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534
Sympathetic Innervation
Alters Growth and Intrinsic Heart
Rate of Fetal Rat Atria Maturing in Oculo
Diane C. Tucker and Richard Gist
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The influence of sympathetic innervation on the growth and intrinsic rate of beating established by
fetal rat heart was studied by culturing fetal atrial tissue in sympathetically innervated and denervated
anterior eye chambers of adult Sprague-Dawley rats. One anterior eye chamber in each host rat was
sympathetically denervated by removing the ipsilateral superior cervical ganglion. In oculo, atrial
grafts were vascularized by blood vessels sprouting from the his and innervated by sympathetic and
parasy mpathetic fibers from the ground plexus of the iris. Innervation was assessed by light-activated
efferent nerve stimulation to the grafts that changed their rates of beating. The norepinephrine
contents of 16 atria cultured for 2.5 months in sympathetically innervated and denervated eye
chambers were 5.7 ± 1.1 ng/implantvs. 0.2 ± 0.07 ng/implant (mean ± SEM), indicating permanent
sympathetic denervation of the anterior eye chamber and the implanted atria. By 8 weeks in oculo,
atria maturing in sympathetically innervated anterior eye chambers were 86% larger than those hi
denervated eye chambers (2.22 ± 0.29 vs. 1.19 ± 0.13 mm2); the weight of innervated transplants was
over 3 tunes that of noninnervated grafts (2.35 ± 0.75 vs. 0.76 ± 0.21 mg). After implanted atria had
ceased growing rapidly (2.5 months in oculo), bipolar electrodes were implanted adjacent to the
cornea to record impulses from atrial grafts while host rats were unanesthetized. The dark-adapted
baseline heart rates of sympathetically innervated and noninnervated atria were virtually identical
(289 vs. 290 bpm). Graft intrinsic heart rate was estimated by combined /3-adrenergic and muscarinic
receptor blockade with atenolol (1.0 mg/kg) and methylatropine (10 /xg/kg). Sympathetically innervated transplants had lower intrinsic heart rates than noninnervated atria (134 ± 25 vs. 213 ± 12 bpm).
These data suggest that sympathetic innervation of the developing heart influences both growth and
intrinsic rate of beating. (Circulation Research 1986;59:534-544)
C
ONTROVERSY exists over the role of sympathetic stimulation in maturation of the heart.
Individual differences in tonic sympathetic
stimulation of the heart are observed in newborn animals and may contribute to subsequent differences in
heart size and function1"3 (and Clubb et al, unpublished
manuscript). Since cardiac function and autonomic innervation each mature rapidly during fetal and perinatal development, it is difficult to separate inherent
from innervation-induced changes in the heart. In vivo
and in vitro experiments suggest that sympathetic stimulation exerts a trophic effect on the heart and alters its
intrinsic pacemaker activity. ' ^ However, other investigations argue against this hypothesis.10"18 Because
sympathetic innervation of the heart occurs prior to
birth,19"21 resolution of this controversy requires a developmental model in which sympathetic innervation
of the heart can be manipulated beginning early in
cardiac development and in which direct consequences
of sympathetic innervation can be observed. To this
From the Department of Psychology (D.C.T.) and Cardiovascular Research and Training Center (R.G.), University of Alabama at
Birmingham, Birmingham, Alabama.
Supported in part by a grant-in-aid from the American Heart
Association, Alabama Affiliate (633786) and a Faculty Reserach
Grant from the University of Alabama at Birmingham (2-12715).
Address for reprints: Diane C. Tucker, PhD, Department of
Psychology, Campbell Hall, University of Alabama at Birmingham, Birmingham, AL 35294.
Received April 4, 1986; accepted July 21, 1986.
end, we compared the growth and intrinsic rate of
beating of fetal rat heart tissue cultured in rat anterior
eye chambers that were sympathetically innervated or
denervated.
Sympathetic influences on cardiac growth depend
on the developmental stage of the heart. In fetal and
newborn rodents, increases in cell number contribute
to cardiac growth. During the second postnatal week,
cardiac cells stop dividing, become binucleated, and
the heart begins to grow by cellular hypertrophy.22 The
transition of the heart to growth by hypertrophy coincides with functional maturation of sympathetic cardiac innervation.23>24 /3-Adrenergic receptor stimulation
of newborn rat hearts through a bolus injection of isoproterenol inhibits further cell division and DNA synthesis,4 suggesting that catecholamine stimulation of
the developing heart may influence its mechanism of
growth. In hearts which have made the transition to
growth by hypertrophy, isoproterenol treatment results
in cardiac hypertrophy which is mediated by an increase in cell size.23 In young rabbits whose hearts are
presumably postmitotic, chronic blockade of )3-adrenergic receptors inhibited cardiac growth. In vitro,
nondividing cells isolated from neonatal hearts showed
hypertrophy when incubated widi )3-adrenergic7 or aadrenergic receptor agonists.
