D. LUND, PHILLIP G. SCHMID,. STEPHEN E. KELLEY, ..... D. Histochemical studies of the relationship of ... L. M., V. L. WILLIAM,. C. R. HANLON,. AND T.
Choline acetyltransferase after transplantation
activity
in rat heart
DONALD D. LUND, PHILLIP G. SCHMID, STEPHEN E. KELLEY, ROBERT J. CORRY, AND ROBERT ROSKOSKI, JR. Cardiovascular Center, Department of Internal Medicine; Transplantation Service, Department of Surgery; and Department of Biochemistry, University of Iowa; and Veterans Administration Hospital, Iowa City, Iowa 52242
LUND, DONALD II., PHJLLIP G. SCHMID, STEPHEN E. KELLEY, ROBERT J. CORRY, AND ROBERT ROSKOSKI, JR. Choline acetyltransferase activity in rat heart after transplantation. Am. J. Physiol. 235(4): H367-H371, 1978 or Am. J. Physiol.: Heart Circ. Physiol. 4(4): H367-H371, 1978. -Choline acetyltransferase (CAT) catalyzes the biosynthesis of acetylcholine according to the chemical equation: Acetyl-CoA + Choline e Acetylcholine + CoA. To demonstrate the neuronal relationship of this enzyme, CAT activity was measured in hearts to which the extrinsic innervation was completely interrupted by extirpation of the heart from a donor animal and transplantation in a heterotopic position into the recipient’s abdominal cavity. After the animals were killed at 8 days, enzyme activity in the transplanted hearts was measured and compared with that of the recipient. Choline acetyltransferase activity decreased 98% in the right atrium of the transplanted denervated hearts, 94% in the SA node, 87% in the left atrium, 80% in the right ventricle, and about 50%> in the interventricular septum and left ventricle. These data show that sectioning the extrinsic parasympathetic innervation to the heart results in variable decreases in CAT activity, a finding which confirms that CAT activity is related to the extrinsic cholinergic innervation. The extrinsic (preganglionic) component of the parasympathetic system is nonuniform and supplies predominantly the specialized regions of the heart with conduction tissue. The residual enzyme activity is related to intrinsic (postganglionic) parasympathetic neurons. The intrinsic component is uniformly distributed to both specialized and contractile regions of the heart.
reported that after surgical denervation of the heart the remaining intrinsic cholinergic neurons in the atria and the ventricles can be activated, and the magnitude of these responses are fairly uniform in the various chambers. This suggests that the distribution of the intrinsic parasympathetic innervation throughout the denervated heart may be uniform. We therefore initiated studies to determine the pattern of the intrinsic parasympathetic innervation in the transplanted rat heart and the pattern of the combined extrinsic and intrinsic innervation in the intact heart by measuring the activity of choline acetyltransferase (CAT). CAT, which mediates the synthesis of acetylcholine, is postulated to be a marker of the parasympathetic innervation (15). Studies in this laboratory have demonstrated the presence of this enzyme in intact innervated rat hearts, but not in myocardial cell cultures derived from this source (16). If transplantation were to eliminate primarily the extrinsic innervation as a result of nerve sectioning, the remaining choline acetyltransferase activity would represent the intact intrinsic innervation (11). A difference in enzyme activity in normal and transplanted hearts could, therefore, represent an estimate of the extrinsic or preganglionic parasympathetic innervation
cholinergic neurons; intri nsic parasympathetic extrinsic parasympathetic innervation
MATERIALS
innervation;
THE PARASYMPATHETIC INNERVATION of the heart has two components. Preganglionic neurons, which originate in the dorsal motor nucleus of the brain stem, comprise the extrinsic parasympathetic innervation to the heart; postganglionic neurons, which originate and terminate within the heart, comprise the intrinsic parasympathetic innervation (6). During cardiac transplantation, the preganglionic neurons are severed; the postganglionic neurons remain intact (11, 14). It would appear from previous work that reflex activation of the parasympathetic nervous system regulates heart rate and to a lesser extent the myocardial contractile state (6). Priola and co-workers (14) have 0363-6135/78/0000-OOOO$Ol.
