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Cardiovascular Research 2004 62(1):112-121; doi:10.1016/j.cardiores.2004.01.012
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Localization of cholinergic innervation in guinea pig heart by immunohistochemistry for high-affinity choline transporters

Donald B Hoover*,a, Charles E Ganoteb,c, Shawn M Fergusond, Randy D Blakelye,f and Rodney L Parsonsg

aDepartment of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA
bDepartment of Pathology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA
cJames H. Quillen Veterans Affairs Medical Center, Mountain Home, TN 37684, USA
dNeuroscience Graduate Program, Center for Molecular Neuroscience, Vanderbilt University, Nashville, TN 37232, USA
eCenter for Molecular Neuroscience, Vanderbilt University, Nashville, TN 37232, USA
fDepartment of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA
gDepartment of Anatomy and Neurobiology, University of Vermont College of Medicine, Burlington, VT 05405, USA

* Corresponding author. Tel.: +1-423-439-6322; fax: +1-423-439-8773. Email address: hoover{at}mail.etsu.edu

Received 7 October 2003; revised 27 December 2003; accepted 8 January 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Previous studies have used acetylcholinesterase (AChE) histochemistry to identify cholinergic nerves in the heart, but this enzyme is not a selective marker for cholinergic neurons. This study maps cholinergic innervation of guinea pig heart using a new antibody to the human high-affinity choline transporter (CHT), which is present only in cholinergic nerves. Methods: Immunohistochemistry was used to localize CHTs in frozen and paraffin sections of heart and whole mount preparations of atrial ganglionated nerve plexus. AChE-positive nerve fibers were identified in sections from separate hearts for comparison. Results: Control experiments established that the antibody to human CHT selectively labeled cholinergic neurons in the guinea pig. CHT-immunoreactive nerve fibers and AChE-positive nerves were very abundant in the sinus and AV nodes, bundle of His, and bundle branches. Both markers also delineated a distinct nerve tract in the posterior wall of the right atrium. AChE-positive nerve fibers were more abundant than CHT-immunoreactive nerves in working atrial and ventricular myocardium. CHT-immunoreactive nerves were rarely observed in left ventricular free wall. Both markers were associated with numerous parasympathetic ganglia that were distributed along the posterior atrial walls and within the interatrial septum, including the region of the AV node. Conclusions: Comparison of labeling patterns for CHT and AChE suggests that AChE histochemistry overestimates the density of cholinergic innervation in the heart. The distribution of CHT-immunoreactive nerve fibers and parasympathetic ganglia in the guinea pig heart suggests that heart rate, conduction velocity, and automaticity are precisely regulated by cholinergic innervation. In contrast, the paucity of CHT-immunoreactive nerve fibers in left ventricular myocardium implies that vagal efferent input has little or no direct influence on ventricular contractile function in the guinea pig.

KEYWORDS Acetylcholine; Autonomic nervous system; AV-node; Innervation; Purkinje fiber; Conduction system; Neurotransmitters; Sinus node; High-affinity choline transporter; Immunohistochemistry; Guinea pig


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Cholinergic nerves control cardiac function by releasing acetylcholine (ACh), which has a direct inhibitory influence on cardiomyocytes and a prejunctional inhibitory effect on sympathetic nerves [1,2]. Additionally, the postjunctional action of ACh can attenuate excitatory responses evoked by cardiac sympathetic nerves and circulating catecholamines [1]. Although both divisions of the autonomic nervous system are crucial for regulation of the heart, the anatomic distribution of cholinergic nerves is less precisely known than that of sympathetic innervation. Histochemical localization of acetylcholinesterase (AChE), the enzyme that hydrolyzes ACh at cholinergic neuroeffector junctions, has been widely used to map cholinergic innervation of tissues. However, the specificity of this method is limited due to expression of AChE by some noncholinergic neurons [3].

Three ACh-related proteins are considered specific markers for cholinergic neurons: choline acetyltransferase (ChAT), the vesicular ACh transporter (VAChT), and the high-affinity choline transporter (CHT). Synthesis of ACh is catalyzed by ChAT, and antibodies to this marker have been used to map central cholinergic pathways [4]. However, ChAT antibodies had limited utility for identifying cholinergic nerves in peripheral tissues until recently [5,6]. VAChT transfers ACh to storage vesicles in nerve terminals or varicosities, and VAChT antibodies have been used to identify cholinergic neurons and nerve fibers in brain and peripheral tissues, including rat and human heart [6–9]. However, the latter studies did not report a thorough mapping of cardiac cholinergic nerves. The CHT protein is highly expressed at cholinergic varicosities where it transports choline into neurons for use in synthesizing ACh [10,11]. The CHT gene was cloned recently [12–14], and antibodies to this cholinergic marker have been used for immunohistochemical detection in multiple species and tissues [15–17].

