effects of autolysis. Accordingly, double immunostaining for DBH/TH and choline acetyltransferase, which would have been ideal for an overview of pattern of sympathetic and parasympathetic innervation, had not been possible in the present study. However, in all cases, serial sections from each area of the conduction tissues were used for immunostaining and enzyme histochemical study, so that the pattern of innervation of different nerve subtypes in the same site can be compared, thereby gaining most, if not all, of the information that can be achieved by double immunostaining.

Immunofluorescence Reactivity

The details of the primary antisera used in the present study are as follows: protein gene product 9.5, a polyclonal rabbit antibody from Ultraclone (UK) used in a dilution of 1:400; dopamine β-hydroxylase, a rabbit polyclonal antibody from Eugene Tech International (Allendale, NJ) used in a dilution of 1:200; and tyrosine hydroxylase, a rabbit polyclonal antibody from Incstar, Inc. (Stillwater, MN) used in a dilution of 1:500. An indirect immunofluorescence procedure, using the avidin-biotin complex technique, was employed (Wharton et al., 1990; Chow et al., 1993). Briefly, the cryostat sections were air dried at room temperature for 1 hr, fixed in 0.4% parabenzoquinone for 3 min, then sequentially incubated at room temperature with the diluted primary antisera for 16 hr, biotinylated anti-rabbit immunoglobulin for 30 min, and finally fluorescein Avidin D for 30 min, with thorough washing in phosphate buffered saline between the steps. The sections were mounted in glycerol mixed in the ratio of 1:1 with buffered saline and examined with a microscope equipped for epi-illumination.

For positive controls, we used tissue sections from a segment of adult human small intestine freshly obtained during surgery. The autonomic nerves in the wall of the intestine showed positive immunostaining with all the antibodies. For negative control sections, the primary antisera were either omitted, replaced with preimmune serum, or preabsorbed with their respective antigens. In these controls, immunofluorescent staining was absent.

Cholinesterase Reactivity

Tissue sections were fixed in formol calcium at 4°C for 20 min. After washing in tap water, they were incubated in Gomori's stock solution containing 2 mg/ml of acetyl thiocholine iodide as substrate, at 37°C, pH 6.0 for 16 hr. Sections were then washed again in tap water, developed for 60 sec in freshly prepared 1% ammonium sulphide solution at 20°C and counterstained in haematoxylin for 2 min. The sections were dehydrated, cleared and mounted in Canada Balsam dissolved in tetrachlorethylene. Control sections were incubated in the absence of substrate. As appropriate, sections were also preincubated at 20°C with tetra-isopropyl-pyrophosphoramide (Sigma Chemical Company, St. Louis, MO) as an inhibitor of pseudocholinesterase, while others were incubated in the presence of 1,5-bis (4-Allyldimethyl-ammoniumphenyl)-pentan-3-one-dibromide (Sigma) as an inhibitor of acetylcholinesterase.

For positive controls, tissue sections from the same segment of adult small intestine freshly obtained from surgery were used. The wall of the intestine showed presence of AChE-positive nerves. For the negative control sections, they were incubated in the absence of substrate.

Semiquantitative Assessment of the Density of Innervation

For semiquantitative assessment, comparable regions of each part of the conduction system of all the hearts were selected, so as to ensure an accurate comparison of the density of innervation among the various age groups. Since the density of nerves was highest in the central region of the sinus node (Crick et al. 1994), we selected three sections from the central portion of each part of the conduction system for each antibody and cholinesterase reaction, and the overall densities of nerves in these regions were assessed visually in a semiquantitative manner.

Using eyepieces of 10× and objective of 20×, the relative number of nerves in this high power field (HPF) were graded from 0 to 5+ according to the following criteria:

0

No immunoreactive nerve detected

±

Immunoreactive nerves rarely detected, less than 3/HPF

1+

Sparse and scattered individual nerves, 3–10/HPF

2+

Moderate number of nerves, 11–30/HPF

3+

Abundant nerves, 31–60/HPF

4+

Numerous nerves, 61–100/HPF

5+

Very numerous nerves, > 100/HPF

The intensity of the enzyme staining reaction of the myocardial cells was also assessed semiquantitatively, and graded as 0, + or ++ according to the following:

0

Negative staining

+

Weak staining

++

Strong staining

Examples of this density grading and intensity of staining are illustrated in the figures and explained in the legends.

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  2. Abstract
  3. RESULTS
  4. DISCUSSION
  5. CONCLUSION
  6. EXPERIMENTAL PROCEDURES
  7. REFERENCES
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