Sympathetic stimulation of developing heart is also
reported to influence cardiac function. Nayler and colleagues8 found that prolonged )3-adrenergic receptor
blockade resulted in & faster pacemaker rate in young
Tucker and Gist
Innervation of Rat Atria in Oculo
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rabbit hearts, as measured in vitro while perfusing with
the Langendorff technique. Ishii and colleagues5 reported that immunological or chemical sympathectomy of neonatal rats caused postjunctional inotropic
supersensitivity to norepinephrine in both atrial and
ventricular myocardium. Beating of quiescent myocytes isolated from newborn rat ventricle was induced
by combined /3-adrenergic and a-adrenergic receptor
stimulation.7 Thus, in vivo and in vitro experiments
suggest that sympathetic innervation may influence
both the growth and function of the developing heart.
In contrast, other investigators suggest that inherent
developmental processes determine the growth and
functional characteristics of the young heart. Kirby
and Stewart14 produced "sympathetically aneural
hearts" by ablating the portion of the neural crest containing presumptive sympathetic neurons in embryonic
chicks and observed normal cardiac morphology.
Similarly, destruction of sympathetic innervation of
the embryonic chick heart by injection of either reserpine or 6-hydroxydopamine in ovo altered neither the
subsequent growth of the heart in ovo nor the developmental changes in its inotropic sensitivity to /3-adrenergic receptor stimulation.15 Postnatal sympathectomy
of rats with either 6-hydroxydopamine or antiserum to
nerve growth factor did not prevent cardiac hypertrophy in two strains of genetically hypertensive rats
(i.e., SHR,
New Zealand G H R ) . These data argue
against a role for sympathetic innervation in determining the growth of the young heart.
Developmental changes in intrinsic rate of beating
have been argued to be inherent to the maturing heart.
Heart rate increases gradually during fetal and early
postnatal development.1011 Because neither acute electrical nor pharmacological manipulations altered heart
rate significantly during this period, Adolph" concluded that this gradual increase in the resting heart rate of
fetal and neonatal rodents was due to changes to "intrinsic controls" rather than autonomic influences. Vlk
and Vincenzi12 corroborated this observation through
in vitro study of hearts isolated from newborn, 1-weekold and adult mammals (i.e., rats, guinea pigs, and
rabbits). Wekstein13 reported that the baseline heart
rate of 1-week-old rats was not altered by treatment
with antiserum to nerve growth factor or by chronic
blockade of sympathetic influences with reserpine.
These data support the hypothesis that changes in the
pacemaker rate of the heart during early development
are independent of sympathetic stimulation.
Catecholamine fibers innervate the heart prior to
birth. In the rat, catecholamine-synthesizing enzymes
are first detected in the primordia of the thoracic sympathetic ganglia of rat embryos on the 11th day of
gestation (i.e., 12 days postconception, 6-mm crownto-rump length26). Catecholamine-induced fluorescence was observed by de Champlain and colleagues19
in the stellate ganglia of 13-day postconception rat
embryos and fluorescent axon bundles were visualized
in the heart at 18 days after conception.20 Thus, the rat
heart is not innervated by sympathetic neurons prior to
12-13 days after conception.
535
To test the hypothesis that sympathetic innervation
influences the growth and intrinsic rate of beating of
the developing mammalian heart, it is necessary to
manipulate sympathetic innervation of the heart beginning in midgestation. We tested this hypothesis by
culturing heart tissue dissected prior to innervation in
situ in the anterior eye chamber of a host rat.
Culture of heart tissue in oculo has several advantages over alternative methods. While fetal hearts cultured in vitro steadily lose weight and are a "failing
preparation,"27"29 embryonic or fetal hearts cultured in
oculo become vascularized by vessels from the ground
plexus of the iris, grow and continue to beat for over a
year.30"33 Sympathetic and parasympathetic fibers that
normally control pupil size sprout and innervate the
transplanted fetal heart. One anterior eye chamber can
be sympathetically denervated in host rats by ipsilateral superior cervical ganglionectomy. Using this procedure, we were able to compare the growth and controls of pacemaker activity of fetal hearts innervated by
the sympathetic nervous system with those never innervated. As the in oculo heart does not pump against a
pressure gradient, hemodynamic consequences of
manipulating sympathetic innervation during development do not confound interpretation of the data.
The present study examined two questions. (1) Does
sympathetic innervation exert a trophic effect on the
growth of fetal heart tissue? (2) Does sympathetic innervation influence the intrinsic rate of beating established by the pacemaker of fetal heart tissue maturing
in oculo?
Materials and Methods
Heart tissue was dissected from Sprague-Dawley
rats at 12 days postconception. Dams and sires were
purchased from Taconic Farms and mated in our laboratory. Successful mating (day 0 of gestation) was
judged by the presence of a vaginal plug. Host rats
were 34 4-week-old Sprague-Dawley males (Taconic
Farms). All rats were maintained on a 12-hour lightdark cycle.