This study was done to delineate the cholinergic innervation of guinea pig heart by immunohistochemistry with antibodies to the CHT and ChAT. The presence and localization of these specific cholinergic markers were evaluated in atrial ganglionated nerve plexus preparations and serial sections of heart. We also used a histochemical procedure to localize AChE in sections from separate hearts for comparison.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Tissue preparation for immunohistochemistry
This study conforms with the Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). Male Hartley guinea pigs (200 to 800 g, n=40) were used. Animals were pretreated with heparin (1000 units/kg, i.p.) and anesthetized with sodium pentobarbital (75 mg/kg, i.p.). Hearts were removed and perfused via the ascending aorta with 10 ml phosphate-buffered saline (PBS, pH 7.4) containing 10 units heparin/ml and 10 ml cold 4% paraformaldehyde in PBS or 10% neutral buffered formalin. Hearts perfused with paraformaldehyde were postfixed overnight at 4 °C, stored for 2 days at 4 °C in PBS with 20% sucrose, and sectioned at –20 °C in a microtome-cryostat. Blocks were cut from two formalin fixed hearts, embedded in paraffin, and sectioned at 5 µm thickness. Serial frozen 30 µm sections were collected along the short axis of the heart beginning superior to the sinus node (SN) and ending inferior to the bundle of His. These sections and others collected from different levels of ventricles were transferred to coated slides and stored at –20 °C. For some experiments, 50 µm ventricular sections were stained free floating. Every third serial section was stained for CHT and half of the other sections were stained with hematoxylin and eosin (H&E). Remaining sections were immunostained for ChAT or tyrosine hydroxylase (TH) or processed using Masson's trichrome stain. For other experiments, whole mount preparations of atrial ganglionated nerve plexus were dissected from the posterior atrial walls and either fixed immediately for immunohistochemistry or maintained in culture for 3–4 days [18,19]. One animal was anesthetized with pentobarbital and a cannula inserted through the heart into the aorta for perfusion with 200 ml PBS containing heparin and 300 ml cold fixative. The brain was removed and processed as described for hearts.

2.2. Antibodies
The peptide VDSSPEGSGTEDNLQ fused to keyhole limpet hemocyanin was used to raise rabbit polyclonal antisera against CHT (Research Genetics, Huntsville, AL). This peptide corresponds to the distal C-terminus (residues 566–580) of CHT and is completely conserved between humans, rats, and mice [12,13,17,20]. Affinity purification of the antisera was performed using an Affigel column (BioRad) coupled to the immunizing peptide. Affinity purified CHT antibody was used at a dilution of 1:500 or 1:1000. The specificity of this antibody has been established in transfected cells and rodent brain [17] as well as choline transporter knockout mice (unpublished data). Mouse monoclonal anti-TH (clone TH-16) was purchased from Sigma-RBI (St. Louis, MO) and used at a dilution of 1:4000. Goat anti-ChAT (AB144P) was purchased from Chemicon International (Temecula, CA) and used at 1:50.

2.3. Immunohistochemistry
Sections and whole mounts were immunostained using the ABC technique (Vector Laboratories, Burlingame, CA) or fluorescent secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) as previously described [19]. Paraffin sections were treated with Citra Plus antigen retrieval solution as described by the manufacturer (BioGenex, San Ramon, CA) before immunostaining. For staining with the ABC technique, tissue was pretreated with 0.3% hydrogen peroxide to suppress endogenous peroxidase activity, and VIP substrate (Vector) was used as the chromogen. Slides were viewed and photographed using an Olympus BX41 microscope equipped with a MagnaFire SP digital camera. Confocal images were obtained using a Leica SP2 laser scanning confocal microscope. Photographs of heart sections are oriented according to the BNA system [21] with anterior at top and right and left in the reader's position as viewed from above.

