Pediatr Cardiol 12:155-160, 1991
Pediatric Cardiology 9 Springer-Verlag New York Inc. 1991
D e t e r m i n a t i o n o f I n f u n d i b u l a r I n n e r v a t i o n a n d A m i n e R e c e p t o r C o n t e n t in C y a n o t i c a n d A c y a n o t i c M y o c a r d i u m : R e l a t i o n to C l i n i c a l E v e n t s in Tetralogy of Fallot Lynn B. McGrath, Chao Chen, Jiang Gu, John Bianchi, and James M. Levett Department of Surgery, Deborah Heart and Lung Center and Deborah Research Institute, Browns Mills, New Jersey; and Department of Surgery, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, New Brunswick, New Jersey, USA
SUMMARY. Right ventricular myocardium was assessed for cholinergic and adrenergic innervation, as well as alpha-adrenergic, beta-adrenergic, and muscarinic receptors, in 18 cyanotic patients with tetralogy of Fallot (TOF) and four acyanotic control patients with ventricular septai defect, each of whom underwent a cardiac repair from June through December 1987. Neurons containing acetylcholine (ACH), neuron-specific enolase (NSE), S-100 protein, neuropeptide-Y (NPY), dopamine-beta-hydroxylase (DBH), and calcitonin gene-related polypeptide (CGRP) were detected surrounding arterioles and myocytes in all specimens. NSE and S-100 immunoreactivities were also identified in the cytoplasm of TOF cardiocytes, possibly indicating a neuroendocrine origin of these cells. Cardiocyte size was increased in TOF (p = 0.05). Acetyicholine (cholinergic) (p = 0.04) and CGRP (cholinergic) positive neurons (p = 0.07) were decreased in the TOF as compared to controls. Adrenergic fiber content (p = 0.15) and beta receptors (p = 0.21) were similar in both groups. There was an increase in muscarinic receptors in the controls (p = 0.002), and a marked increase in alpha receptors in TOF (p = 0.019). There were no intragroup differences in the TOF patients according to degree of cyanosis. In conclusion, there were important differences in neuronal and amine receptor content between TOF and control patients. Increased alpha receptors in TOF could account for differences in clinical and hemodynamic events. KEY WORDS: Alpha receptors--Beta receptors--Muscarinic receptors--Infundibular innervation
Tetralogy of Fallot (TOF) patients may present with a wide variation in cyanotic clinical events. Various maneuvers have been shown to accentuate cyanosis in TOF and to predispose to cyanotic spells [10, 62]. The effects of beta-adrenergic blockade preoperatively to alleviate cyanosis [11, 12, 30, 54, 57, 58, 60, 61], and intraoperatively to lower right ventricular pressure [48], have been the subject of a number of reports. In general, these studies have indicated a beneficial effect with amelioration of clinical events. However, the innervation and amine receptor content of ventricular myocardium in TOF, to Address offprint requests to: Dr. Lynn B. McGrath, Department of Surgery, Deborah Heart and Lung Center, 200 Trenton Road, Browns Mills, NJ 08015, USA.
our knowledge, has not previously been documented. The purpose of this report was to characterize the infundibular neuronal and amine receptor content in TOF, to compare it with acyanotic control patients, and to determine if variations in innervation patterns and amine receptor content are risk factors for clinical events in TOF.
Materials and Methods Patients From June 1 through December 31, 1987, 18 cyanotic patients with TOF and four control patients underwent an intracardiac repair, which included resection of infundibular myocardium. All
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procedures were performed by one surgeon (LBM). In the TOF patients a specimen for analysis was obtained from the resected muscle of the parietal extension of the infundibular septum. In the four control patients with simple large perimembranous ventricular septal defect and no right ventricular outflow tract obstruction, tissue was obtained from a biopsy of the right ventricular side of the infundibular septum. There were no hospital deaths. The tetralogy patients' ages ranged from 11 months to 240 months, with a mean 64.6 months. There were nine males and nine females. All tetralogy patients were cyanotic preoperatively, nine were having active spells and eight had a prior systemic pulmonary shunt. No patient had been taking a beta-adrenergic receptor blocking agent. Six of the tetralogy patients were repaired using a formal ventriculotomy incision. Twelve were repaired using a transatrial and transpulmonary approach, and six of the latter also required a limited transannular patch. In the four control patients with ventricutar septal defect closure the mean age was 105 months, with a range of 12 to 192 months. There were three males and one female.