25 Copyright
CG 1978 the American
Physiological
AND
METHODS
All studies were performed on male Sprague-Dawley rats weighing ZOO-250 g. Rats were given Purina rat chow and water ad libitum. They were anesthetized with chloral hydrate, 10 ml/kg (3.6% solution), by an intraperitoneal injection. Transplantation was carried out as reported previously (1, 3). Each recipient animal was prepared under x16 magnification by a midline abdominal incision exposing the inferior vena cava and abdominal aorta distal to the renal vessels. The donor heart was removed by transecting the main pulmonary artery and the ascending aorta and ligating and dividing the vena cava and pulmonary veins. The heart was then placed in Ringer lactate at 4°C. The donor heart was transplanted by joining the donor aorta to the abdominal aorta of the recipient and the donor pulmonary artery to the inferior vena cava of Society
H367
LUND
r-11368 the recipient. The interval between removal of the heart from the donor animal and its transplantation was less than 45 min. The anastomoses were created using x25 magnification and 10-O monofilament nylon suture tipped with a W-5 needle (Ethicon). Vascular occluding ties were release& permitting the delivery of oxygenated blood from the recipient’s aorta through the donor coronary arteries. The pulmonary arteryvena cava anastomosis permitted blood from the coronary sinus, right atrium, and right ventricle to return to the recipient’s circulation. A regular rhythm ensued in the donor heart after perfusion of the coronary arteries with oxygenated blood. Pilot studies showed that choline acetyltransferase activity declined 70% in 2 days and was undetectable at 4 and 8 days in the distal portion of sectioned vagus nerves. Therefore, to provide time for the degeneration the animals were killed 8 of preganglionic neurons, days after transplantation. During this time, beating donor hearts could be palpated easily in the abdominal cavity of the recipient animal. Hearts maintained a regular rhythm and did not enlarge detectably. The recipient rats were killed by spinal-cervical dislocation and tissues were obtained from the regions illustrated in Fig. l* All samples were taken first from the donor heart and then from the normal host heart. Tissue samples were lightly blotted, weighed, immediately frozen in liquid nitrogen, and stored. Choline acetyltransferase activity. To prepare stored tissues for assay of choline acetyltransferase, samples were disrupted in a tight-fitting stainless steel pulverizer cooled with liquid nitrogen (15-17). The powdered samples were further disrupted with a Teflon-to-glass, motor-driven, homogenizer in a minimum amount (less than 3 ml) of buffer (5 mM potassium phosphate, 0.1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.4). The assay of acetylcholine biosynthesis in myocardial tissues was carried out as described previously (15-17, 19) by quantifying the formation of [ Wlacetylcholine from [I% jacetylcoenzyme A. A portion of the left ventricle was also taken for histological examination. Sections 6 pm thick were stained with hematoxylin and eosin for routine microscopic evaluation. Statistical comparisons of mean values were made using Student’s independent t test (20). A P value G.05 was selected to denote statistical significance for all group comparisons.
FIG.
1. Schematic
of choline
TABLE
1. Choline Right n=
Control 45min age t values
Stor-
TWO of the eight transplanted hearts from animals that survived the operation were not beating when the animals were killed at 8 days; these were excluded from the study. The weights of the transplanted hearts (mean k SE, 616 t 34 mg) were not different from the weights of the recipients’ hearts (605 2 17 mg), nor was there a difference in the myocardial water content. To investigate the possibility that the operation and storage might alter enzyme activity, hearts were assayed immediately after the animal was killed and after 45 min in Ringer lactate at 4°C. No change was
regions activity.
sampled
acetyltrunsferase
activity
Atrium 3
Right
Left Atrium n=3
Ventride n=3
for deter-
Left Ventride n=3
20.1 t_ 1.3 18.8 k 1.4
4.9 t 0.6 4.9 2 0.7
7.7 + 2.0 6.7 k 1.2
2.3 +_ 0.2 2.2 2 0.3
0.88
0.01
0.40
0.31
Values are means 2 SE, expressed in nmol 8-I *minP. Control specimens were placed in liquid nitrogen immediately. Stored specimens were kept in Ringer lactate at 4°C for 45 min before being placed in liquid nitrogen. These specimens represented aliquots of the same hearts after samples were obtained, cut into small pieces, and the pieces mixed thoroughly. The interval in solution did not alter the activity of choline acetyltransferase in tissues. l
noted in choline acetyltransferase activity after storage (Table I). Histological evaluation of the six beating transplanted hearts revealed that myocardial cells were in good condition, with only limited areas of ischemic changes. Focal areas of acute epicardial necrosis with minimal subepicardial changes were found, but these areas were limited to the subepicardial muscle; the myocardium and endocardium were normal. The morphology of the transplanted hearts did not differ from the host hearts or normal rat hearts (9). Data on the cholinergic innervation and the regional distribution of CAT activity for host (control) and transplanted hearts is presented in Fig. 2. Control CAT activity was highest in the region of the sinoatrial node (30.8 nmol min-’ 9g-l). Enzyme activity was high also in the right atrium and interatrial septum. Intermediate levels of enzyme activity were found in the left atria1 appendage and in tissue near the conduction pathways, the superior interventricular septum, and the moderator band and the papillary muscle in the anterior right ventricle. Lower levels of CAT activity occurred in tissues more remote from the conduction pathways, including the inferior interventricular septum and the left ventricle. In contrast to the host heart, which had nonuniform CAT activity, the transplanted hearts had uniformly low CAT activity in all the regions that were examined (Fig. 2). l
RESULTS
of heart illustrating acetyltransferase
minations
ET AL.