2.4. AChE histochemistry
The histochemical method of Koelle [3] was used to identify AChE in 30 µm sections from three hearts (two unfixed and one fixed). Nonspecific cholinesterase was inhibited irreversibly by preincubation of sections in buffer with 1 µM tetraisopropylpyrophosphoramide (Sigma) for 30 min at 37 °C.

2.5. Western blot analysis
The brain was removed from an anesthetized guinea pig and dissected to obtain samples of caudate nucleus, hippocampus, and cerebellum. Samples were homogenized in 20 volumes of cold 0.32 M sucrose, 10 mM HEPES-NaOH (pH 7.4) with 10 strokes of a Potter Elvejhem homogenizer. Homogenates were centrifuged at 4 °C for 10 min at 1000 x g, and resulting supernatants were centrifuged 20 min at 13,000 x g. Pellets were suspended in PBS containing 1% Triton X 100 and evaluated for the presence of CHT immunoreactivity by standard Western blotting methods.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Characterization of CHT antibody
Western blot analysis of extracts from guinea pig brain with the CHT antibody demonstrated a broad band at approximately 60 kDa (Fig. 1A), which is the molecular mass reported for CHTs in other species [15,17]. This band was present in extracts from regions of brain with dense cholinergic innervation (i.e., hippocampus and caudate nucleus) and absent in extract from cerebellum, where cholinergic innervation is extremely sparse. Immunohistochemical evaluation of sections from guinea pig brain using CHT antibody demonstrated intense labeling of varicose nerve fibers in regions known to receive cholinergic innervation and light to moderate staining of cholinergic cell bodies (Fig. 1B–D). Immunolabeling of whole mounts revealed intense CHT-immunoreactivity of varicose nerve fibers that surrounded the postganglionic parasympathetic neurons (Fig. 1E). Staining was absent if CHT antibody was omitted from the incubation or preabsorbed with immunizing peptide (Fig. 1F). In preparations double-immunolabeled for CHT and ChAT, both cholinergic markers were present in varicose nerve processes within the ganglia (Fig. 2A and B).


Figure 1
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  		Fig. 1 Characterization of CHT antibody in guinea pigs. (A) Western blot with CHT antibody showing a broad 60 kda band present only in regions of brain with cholinergic nerves. (B–D) CHT-IR nerve fibers in interpeduncular nucleus (B), dorsal motor nucleus of vagus (C), and nucleus ambiguus (D). Cholinergic cell bodies are also stained lightly (C and D). These nuclei contain preganglionic vagal efferent neurons. (E) CHT-IR nerve fibers in intracardiac ganglia of whole mount preparation. (F) Absence of labeling in whole mount processed using antibody that was preabsorbed with immunogen. Immunodetection was by the ABC technique (B–D) or FITC-conjugated secondary (E and F). Scale bar indicates 50 µm (C and D), 100 µm (E and F), or 200 µm (B).

 

Figure 2
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  		Fig. 2 Localization of ChAT and CHT immunoreactivity in atrial ganglion whole mounts. Double labeling was done with secondary antibodies conjugated to FITC (ChAT) or Cy3 (CHT). (A and B) Intracardiac neurons, varicose nerve fibers around these neurons, and nerve bundles are ChAT-IR. CHT immunoreactivity is present only in nerve fibers and colocalized with ChAT. (C and D) Double labeling in 4 day cultured whole mount. (E and F) Double labeling in whole mount cultured 4 days with 10 µM colchicine. Scale bars indicate 25 µm.

 
3.2. Localization of CHT and ChAT in intracardiac ganglia
CHT-IR nerve fibers were observed in all ganglia in atrial whole mount preparations and surrounded virtually all neurons. CHT immunoreactivity was not evident in nerve bundles that interconnect ganglia within a preparation, and staining of neuronal cell bodies was either weak or absent (Figs. 1E and 2B)Go. This contrasts with ChAT immunoreactivity, which was strong in ganglion cell bodies and varicose nerve processes around postganglionic neurons (Fig. 2A). ChAT-IR nerve fibers were also found in nerve bundles as reported previously [19,22,23]. In whole mounts maintained in culture for 4 days so extrinsic nerve fibers would degenerate, a vast majority of the CHT- and ChAT-IR nerve fibers were absent (Fig. 2C and D). Postganglionic neurons remained intensely ChAT-IR and negative for CHT. However, if 10 µM colchicine was added to the culture medium to block axoplasmic transport, then neurons exhibited strong immunoreactivity for CHT and ChAT (Fig. 2E and F). Identical patterns of CHT and ChAT immunoreactivity were observed in tissue sections with ganglia located adjacent to the posterior edge of the right and/or left atrium, around the pulmonary artery, in the interatrial septum, and around the AV node (AVN; Figs. 4D and 5E–FGo). CHT immunoreactivity of cholinergic cell bodies was more evident with the ABC method than fluorescence.