Tissue Processing Resected tissue was assessed using histologic and immunocytochemistry [23] methods for the presence, distribution, and degree of cholinergic and adrenergic innervation [13, 32, 34, 37, 41, 46, 53]. Alpha-adrenergic [7, 22, 29, 35, 59, 67], beta-adrenergic [6, 27, 36, 45, 49-52, 56], and muscarinic receptor [4, 8, 18] content was determined in each specimen. The specimens were processed immediately upon removal from the patient. Tissue was fixed with freshly prepared 4% paraformaldehyde in phosphate-buffered saline (PBS), with pH 7.3 for 3 h at room temperature. The specimens were washed overnight in PBS containing 17% sucrose at 4~ After dehydration with graded alcohol and xylene, the samples were imbedded in paraffin and cut into paraffin blocks. Five-micrometer thick consecutive sections were cut. Two sections from each piece of tissue were stained with hematoxylin and eosin to assess the morphology. The rest of the sections were processed for light microscopic immunocytochemistry. Five sections of each specimen were graded 0 to 4+ per high-power field by a single-blinded observer (JG) to denote the presence and abundance of neuronal structures. The specimens were analyzed for neuronal markers and were investigated utilizing antibodies to specific tissue antigens. These included dopamine-beta-hydroxylase (DBH, a marker for adrenergic and nonadrenergic nerves) [21], neuropeptide-Y (NPY, a neuromodulator present in adrenergic and nonadrenergic nerves) [24], S-100 protein (a protein present in perineuronal structures, such as Schwann's cells) [47], acetylcholine (ACH, a neurotransmitter for cholinergic nerves), neuron-specific enolase (NSE, a marker for all nerve types) [5, 25], and calcitonin gene-related polypeptide (CGRP, a marker of subpopulations of cholinergic nerves) [20]. The antibodies used were rabbit polyclonal and were purchased from Accurate Laboratories (Accurate Chemical and Scientific Corporation, Sand Diego, CA, USA). They were diluted in appropriate dilutions in PBS, generally from 1 : 1000 to 1 : 4000. The avidin-biotin-peroxidase complex immunostaining technique was employed as described by Hsu et al. [31]. The first layer incubation lasted for 12 h at room temperature. The slides were counterstained with hematoxylin. The controls included the use of normal rabbit serum as
Pediatric Cardiology Vol. 12, No. 3, 1991
the first layer and the preabsorption test with respective antigens at a concentration of 10 nmol/ml of diluted antisera.
Adrenoceptor Content The adrenoceptor content of each specimen was analyzed by disruption of cells with proteinase inhibitors present and differential centrifugation to produce a final subcellular pellet of enriched sarcolemmal membrane. The receptor assay was performed by standard rapid filtration of radioligand binding to determine adrenoreceptor density and to characterize the receptors. The techniques are standard and well-described by Khatter et al. [38]. The technique for studying beta-adrenergic receptors involved radioligand binding using (-)3H-dihydroalprenolol (3HDHA) [39]. The binding of 3H-DHA to membranes provides a simple, convenient, rapid and reliable method of assessing the density of beta-adrenergic receptors. The density of alphaadrenoreceptors was measured using 3H-clonidine as the radioliogand. Muscarinic receptors were measured using 3H-quinuclidinylbenzylate as the radiolabeled antagonist. The counts per minute were expressed as moles of ligand and, after subtracting nonspecific binding, the density of receptors was expressed as moles per milligram protein. Protein levels were determined by the method of Lowry et al. [42].
Statistical Analysis Usual methods were used to determine whether differences between the groups were due to chance, including calculation o f p values by X-', Fisher methods, liklihood ratio, two-tailed t tests, Kruskal-Wallis, and Bartlett methods.