PARASYMPATHETIC
THE
IV Septum
186 r20
IA Sept
Ap
Right
H369
HEAR?
1
8
RA
OF
30 8 ?5 0
I Atrium
rl
INNERVATION
Ventricle
LA
Ap
a
Control
q
Transplant FIG. 2. Choline acetyltransferase activity in atria, interventricular septum, and ventricles in control and transplanted rat hearts. RA Ap, right atria1 appendage; SA Node, sinoatrial node; IA Sept, interatrial septum; LA Ap, left atria1 appendage; Pap Mus, papillary muscle; Sup, superior; Inf, inferior; Post, posterior; Ant, anterior. Numbers represent means * SE, expressed as nmol-rnin-‘.g-l.
0
Sup Ant
Left
SupPost
Inf
Ant
Id
Post
Vent ride
50 ?O 5
38
,rppJ+Jk ant
oLL Post
Pap Mus
To obtain an estimate of the sympathetic innervation, total catecholamine content was determined by a modification of the Anton-Sayre alumina-trihydroxyindole procedure (10). Mean values declined from 780 t 80 ngn g-l in the normal hearts to 90 t 10 ng* g-l, indicating a substantial reduction of this neurotransmitter. DISCUSSION
These studies demonstrate that choline acetyltransferase activity is distributed nonuniformly in the innervated host rat heart. The distribution of CAT activity in rat heart follows the same relative pattern as in the guinea pig heart (15, 17, 19) and is similar to the regional pattern of acetylcholine concentrations found in the rat (18) and the cat (2). Surgical removal of the heart severs all nerves to it and results in degeneration of all fibers of extrinsic origin (preganglionic parasympathetic neurons). To document degeneration of the extrinsic (preganglionic) innervation, we confirmed that choline acetyltransferase activity became undetectable in the distal portion of sectioned vagus nerves in rats at 4 and 8 days after vagotomy (unpublished results). The intrinsic (postganglionic) neurons, on the other hand, should have remained intact (11). In the present study, total myocardial denervation was confirmed by the marked decrease in the total catecholamine content. The small amount of residual catecholamines probably represents nonspecific binding of circulating catecholamines (4, 12). Furthermore, histological evidence suggested the myocardial cells were unaltered, yet the choline acetyltransferase activity decreased significantly. The magnitude of our values should not have been changed by factors such as the operative procedure and temporary storage in Ringer lactate. No difference in enzyme activity was noted in hearts removed immediately and hearts that had been stored in 4°C Ringer lactate solution before transplantation. Thus, a reduc-
tion in enzyme activity did not result from the surgical procedure. Myocardial alterations are an unlikely cause of the changes because no fiber atrophy was noted upon histological examination. In addition, no differences were found between the control and transplanted heart weights. The transplanted heart does not function exactly like the normal heart in terms of cardiac output, but the left ventricle does eject small quantities of blood against a normal arterial systemic pressure. The working state thus is maintained. Although the work load of the transDlanted hearts is decreased, 8 days in this state does lnot appear to be sufficient for detectable atrophy to develop. Tissue rejection would not be expected since the donors and recipients were from identical strains (isographs). Transplanted (isograph) rat hearts similar to the present model have survived as long as 290 days with relatively normal electrocardiograms and no further histupathic changes (1). Therefore, from the normal histology, weights, water content, and the expected maintenance of a working state, we conclude that the hearts, although transplanted, remain viable and reasonably normal. In previous studies, we have found choline acetyltransferase activity in intact innervated neonatal rat hearts, but not in myocardial cell tissue cultures derived from these hearts (16). The present data, along with the previous experiments, indicates that choline acetyltransferase has a neuronal rather than a myocardial origin. The present data on choline acetyltransferase activity suggest a rather uniform postganglionic parasympathetic innervation throughout the transplanted heart. This result conforms with the recently published data on uniform functional responses to the activation of cholinergic neurons in denervated and transplanted canine hearts (14). The differences in choline acetyltransferase activity between the transplanted and host rat hearts varied