Figure 4
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  		Fig. 4 CHT-IR nerve tract localized to posterior wall of right atrium. Short axis sections were stained by the ABC technique. (A–E) Photomicrographs beginning below the SN (A) and progressing through inferior levels (B–E). Dashed lines indicate approximate boundaries of CHT-IR nerve fiber tract. Arrows and letters signify regions shown at higher magnification in corresponding lettered panels. RA—right atrium, Ao—aorta, RBB—right bundle branch, LBB—left bundle branch, TV—tricuspid valve, and VS—ventricular septum. Asterisks indicate ganglia (D). Star marks inferior margin of coronary sinus ostium. Scale bars indicate 0.5 mm (A–E), 50 µm (F, G, I), and 100 µm (H).

 

Figure 5
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  		Fig. 5 Cholinergic innervation of selected atrial regions. (A and B) CHT-IR nerves in anterior wall of right atrium (A) and near ostium of right atrial appendage (B). (C and D) Adjacent paraffin sections (long axis) through annulus of TV showing trichrome staining (C) and CHT-IR nerves (D). (E and F) Ganglia near AVN in different sections stained for CHT (E) or ChAT (F). (G) Trichrome stain (same section as C) showing AVN and retroaortic specialized atrial muscle (arrowheads). Arrows indicate connective tissue (blue). (H and I) Adjacent paraffin section showing CHT-IR nerves around specialized myocytes (H) and AV nodal cells (I). Scale bars indicate 50 µm (A, D–F), 100 µm (B), or 200 µm (C and G).

 
3.3. Cholinergic innervation of atrial tissue
Fluorescent and bright-field immunohistochemistry with CHT antibody revealed significant regional differences in the density of cholinergic innervation in the heart (Table 1). Labeled nerve fibers consistently had a varicose appearance indicative of neurotransmitter release sites.


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  		Table 1 Distribution and relative abundance of CHT-IR and AChE-positive nerve fibers in the heart

 
The sinus node (SN) contained a dense plexus of nerve fibers that formed a meshwork supplying nodal cells (Fig. 3A–C). Double staining for CHT and ChAT demonstrated that these markers were located within the same nerve fibers in the SN (Fig. 3C) and other regions of the heart. CHT- and ChAT-IR nerve fibers likewise had similar distributions in sections stained by the ABC technique.


Figure 3
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  		Fig. 3 Confocal images showing colocalization of CHT and ChAT immunoreactivities in nerve fibers in the sinus node. Images were obtained from sequential scans of SN in section double-labeled for CHT (A: Cy3, red) and ChAT (B: FITC, green). Yellow-orange in overlay channel (C) signifies colocalization. Scale bar indicates 80 µm.

 
A prominent tract of cholinergic nerve fibers began in subepicardial muscle at the lateral aspect of the SN and extended down the posterior wall of the right atrium (Fig. 4). At its origin and superior levels along the posterior wall, the density of cholinergic innervation was indistinguishable from that in the SN (Fig. 4A). In the inferior direction, this nerve tract broadened and gradually shifted to the inferolateral border of the inferior vena cava (Fig. 4B–D). Over this course, density of CHT-IR nerve fibers gradually decreased but remained greater than in surrounding atrial muscle (Fig. 4F–H). The density of CHT-IR nerves then increased in posterior right atrial muscle between the inferior vena cava and the annulus of the posterior tricuspid leaflet. This nerve tract continued beneath the coronary sinus ostium (Fig. 4E and I) and extended to the region of the posterior AVN.

Most other regions of right atrium contained a low density of CHT-IR nerves (Fig. 5A), but a moderately dense plexus occurred around the ostium of the atrial appendage (Fig. 5B). The density of CHT-IR nerves also increased significantly as the annulus of the tricuspid valve was approached (Fig. 5C and D). The density of CHT-IR nerve fibers in left atrium was generally less than observed in right atrium but increased in the vicinity of the mitral valves. Interatrial septum contained a low density of CHT-IR nerve fibers that became more abundant at inferior levels near the AVN. This area was also characterized by the presence of ganglia within and posterior to the septum (Fig. 5E and F).