Results
Infundibular Innervation Overall. N e r v e s t r u c t u r e s c o n t a i n i n g N S E , S - 1 0 0 protein, NPY, CGRP, ACH, and DBH immunor e a c t i v i t i e s w e r e d e t e c t e d t h r o u g h o u t all o f t h e s p e c i m e n s o b t a i n e d . N e r v e f i b e r s w e r e m a i n l y located surrounding arterioles and associated with c a r d i a c m u s c l e c e l l s . I n a d d i t i o n t o b e i n g p r e s e n t in the nerve structures, NSE and S-100 protein immunoreactivity was weakly but postively demonstrated in the cytoplasm of certain TOF myocytes ( F i g . 1A a n d B ) . T h i s w a s n o t f o u n d in t h e c o n t r o l patients. In the TOF tissue, the combined S-100 protein and NSE data indicated a high overall degree of i n n e r v a t i o n o f 3 + t o 4 + i n all o f t h e s p e c i m e n s e x amined. There were no differences in the overall concentration of neuronal structures between the TOF and the control samples [p (likelihood ratio) = 0.39]. Adrenergic. in T O F
Examination of adrenergic innervation using the combined NPY and DBH data
McGrath et al.: Adrenergic Receptors in Right Ventricular Outflow Tract
Fig. 1. (A) NSE immunoreactivity in the cytoplasm of myocardiocytes of the infundibular muscle of Fallot's tetralogy as detected by light microscopic immunocytochemistry. Original magnification, x600. (B) S-100 immunoreactivity in the cytoplasm of myocardiocytes of the infundibular muscle of Fallot's tetralogy as detected by light microscopic immunocytochemistry. Original magnification, •
revealed that there was a low total adrenergic neuronal structure count. In all 35 specimens examined, 0 to 2+ adrenergic nerve fibers were identified. Similar findings were obtained in the control patients with small numbers of adrenergic fibers noted in all specimens [p (X 2) ~" 0.15]. NPY-containing neurons identified per field in T O F were a mean of 1.41 [standard error (SE) -+ 0.17] and 1.60 (SE +- 0.24) in the controls [p (2sample t test) = 0.24]. Similarly, the D B H data indicated a mean number of neuronal structures per field in the T O F group to be 1.11 (SE + 0.18) and 1.00 (SE -+ 0.31) in the control group [p (2-sample t test) = 0.77].
Cholinergic. Cholinergic fibers predominated in both T O F and control specimens. Comparing the cholinergic neuronal content of T O F and control patients revealed a small but significant increase in the number of cholinergic structures in the controls. The mean alpha-receptor level in T O F patients was 355.5 pmol/mg of protein (SE -+ 72.4); in the controis no alpha-receptor activity was detected [p (Kruskal-Wallis) = 0.019]. Mean n u m b e r of cholinergic fibers per field in the controls was 1.8 (SE -+ 0.20) versus 1.00 (SE + 0.16) in the T O F group [p (2-sample t test) = 0.02]. Cholinergic-associated CGRP was also increased in the control patients. The mean was 2.80 in the control (SE -+ 0.20) and
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2.12 (SE + 0.19) in the T O F group [p (2-sample t test) = 0.07].
Amine Receptors There was not difference between T O F and control groups for beta-adrenergic receptor concentration. The mean beta-receptor level in the T O F patients was 109.6 pmol/mg of protein (SE -+ 18.6); in the controls it was 53.7 pmol/mg of protein (SE -+ 20.5) [p (2-sample t test) = 0.21]. Alpha-adrenergic receptors were increased in the T O F patients. The mean concentration in the controls was 0 (SE + 0) and 355.5 (Se + 72.4) in the T O F patients with a range of 0-942 [p (KruskalWallis) = 0.019]. Muscarinic r e c e p t o r content was increased in the control patients. The mean concentration in the controls was 968.7 pmol/mg of protein (SE -+ 899.4) and was 244.1 (SE + 118.8) in the T O F group [p for variance (Bartlett) = 0.002].