H370 from region to region. This suggests strongly that the preganglionic parasympathetic innervation of the rat heart is nonuniform. In the atria, the greater than 90% decrease in choline acetyltransferase activity suggests that substantial enzyme activity may be associated with preganglionic neurons. In the left and right ventricles, on the other hand, the 50-60% decline in CAT after transplantation suggests that relatively less enzyme activity may have been associated with preganglionic fibers. I In agreement with our conclusion that the preganglionic parasympathetic innervation is nonuniform, Brown (2) found, in bilaterally vagotomized cats, a variable reduction in acetylcholine in the right atrium, sinoatrial node, papillary muscle, and right ventricle, but no change in left atrium, interventricular septum, or left ventricle. The minor differences between results of the present study and that of Brown (2) may be attributed to species specificity (rat vs. cat), methodology (transplant vs. bilateral vagotomy), or the component measured (choline acetyltransferase vs. acetylcholine). We considered the possibility that the preganglionic parasympathetic innervation to the rat heart might exert a trophic influence on enzyme activity in the postganglionic neurons (13). If this were true, a portion of the observed reduction in CAT activity could have been associated with atrophic changes in the postganglionic innervation. Such a decrease would lead to an overestimation of the preganglionic component of the parasympathetic system. We cannot exclude this possibility in the rat heart. However, the possibility of trophic influences should not detract from the major conclusion that the preganglionic parasympathetic innervation to the rat heart is nonuniform. It is possible that a large fraction of CAT in control hearts is derived from the intrinsic innervation that is maintained by a trophic influence of the preganglionic (extrinsic) innervation. If this is true, CAT activity after denervation (transplantation) might decline in the absence of a trophic influence from the extrinsic innervation. It is therefore possible that CAT activity in the intrinsic parasympathetic system of control hearts may differ from that observed in the transplanted hearts. We cannot exclude this possibility in
LUND
ET AL.
the rat heart. In the transplanted hearts in this study, however, the interruption of extrinsic innervation and cessation of possible trophic influences produces a uniform pattern of CAT activity which presumably represents the nonstimulated postganglionic parasympathetic innervation. In summary, these experiments demonstrate an intervention that successfully alters heart choline acetyltransferase activity in a predictable manner. Cardiac transplantation and earlier work in our laboratory indicate that choline acetyltransferase activity is a reliable marker for the parasympathetic innervation of the heart (16). The extrinsic parasympathetic innervation to the heart (preganglionic component) is nonuniform and supplies predominantly the specialized regions with conduction tissue. In contrast, the nonstimulated intrinsic innervation (postganglionic) is rather uniformly distributed to all the regions of the transplanted heart and comprises 50% or less of the total parasympathetic innervation of any heart region. These biochemical results are somewhat at variance with the reported absence of histological evidence for cholinergic ganglia in rat ventricles (7, 21). It is possible that the biochemical criterion of cholinergic innervation is the more sensitive. However, these results parallel the physiological findings that reflex activation of the parasympathetic system regulates heart rate and to a lesser extent the myocardial contractile state (5, 6, 8). Also, in the present study the direct evidence of a uniform postganglionic parasympathetic innervation to the heart parallels the physiological findings that activation of the intrinsic innervation ramacologically tends to produce uniform contracPha tile responses in atr 1a and ventricles (14). We thank Albert0 R. Subieta and Robert P. Oda for technical assistance, and Barbara Nolte and Carol Huffman for secretarial assistance. This study was supported by Program Project Grant HL-014388, Public Health Service Grant HL-20768, and Grant MRIS-7737.03 from the Veterans Administration. The work was done during the tenure of Institutional Research Fellowship HL-07121 and Special National Institutes of Health Postdoctoral Fellowship HL-05529 for D, D. Lund. Received
16 January
1978; accepted
in final
form
18 May
1978.