Extranodal atrial specialized tissue [24] received prominent input by CHT-IR nerve fibers (Fig. 5G–I). This retroaortic tissue includes a node-like structure located above the AVN at the anterior margin of the interatrial septum and side extensions that run in fibrous tissue between the aortic root and anterior atrial walls. The lateral extensions appeared to merge with atrial muscle cells at the annulus of the tricuspid and mitral valves. CHT-IR nerve fibers were present in AV values but not the aortic or pulmonary valve.

3.4. CHT-IR nerves supplying the atrioventricular conducting system
Cells of the AVN, bundle of His, and bundle branches were readily identified in paraffin sections because their myocytes stained lighter than working myocytes when processed with H&E or Masson's trichrome stain (Figs. 5G and 6A–B)Go. The AVN was separated from neighboring atrial myocytes at the superior approach by a thin sheet of connective tissue (Fig. 5G). Nevertheless, nodal cells received a rich cholinergic innervation, which is likely supplied by adjacent ganglia and those located in the interatrial septum. The node extended anteriorly to the bundle of His but also extended posteriorly and inferiorly as a distinct tract of pale cells (Fig. 6D–F). The posterior extension of the AVN appeared to join with atrial myocytes above the posterior tricuspid leaflet. All elements of the atrioventricular conducting system were heavily innervated by CHT-IR nerve fibers (Figs. 5–8)GoGoGo. Staining of adjacent sections for TH showed that adrenergic innervation was dense in the AVN but sparse or absent in the bundle of His and bundle branches (Fig. 7).


Figure 6
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  		Fig. 6 Conducting system and CHT-IR nerve fibers in short axis paraffin sections. (A) Trichrome stain showing AVN and bundle of His. (B) Bundle of His at higher magnification. (C) CHT stain of same region in adjacent section. (D–F) Posterior extension of AVN (pAVN). (D and E) Trichrome-stained section at low and higher magnification. Specialized myocytes of pAVN stained lighter than working myocytes of VS. (F) Adjacent section with CHT-IR nerves concentrated in pAVN. (G and I) Trichrome stain showing specialized myocytes of LBB and RBB. (H and J) CHT stain of adjacent section showing cholinergic innervation of LBB (H) and RBB (J). Scale bars signify 0.5 mm (A and D) or 50 µm (B, C, and E–J).

 

Figure 7
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  		Fig. 7 CHT and TH immunoreactivity at AVN, bundle of His, and left bundle branch. (A) H&E stained section. Rectangle indicates region shown in adjacent sections stained for CHT (B) and TH (C). (B) CHT-IR nerve fibers are abundant in AVN, His, and LBB. (C) TH-IR nerve fibers are largely confined to AVN. (D and E) Higher magnification of AVN showing cholinergic (D) and noradrenergic (E) innervation. (F and G) Higher magnification of LBB and His regions stained for CHT (F) or TH (G). Some CHT-IR nerve fibers continue directly into the VS (thick arrow). FS—fibrous septum, LV—left ventricle, MV—mitral valve, and RV—right ventricle. Scale bar indicates 50 µm (D and E), 100 µm (F and G), or 250 µm (B and C).

 

Figure 8
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  		Fig. 8 Cholinergic innervation of ventricular muscle. (A) CHT-IR nerve fibers in RV subendocardium. (B) CHT-IR nerve fibers in trabecular muscle of left septal wall. (C) CHT-stained long-axis section through LV papillary muscle. Rectangle indicates region shown at higher magnification in adjacent section stained with H&E (D) and same CHT-stained section (E). (F) Arrow indicates rare CHT-IR nerve fiber observed in left ventricular free wall. Insert shows this nerve fiber at higher magnification. (G) TH-IR nerve fibers in left ventricular free wall of section from same heart as F. Scale bar signifies 0.5 mm (C), 50 µm (A, B, F, G), or 100 µm (D and E).