Clinical Events Among the T O F patients, a n u m b e r of variables were evaluated to determine whether various clinical subgroups exhibited differences in infundibular innervation or amine receptor content. Overall infundibular neuronal content was assessed to determine its association with severity of cyanosis on admission. Tetralogy patients with severe preoperative cyanosis (n = 11) were found to have infundibular neuronal counts similar to those patients with mild to moderate cyanosis preoperatively (n = 7) [p (X 2) = 0.27]. Similarly, there was no difference in adrenergic neuronal content [p (X 2) = 0.37] nor in patterns of cholinergic innervation [p (X z) = 0.32] in those patients with severe preopera-
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rive cyanosis, as c o m p a r e d to those with mild to moderate cyanosis. A history o f cyanotic spells (n = 9) was not associated with an increase in overall infundibular neuronal content [p (X 2) = 0.51], adrenergic innervation [p (X 2) = 0.41], or cholinergic innervation [p (X 2) = 0.22]. An assessment was made to determine the liklihood that a patient undergoing T O F repair with the requirement for a formal right ventricular patch (n = 6) would exhibit changes in infundibular innervation in comparision to those patients not needing a right ventricular patch (n = 12). There was no difference in overall infundibular innervation [p (X 2) = 0.22], adrenergic innervation [p (X 2) = 0.11], or cholinergic innervation [p (X 2) = 0.17], according to the type of repair performed. There were also no differences between these two tetralogy repair subgroups for concentration of alpha receptors [p (X 2) = 0.31], beta receptors [p (X 2) = 0.72], or muscarinic receptors [p (X'-) = 0.19]. Comparing levels of postrepair right ventricular to left ventricular pressure ratio in patients with (n = 6) or without (n = 12) a formal right ventricular patch revealed no difference according to overall infundibular neuronal content [p (one-way analysis of v a r i a n c e - - A N O V A ) = 0.89], adrenergic innervation [p (ANOVA) = 0.71], or cholinergic innervation [p (ANOVA) = 0.62]).
Discussion The etiology of cyanosis in T O F has been of interest since Fallot first analyzed this condition in 1888 [17]. Bing and coworkers in 1947 indicated the importance of obstruction to right ventricular outflow [3]. Shah and Kidd outlined the fine balance between right ventricular outflow resistance and systemic vascular resistance in determining the relative flows in any given patient [57]. Wood in 1958 indicated that infundibular stenosis could be extremely labile, and he attributed the o c c u r r e n c e of cyanotic spells to spasm of the infundibular septum [68]. Previously d o c u m e n t e d alterations in cellular [1, 2, 28] and myocardial metabolism [55, 56, 66] in cyanotic subjects do not appear to account for variations in cyanotic events. The present study was undertaken to determine the innervation of the right ventricular outflow tract and its amine receptor content in T O F . The aim was to decide whether there was a quantitative or qualitative difference in those patients presenting with more severe degrees of cyanosis and cyanotic spells and to compare them to acyanotic controls. Our findings suggest that there were significant
Pediatric Cardiology Vol. 12. No. 3. 1991
differences in neuronal and amine receptor content between the T O F and control patients. The increased levels of alpha-adrenergic receptor content in T O F is an interesting finding. Although there were no intragroup differences in T O F patients according to clinical events, this may be related to the small sample size. Furthermore, the sensitivity of plasma membrane receptors can vary widely [51]. The importance of the relative decrease in muscarinic receptor levels in the T O F patient is not apparent. The clinical implications of the findings of abnormal infundibular myocardium in T O F are also not clear. H o w e v e r , the presence of NSE and S-100 immunoreactivity in these cells, and the increased levels of alpha-adrenergic receptors may have an important influence on the preoperative clinical condition of the patient. In addition, the use of alpha-adrenergic receptor blockers may be potentially beneficial in decreasing the requirement for shunting in infants [9, 14, 16, 33]. Of course there would be potential for increased right-to-left shunting if a concentration of alpha-receptor blocking agents sufficient to lower systemic arterial resistance were used. The discovery of neuropeptides in the heart has changed the traditional concept of biparted cardiac innervation [15, 19, 26, 40, 43, 44, 63-65]. Among the many peptides reported to be present in the heart, NPY and C G R P appear to be the most abundant and probably also the most functionally important neuropeptides. NPY is stored at least partially in adrenergic nerves and CGRP, at least partially, in sensory nerves. The physiologic and pathologic significance of these peptides in the heart is still a matter of debate [15, 19, 26, 40, 43, 44, 63-65]. The observations made in this study provide a morphologic basis for further evaluation of the roles that these peptides may play in both normal and ischemic conditions. Future work will focus on the assessment of variations in sensitivity of amine receptors in TOF, clarification of the origin of the intracellular NSE and S-100 immunoreactivity in these patients, and evaluation of other anomalies with right ventricular outflow tract obstruction in both cyanotic and acyanotic patients.