REFERENCES 1. ABBOTT, C. P., W. E. DEWITT, AND 0. CREECH, JR. The transplanted rat heart: histologic and electrocardiographic changes. Trnnsplantation 3: 432-445, 1965. 2. BROWN, 0. M. Cat heart acetylcholine: structural proof and distribution. Am. J. Physiol. 231: 781-785, 1976. 3. CORRY, R. M., AND S. E. KELLEY. Immunological enhancement of primarily vascularized rat heart xenotransplants in mice. Transplantation 18: 503-507, 1974. 4. GEIS, W. P., C. J. TAT~~LS, M. P. KAYES, AND W. C. RANDALL. Complete cardiac denervation without transplantation: a simple and reliable technique. J. Appl. Physiol. 30: 289-293, 1971. 5. GELBAND, H., R. J. MYERBURG, B. F. HOFFMAN, AND A. L. BASSETT. Acetylcholine-induced reversal of canine and feline atria1 myocardial depression during stretch, cardiac failure, and drug toxicity. Circulation Res. 37: 542-549, 19%. 6. HIGGINS, C. B., S. F. VATNER, AND E. BRAUNWALD. Parasympathetic control of the heart. Pharmacol. Reu. 25: 119-155, 1973.
7. JACOBOWITZ, D. Histochemical studies of the relationship of chromaffm cells and adrenergic nerve fibers to the cardiac ganglia of several species. J. Pharmacol. ExptZ. Therap. 158: 227-240, 1967. 8. KENT, D. M., S. E. EPSTEIN, T. COOPER, AND D. M. JACOBOWITZ. Cholinergic innervation of canine and human ventricular conduction system: anatomic and electrophysiologic correlations. Circulation 50: 948-955, 1974. 9. LUND, D. D., R. J. TOMANEK, AND J. W. DAVIS. Cellular and subcellular basis of cardiac hypertrophy in the spontaneously hypertensive rat. In: Spontaneous ffypertension: Its Pathogenesis and Complications, edited by K. Okomoto and F. M. Bumpus. Washington, D. C.: US Gov. Printing Office, 1977, vol. II, p. 41-50. 10. MAYER, H. E., F. M. ABBOUD, D. R. BALLARD, AND J. W. ECKSTEIN. Catechol amines in arteries and veins of the foreleg of the dog. Circulation Res. 23: 653-661, 1968.
PARASYMPATHETIC
INNERVATION
OF
THE
H371
HEART
11. NAPOLITANO, L. M., V. L. WILLIAM, C. R. HANLON, AND T. COOPER. Intrinsic innervation of the heart. Am. J. Physiol. 208: 455-458, 1965, 12. O’KEEFE, D. D., R. T. LEFKOWITZ, R. N. GRANTHAM, E. A. BEIERHOLM, AND W. M. DAGGETT. Absence of effect of cardiac denervation and induced right ventricular failure on canine myocardial norepinephrine binding sites. Cardiovascular Res. 9: 219-223, 1975. 13. OSECH, F. Trans-synaptic induction of choline acetyltransferase in the preganglionic neuron of the peripheral sympathetic nervous system. J. PharmacoZ. Exptl. Therap. 188: 439-446, 1974. 14. PRIOLA, D. V., H. A, SPURGEON, AND W. P. GELS. The intrinsic innervation of the canine heart: a functional study. Circulation Res. 40:50-56, 1977. 15. ROSKOSKI, R,, JR., H. E. MAYER, AND P. G. SCHMID. Choline acetyltransferase activity in guinea pig heart in vitro. J. Neurothem. 23: 1197-1200, 1974.
16.
17.
18.
19.
20.
21.
JR., R. J. MCDONALD, L. M. ROSKOSKI, W. J. K. HERMSMEYER. Choline acetyltransferase activity in heart: evidence for neuronal and not myocardial origin. Am. J. Physiol. 233: H642-H646, 1977 or Am. J. Physiol.: Heart Circ. PhysioZ. 2: H642-H646, 1977. ROSKOSKI, R., JR., P. G. SCHMID, H. E. MAYER, AND F. M. ABBOUD. In vitro acetylcholine biosynthesis in normal and failing guinea pig hearts. Circulation Res. 36: 547-552, 1975. ROTHSCHUH, D. C. Das herzmuskeleigene Acetylcholin. PfZuegers Arch. 258: 481-488, 1954. SCHMID, P. G., B. J. GREIF, D. D. LUND, AND R. ROSKOSKI, JR. Regional choline acetyltransferase activity in the guinea pig heart. CircuZation Res. 42: 547-660, 1978. STEEL, R. B. D., AND J. H. TORRIE. Principles and Procedures of Statistics. New York: McGraw, 1960, p. 67-87. TRUEX, R. C. Chromaffm tissue of the sympathetic ganglia and the heart. Anat. Record 108: 687-697, 1950. ROSKOSKI, MARVIN,
R.,
AND