 
3.5. CHT-IR nerves in ventricular myocardium
The highest density of CHT-IR nerves in ventricular myocardium was localized to the conducting system. At superior levels, CHT-IR nerves were concentrated in a thin subendocardial band spanning much of the left septal wall and to the right bundle branch. Abundant CHT-IR nerve fibers were localized to the right ventricular subendocardium (Fig. 8A), and a low-to-moderate innervation occurred in trabecular muscle of the right ventricle and left ventricular septum (Fig. 8B). Cholinergic nerves were also identified at the base of left ventricular papillary muscle (Fig. 8C–E). CHT-IR nerve fibers were extremely sparse in the left ventricular free wall (Fig. 8F), while sections stained for TH demonstrated a dense adrenergic innervation in all regions of ventricular myocardium (Fig. 8G).

3.6. Innervation by AChE-containing nerves
AChE histochemistry labeled a larger population of nerve fibers than revealed by immunohistochemistry for CHT or ChAT (Table 1). This was most obvious for the left ventricular wall (Fig. 9A), but was also evident for other regions that contained a low density of CHT-IR nerves (Fig. 9B–D). Nodal and conducting tissue, which contained the highest density of CHT-IR nerves, also had the most abundant innervation by AChE-positive nerve fibers.


Figure 9
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  		Fig. 9 AChE-positive nerve fibers in working atrial and ventricular myocardium. CHT-IR nerve fibers are less abundant than AChE-positive nerves in these regions. Scale bar indicates 100 µm.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
This study is the first to provide a detailed localization of cholinergic nerve fibers in a mammalian heart using an antibody to a specific cholinergic marker protein, the CHT. Our results suggest that cholinergic nerve fibers have a more focused distribution than revealed by histochemical staining for AChE and provide structural evidence to support a primary influence of parasympathetic cholinergic innervation on chronotropic and dromotropic functions of the guinea pig heart. We have also obtained evidence that the guinea pig AVN has a posterior extension, which receives a dense cholinergic innervation.

Recent studies using CHT antibodies have confirmed that CHTs are specifically localized to cholinergic neurons in the brain [15–17]. Our control experiments demonstrated that the human CHT antibody also recognizes CHT in guinea pig brain and heart and that CHTs are associated with cholinergic neurons in guinea pig. The co-localization of CHT and ChAT to nerve fibers in the heart and intracardiac ganglia further substantiates the cholinergic phenotype of these fibers.

The pattern of cholinergic innervation demonstrated by immunohistochemical localization of CHTs was similar to that observed with AChE histochemistry in this study and others [5,24]. Results for both markers indicate that cholinergic innervation of guinea pig heart is most abundant in the nodal tissue, the AV bundle, and the ventricular conducting system. However, prominent differences are evident in working atrial and ventricular myocardium where AChE histochemistry consistently labels more nerve fibers than immunohistochemistry for CHT or ChAT. This observation suggests that AChE histochemistry stains some noncholinergic nerve fibers as well as those that are cholinergic. Vagal afferent nerve fibers are likely candidates since AChE-positive neurons are present in the nodose ganglia [3]. Furthermore, treatment of guinea pigs with the sensory nerve toxin capsaicin decreases the number of AChE-positive nerve fibers in some regions of the heart [25]. The latter finding may underestimate the total number of AChE-positive sensory nerves in the heart since capsaicin only affects sensory neurons that contain substance P. Although AChE histochemistry may stain some noncholinergic nerves in the conducting system as well, the impact of such staining is likely diluted by the abundance of AChE-positive cholinergic nerves.

Dense innervation of the SN and AVN by AChE-containing nerves has been a consistent finding in other species including humans [26], dogs [27], pigs [28], rabbits [29], and rats [30]. Experimental and clinical studies have established that vagal nerve stimulation decreases heart rate and AV conduction velocity by activating cholinergic input to the SN and AVN [1,31–33]. Studies with most species have found that AChE-positive nerve fibers are also abundant in the bundle of His and bundle branches. Cholinergic nerves in the ventricular conducting system decrease automaticity of Purkinje fibers through postjunctional effects of ACh and possibly by prejunctional inhibition of sympathetic nerves. Ventricular Purkinje fibers may be under less vagal control in the human heart since a recent study detected fewer AChE-positive nerves in the bundle of His and none in bundle branches [26]. However, this report contrasts with an earlier study that observed numerous AChE-containing nerves in the conducting system [27]. In contrast to the dense cholinergic innervation of the ventricular conducting system in the guinea pig, TH immunoreactivity in the bundle of His and bundle branches was restricted primarily to nerve bundles. This observation concurs with a previous report that catecholamine-containing nerves are sparse in these regions of guinea pig heart [24]. This differs from human heart where TH-IR nerves are abundant in the AV bundle and bundle branches [26].