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Pediatric Cardiology 9 Springer-Verlag New York Inc. 1991
D e t e r m i n a t i o n o f I n f u n d i b u l a r I n n e r v a t i o n a n d A m i n e R e c e p t o r C o n t e n t in C y a n o t i c a n d A c y a n o t i c M y o c a r d i u m : R e l a t i o n to C l i n i c a l E v e n t s in Tetralogy of Fallot Lynn B. McGrath, Chao Chen, Jiang Gu, John Bianchi, and James M. Levett Department of Surgery, Deborah Heart and Lung Center and Deborah Research Institute, Browns Mills, New Jersey; and Department of Surgery, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, New Brunswick, New Jersey, USA
SUMMARY. Right ventricular myocardium was assessed for cholinergic and adrenergic innervation, as well as alpha-adrenergic, beta-adrenergic, and muscarinic receptors, in 18 cyanotic patients with tetralogy of Fallot (TOF) and four acyanotic control patients with ventricular septai defect, each of whom underwent a cardiac repair from June through December 1987. Neurons containing acetylcholine (ACH), neuron-specific enolase (NSE), S-100 protein, neuropeptide-Y (NPY), dopamine-beta-hydroxylase (DBH), and calcitonin gene-related polypeptide (CGRP) were detected surrounding arterioles and myocytes in all specimens. NSE and S-100 immunoreactivities were also identified in the cytoplasm of TOF cardiocytes, possibly indicating a neuroendocrine origin of these cells. Cardiocyte size was increased in TOF (p = 0.05). Acetyicholine (cholinergic) (p = 0.04) and CGRP (cholinergic) positive neurons (p = 0.07) were decreased in the TOF as compared to controls. Adrenergic fiber content (p = 0.15) and beta receptors (p = 0.21) were similar in both groups. There was an increase in muscarinic receptors in the controls (p = 0.002), and a marked increase in alpha receptors in TOF (p = 0.019). There were no intragroup differences in the TOF patients according to degree of cyanosis. In conclusion, there were important differences in neuronal and amine receptor content between TOF and control patients. Increased alpha receptors in TOF could account for differences in clinical and hemodynamic events. KEY WORDS: Alpha receptors--Beta receptors--Muscarinic receptors--Infundibular innervation
Tetralogy of Fallot (TOF) patients may present with a wide variation in cyanotic clinical events. Various maneuvers have been shown to accentuate cyanosis in TOF and to predispose to cyanotic spells [10, 62]. The effects of beta-adrenergic blockade preoperatively to alleviate cyanosis [11, 12, 30, 54, 57, 58, 60, 61], and intraoperatively to lower right ventricular pressure [48], have been the subject of a number of reports. In general, these studies have indicated a beneficial effect with amelioration of clinical events. However, the innervation and amine receptor content of ventricular myocardium in TOF, to Address offprint requests to: Dr. Lynn B. McGrath, Department of Surgery, Deborah Heart and Lung Center, 200 Trenton Road, Browns Mills, NJ 08015, USA.
our knowledge, has not previously been documented. The purpose of this report was to characterize the infundibular neuronal and amine receptor content in TOF, to compare it with acyanotic control patients, and to determine if variations in innervation patterns and amine receptor content are risk factors for clinical events in TOF.
Materials and Methods Patients From June 1 through December 31, 1987, 18 cyanotic patients with TOF and four control patients underwent an intracardiac repair, which included resection of infundibular myocardium. All
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procedures were performed by one surgeon (LBM). In the TOF patients a specimen for analysis was obtained from the resected muscle of the parietal extension of the infundibular septum. In the four control patients with simple large perimembranous ventricular septal defect and no right ventricular outflow tract obstruction, tissue was obtained from a biopsy of the right ventricular side of the infundibular septum. There were no hospital deaths. The tetralogy patients' ages ranged from 11 months to 240 months, with a mean 64.6 months. There were nine males and nine females. All tetralogy patients were cyanotic preoperatively, nine were having active spells and eight had a prior systemic pulmonary shunt. No patient had been taking a beta-adrenergic receptor blocking agent. Six of the tetralogy patients were repaired using a formal ventriculotomy incision. Twelve were repaired using a transatrial and transpulmonary approach, and six of the latter also required a limited transannular patch. In the four control patients with ventricutar septal defect closure the mean age was 105 months, with a range of 12 to 192 months. There were three males and one female.
Tissue Processing Resected tissue was assessed using histologic and immunocytochemistry [23] methods for the presence, distribution, and degree of cholinergic and adrenergic innervation [13, 32, 34, 37, 41, 46, 53]. Alpha-adrenergic [7, 22, 29, 35, 59, 67], beta-adrenergic [6, 27, 36, 45, 49-52, 56], and muscarinic receptor [4, 8, 18] content was determined in each specimen. The specimens were processed immediately upon removal from the patient. Tissue was fixed with freshly prepared 4% paraformaldehyde in phosphate-buffered saline (PBS), with pH 7.3 for 3 h at room temperature. The specimens were washed overnight in PBS containing 17% sucrose at 4~ After dehydration with graded alcohol and xylene, the samples were imbedded in paraffin and cut into paraffin blocks. Five-micrometer thick consecutive sections were cut. Two sections from each piece of tissue were stained with hematoxylin and eosin to assess the morphology. The rest of the sections were processed for light microscopic immunocytochemistry. Five sections of each specimen were graded 0 to 4+ per high-power field by a single-blinded observer (JG) to denote the presence and abundance of neuronal structures. The specimens were analyzed for neuronal markers and were investigated utilizing antibodies to specific tissue antigens. These included dopamine-beta-hydroxylase (DBH, a marker for adrenergic and nonadrenergic nerves) [21], neuropeptide-Y (NPY, a neuromodulator present in adrenergic and nonadrenergic nerves) [24], S-100 protein (a protein present in perineuronal structures, such as Schwann's cells) [47], acetylcholine (ACH, a neurotransmitter for cholinergic nerves), neuron-specific enolase (NSE, a marker for all nerve types) [5, 25], and calcitonin gene-related polypeptide (CGRP, a marker of subpopulations of cholinergic nerves) [20]. The antibodies used were rabbit polyclonal and were purchased from Accurate Laboratories (Accurate Chemical and Scientific Corporation, Sand Diego, CA, USA). They were diluted in appropriate dilutions in PBS, generally from 1 : 1000 to 1 : 4000. The avidin-biotin-peroxidase complex immunostaining technique was employed as described by Hsu et al. [31]. The first layer incubation lasted for 12 h at room temperature. The slides were counterstained with hematoxylin. The controls included the use of normal rabbit serum as
Pediatric Cardiology Vol. 12, No. 3, 1991
the first layer and the preabsorption test with respective antigens at a concentration of 10 nmol/ml of diluted antisera.
Adrenoceptor Content The adrenoceptor content of each specimen was analyzed by disruption of cells with proteinase inhibitors present and differential centrifugation to produce a final subcellular pellet of enriched sarcolemmal membrane. The receptor assay was performed by standard rapid filtration of radioligand binding to determine adrenoreceptor density and to characterize the receptors. The techniques are standard and well-described by Khatter et al. [38]. The technique for studying beta-adrenergic receptors involved radioligand binding using (-)3H-dihydroalprenolol (3HDHA) [39]. The binding of 3H-DHA to membranes provides a simple, convenient, rapid and reliable method of assessing the density of beta-adrenergic receptors. The density of alphaadrenoreceptors was measured using 3H-clonidine as the radioliogand. Muscarinic receptors were measured using 3H-quinuclidinylbenzylate as the radiolabeled antagonist. The counts per minute were expressed as moles of ligand and, after subtracting nonspecific binding, the density of receptors was expressed as moles per milligram protein. Protein levels were determined by the method of Lowry et al. [42].
Statistical Analysis Usual methods were used to determine whether differences between the groups were due to chance, including calculation o f p values by X-', Fisher methods, liklihood ratio, two-tailed t tests, Kruskal-Wallis, and Bartlett methods.