The major atrial inputs to the human AVN arrive by superoanterior and inferoposterior approaches [34]. Other investigators have reported that connective tissue around the AVN prevents input from atrial myocytes at the superoanterior approach in guinea pigs [5,24], and our findings with Masson's trichrome stain confirm this view. A posterior extension of the AVN, which receives input from the inferior approach, has been identified in human hearts and may be a substrate for slow conduction [34,35]. A posterior extension of the AVN was likewise identified in our study and contained many CHT-IR nerve fibers.

Although the density of CHT-IR nerves was low in most areas of internodal myocardium, higher cholinergic nerve density was evident at a few locations. The tract of CHT-IR nerve fibers identified in the posterior right atrial wall appears to correspond topographically with the posterior conduction pathway in humans [34]. Although it is controversial whether internodal muscle cells are specialized for preferential conduction by histological criteria [34], this pathway appears to be the shortest route for activation of the AVN in the guinea pig and would be a logical site for neural control. Numerous ganglia identified along the posterior margin of the atria, in this and previous studies [22,36], are likely sources of cholinergic nerve fibers comprising this posterior atrial nerve tract. It is also notable that CHT-IR nerve fibers are concentrated in retroaortic specialized muscle and atrial muscle near the AV valves. Retroaortic specialized tissue of the guinea pig may have a common embryonic origin with the AV conducting system, and this linage could explain its prominent cholinergic innervation [5]. The proximity of this tract and CHT innervated muscle of the inferior atrial walls to AV valves could also be significant. Atrial arrhythmias can originate from muscle around the annulus of the mitral and tricuspid valves [37,38], and myocytes capable of automaticity have been identified in these regions [38]. Accordingly, cholinergic innervation could act to suppress automaticity at these sites.

Although the density of CHT-IR nerves was low in the left atrium, this appears to be sufficient to influence contractility since transmural stimulation of guinea pig left atrium causes an atropine-sensitive decrease in force of contraction [39]. The greater abundance of CHT-IR nerves in right atrium would argue that cholinergic nerves could mediate negative inotropic effects there as well. However, the relative absence of CHT-IR nerves in guinea pig left ventricle suggests that direct vagal efferent effects on ventricular contractility are unlikely in this species. Data addressing this issue are not available for guinea pig, but recent work has demonstrated that vagal stimulation does not affect ventricular contractility in the rat [40]. In contrast, vagal stimulation decreases ventricular contractility in the dog [31], pig, and human [41]. These findings imply that cholinergic innervation of ventricular myocardium may vary significantly between species. The distribution and abundance of cholinergic nerves in ventricular muscle warrant further investigation in other species using a specific cholinergic probe since recent experimental and clinical studies have shown that vagal nerve activity can suppress some ventricular arrhythmias and has a protective effect against sudden cardiac death [42–44].

In summary, our findings demonstrate that CHTs are specifically localized to cholinergic nerve fibers in the guinea pig heart and suggest that AChE histochemistry overestimates cholinergic nerve density. Cholinergic nerve fibers identified on the basis of CHT expression are most abundant in the conducting system and in regions of heart that contain specialized cardiomyocytes but are very sparse in the left ventricular free wall. This distribution pattern is consistent with vagal cholinergic effects on heart rate, cardiac conduction velocity, and automaticity but suggests that little or no direct cholinergic effect on ventricular performance is likely in the guinea pig. The presence of many parasympathetic ganglia along the length of the atria, from the level of the SN to the AV junction, suggests that cholinergic input to specific atrial sites and the conducting system may be highly regulated. Localized disruption of this cholinergic network within the heart could be a factor that contributes to the development of arrhythmias. Further studies are needed to map CHT-IR nerves in other mammalian hearts since previous studies have identified differences in cardiac innervation between species.


   Acknowledgements
 
This work was supported by NIH grants HL54633 (D.B.H.), 2P01HL056693 (R.D.B.), HL65481 (R.L.P.), and a predoctoral fellowship from the Vanderbilt Brain Institute (S.M.F.). We thank Mary Howell and Chris Sosinski for excellent histological assistance, Dr. Lili Zhang for providing atrial whole mount preparations, and Dr. Stephen A. Fahrig for valuable comments on a draft of the manuscript. Dr. Ganote is Director of Autopsy Service at the VAMC, Mountain Home, TN.


   Notes
 
Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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