Results
Infundibular Innervation Overall. N e r v e s t r u c t u r e s c o n t a i n i n g N S E , S - 1 0 0 protein, NPY, CGRP, ACH, and DBH immunor e a c t i v i t i e s w e r e d e t e c t e d t h r o u g h o u t all o f t h e s p e c i m e n s o b t a i n e d . N e r v e f i b e r s w e r e m a i n l y located surrounding arterioles and associated with c a r d i a c m u s c l e c e l l s . I n a d d i t i o n t o b e i n g p r e s e n t in the nerve structures, NSE and S-100 protein immunoreactivity was weakly but postively demonstrated in the cytoplasm of certain TOF myocytes ( F i g . 1A a n d B ) . T h i s w a s n o t f o u n d in t h e c o n t r o l patients. In the TOF tissue, the combined S-100 protein and NSE data indicated a high overall degree of i n n e r v a t i o n o f 3 + t o 4 + i n all o f t h e s p e c i m e n s e x amined. There were no differences in the overall concentration of neuronal structures between the TOF and the control samples [p (likelihood ratio) = 0.39]. Adrenergic. in T O F
Examination of adrenergic innervation using the combined NPY and DBH data
McGrath et al.: Adrenergic Receptors in Right Ventricular Outflow Tract
Fig. 1. (A) NSE immunoreactivity in the cytoplasm of myocardiocytes of the infundibular muscle of Fallot's tetralogy as detected by light microscopic immunocytochemistry. Original magnification, x600. (B) S-100 immunoreactivity in the cytoplasm of myocardiocytes of the infundibular muscle of Fallot's tetralogy as detected by light microscopic immunocytochemistry. Original magnification, •
revealed that there was a low total adrenergic neuronal structure count. In all 35 specimens examined, 0 to 2+ adrenergic nerve fibers were identified. Similar findings were obtained in the control patients with small numbers of adrenergic fibers noted in all specimens [p (X 2) ~" 0.15]. NPY-containing neurons identified per field in T O F were a mean of 1.41 [standard error (SE) -+ 0.17] and 1.60 (SE +- 0.24) in the controls [p (2sample t test) = 0.24]. Similarly, the D B H data indicated a mean number of neuronal structures per field in the T O F group to be 1.11 (SE + 0.18) and 1.00 (SE -+ 0.31) in the control group [p (2-sample t test) = 0.77].
Cholinergic. Cholinergic fibers predominated in both T O F and control specimens. Comparing the cholinergic neuronal content of T O F and control patients revealed a small but significant increase in the number of cholinergic structures in the controls. The mean alpha-receptor level in T O F patients was 355.5 pmol/mg of protein (SE -+ 72.4); in the controis no alpha-receptor activity was detected [p (Kruskal-Wallis) = 0.019]. Mean n u m b e r of cholinergic fibers per field in the controls was 1.8 (SE -+ 0.20) versus 1.00 (SE + 0.16) in the T O F group [p (2-sample t test) = 0.02]. Cholinergic-associated CGRP was also increased in the control patients. The mean was 2.80 in the control (SE -+ 0.20) and
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2.12 (SE + 0.19) in the T O F group [p (2-sample t test) = 0.07].
Amine Receptors There was not difference between T O F and control groups for beta-adrenergic receptor concentration. The mean beta-receptor level in the T O F patients was 109.6 pmol/mg of protein (SE -+ 18.6); in the controls it was 53.7 pmol/mg of protein (SE -+ 20.5) [p (2-sample t test) = 0.21]. Alpha-adrenergic receptors were increased in the T O F patients. The mean concentration in the controls was 0 (SE + 0) and 355.5 (Se + 72.4) in the T O F patients with a range of 0-942 [p (KruskalWallis) = 0.019]. Muscarinic r e c e p t o r content was increased in the control patients. The mean concentration in the controls was 968.7 pmol/mg of protein (SE -+ 899.4) and was 244.1 (SE + 118.8) in the T O F group [p for variance (Bartlett) = 0.002].
Clinical Events Among the T O F patients, a n u m b e r of variables were evaluated to determine whether various clinical subgroups exhibited differences in infundibular innervation or amine receptor content. Overall infundibular neuronal content was assessed to determine its association with severity of cyanosis on admission. Tetralogy patients with severe preoperative cyanosis (n = 11) were found to have infundibular neuronal counts similar to those patients with mild to moderate cyanosis preoperatively (n = 7) [p (X 2) = 0.27]. Similarly, there was no difference in adrenergic neuronal content [p (X 2) = 0.37] nor in patterns of cholinergic innervation [p (X z) = 0.32] in those patients with severe preopera-
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rive cyanosis, as c o m p a r e d to those with mild to moderate cyanosis. A history o f cyanotic spells (n = 9) was not associated with an increase in overall infundibular neuronal content [p (X 2) = 0.51], adrenergic innervation [p (X 2) = 0.41], or cholinergic innervation [p (X 2) = 0.22]. An assessment was made to determine the liklihood that a patient undergoing T O F repair with the requirement for a formal right ventricular patch (n = 6) would exhibit changes in infundibular innervation in comparision to those patients not needing a right ventricular patch (n = 12). There was no difference in overall infundibular innervation [p (X 2) = 0.22], adrenergic innervation [p (X 2) = 0.11], or cholinergic innervation [p (X 2) = 0.17], according to the type of repair performed. There were also no differences between these two tetralogy repair subgroups for concentration of alpha receptors [p (X 2) = 0.31], beta receptors [p (X 2) = 0.72], or muscarinic receptors [p (X'-) = 0.19]. Comparing levels of postrepair right ventricular to left ventricular pressure ratio in patients with (n = 6) or without (n = 12) a formal right ventricular patch revealed no difference according to overall infundibular neuronal content [p (one-way analysis of v a r i a n c e - - A N O V A ) = 0.89], adrenergic innervation [p (ANOVA) = 0.71], or cholinergic innervation [p (ANOVA) = 0.62]).
Discussion The etiology of cyanosis in T O F has been of interest since Fallot first analyzed this condition in 1888 [17]. Bing and coworkers in 1947 indicated the importance of obstruction to right ventricular outflow [3]. Shah and Kidd outlined the fine balance between right ventricular outflow resistance and systemic vascular resistance in determining the relative flows in any given patient [57]. Wood in 1958 indicated that infundibular stenosis could be extremely labile, and he attributed the o c c u r r e n c e of cyanotic spells to spasm of the infundibular septum [68]. Previously d o c u m e n t e d alterations in cellular [1, 2, 28] and myocardial metabolism [55, 56, 66] in cyanotic subjects do not appear to account for variations in cyanotic events. The present study was undertaken to determine the innervation of the right ventricular outflow tract and its amine receptor content in T O F . The aim was to decide whether there was a quantitative or qualitative difference in those patients presenting with more severe degrees of cyanosis and cyanotic spells and to compare them to acyanotic controls. Our findings suggest that there were significant
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differences in neuronal and amine receptor content between the T O F and control patients. The increased levels of alpha-adrenergic receptor content in T O F is an interesting finding. Although there were no intragroup differences in T O F patients according to clinical events, this may be related to the small sample size. Furthermore, the sensitivity of plasma membrane receptors can vary widely [51]. The importance of the relative decrease in muscarinic receptor levels in the T O F patient is not apparent. The clinical implications of the findings of abnormal infundibular myocardium in T O F are also not clear. H o w e v e r , the presence of NSE and S-100 immunoreactivity in these cells, and the increased levels of alpha-adrenergic receptors may have an important influence on the preoperative clinical condition of the patient. In addition, the use of alpha-adrenergic receptor blockers may be potentially beneficial in decreasing the requirement for shunting in infants [9, 14, 16, 33]. Of course there would be potential for increased right-to-left shunting if a concentration of alpha-receptor blocking agents sufficient to lower systemic arterial resistance were used. The discovery of neuropeptides in the heart has changed the traditional concept of biparted cardiac innervation [15, 19, 26, 40, 43, 44, 63-65]. Among the many peptides reported to be present in the heart, NPY and C G R P appear to be the most abundant and probably also the most functionally important neuropeptides. NPY is stored at least partially in adrenergic nerves and CGRP, at least partially, in sensory nerves. The physiologic and pathologic significance of these peptides in the heart is still a matter of debate [15, 19, 26, 40, 43, 44, 63-65]. The observations made in this study provide a morphologic basis for further evaluation of the roles that these peptides may play in both normal and ischemic conditions. Future work will focus on the assessment of variations in sensitivity of amine receptors in TOF, clarification of the origin of the intracellular NSE and S-100 immunoreactivity in these patients, and evaluation of other anomalies with right ventricular outflow tract obstruction in both cyanotic and acyanotic patients.
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