SYMPATHETIC DENERVATION OF THE ANTERIOR EYE
CHAMBER. Two days prior to implantation of heart
tissue, one anterior eye chamber of each host rat was
sympathetically denervated by removal of the ipsilateral superior cervical ganglion (SCG). Both the preganglionic and postganglionic nerve trunks were severed and the entire ganglion was removed to prevent
regrowth of sympathetic fibers into the anterior eye
chamber. Other targets of the SCG receive bilateral
innervation (e.g., pineal and thyroid glands) and unilateral superior cervical ganglionectomy does not compromise their function.34
DISSECTION AND IMPLANTATION OF ATRIAL TISSUE.
At 12 days postconception, both uterine horns were
removed aseptically from 8 ether-anesthetized dams.
Dissections were done in a sterile Tris-Tyrode's solution (pH 7.40) at room temperature.. The beating atria
were dissected from each fetus (10-12 fetuses per
dam) using an Aus Jena surgical microscope. Measurements of fetal crown-to-rump length, heart size,
536
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and atrial size were made using a calibrated micrometer in the microscope eyepiece. Crown-to-rump length
of fetuses from the 8 litters ranged from 5.9 to 8.4 mm
(mean 6.3 ± 0.3 mm). Fetuses had both forelimb and
hindlimb buds. Hearts were four-chambered and 1.6
± 0.09 mm2 in size; heart size was measured as the
product of length and width.
Atria rather than whole fetal hearts were cultured to
avoid confusing slow pacemaker activity by the sinoatrial node with rates controlled by the atrioventricular
node. In a pilot study, 7 whole fetal hearts and 7 atria
were cultured in the same host rats. While whole hearts
were larger than atria after 6 weeks in oculo (3.61 ±
0.50 vs. 1.73 ± 0.22 mm2; mean ± SEM), the rate of
beating did not differ (219 ± 45 vs. 270 ± 28 bpm),
suggesting that chronotropic characteristics of atrial
grafts reproduce those of the whole fetal heart grafts.
Prior to implantation of fetal atria, host rats were
anesthetized with ether and the iris was constricted by
topical application of methylatropine (1.0 mg/ml) to
the cornea. Implantation was accomplished by gently
drawing spontaneously beating atrial tissue into a beveled pipette and injecting it into the anterior eye chamber of a host rat through a small cut made in the cornea.
Atrial tissue rapidly adhered to the iris over which it
was positioned. Neosporin ophthalmic ointment was
placed on the eye after surgery to prevent infection.
Healing of the cornea began almost immediately, and
no scar was evident within a week in most cases. The
behavioral competence of the host rat was not compromised by the implantation of heart tissue into the anterior eye chamber and there was no evidence of chronic
irritation (e.g., evidence of excessive grooming or
scratching around the eyes).
MEASUREMENT OF TRANSPLANT GROWTH. The size of
transplants was measured weekly using a calibrated
micrometer in the surgical microscope. In oculo, atria
assume a round to oval shape. The two-dimensional
area of transplants was calculated as the product of the
longest dimension of the transplant and its width perpendicular to this axis. Atria cultured in the sympathetically innervated and denervated anterior eye
chambers of 8 host rats were weighed after 3 months in
oculo to determine differences in mass.
MEASUREMENT OF RATE OF BEATING. During weekly
size measurements, the rate of beating was counted by
visual observation. Rate was calculated from the time
required for 20 sequential contractions.
After 2.5 months in oculo, electrodes were implanted to allow recording of electrograms from implanted
atria while the host was awake and freely moving.
Specifically, two fine silver wires (0.003 in. diam.
enamel-coated wire) were tunnelled through the conjunctiva and the bared tips positioned near the edge of
the comea on opposite sides of the beating atrial tissue.
Neosporin ophthalmic ointment was applied to the
conjunctiva and under the eyelid after surgery to prevent infection. Host rats adjusted well to these recording electrodes and gained weight at a normal rate.
Evidence of irritation around the eye after implantation
of recording electrodes was rare and easily corrected
Circulation Research
Vol 59, No 5, November 1986
by repositioning of the tips of the wires. Prior to surgery the wires were soldered to male Amphenol microconnectors. The microconnectors were fixed in a plastic spacing bar which was anchored permanently to the
animal's skull. Heart rate of the host was measured
from two additional wires tunnelled under the skin of
the back. Recordings of ECG from both implanted
hearts and the host were made by attaching recording
leads soldered to female Amphenol connectors and
placing the unrestrained host into a light-shielded
chamber.-Signals were filtered and amplified using
Grass P511 preamplifiers and high-impedence input
probes and then fed into Beckman Dynograph (R511)
cardiotachometers. Example recordings are presented
in Figure 1.
MEASUREMENT OF AUTONOMIC CONTROLS AND INTRINSIC HEART RATE. Table 1 presents the protocol for
determining autonomic controls of in oculo heart rate,
maximal and intrinsic heart rate. In preparation for
testing, recording leads were attached, host rats were
placed in an individual cage containing shavings from
their home cage and then adapted to a darkened test
chamber for 20 minutes. Baseline in oculo heart rates
were collected from dark-adapted host animals. Heart
rate responses to the onset and offset of a 5-second
light stimulus (40-foot candles) were then measured.
Pronounced bradycardia (greater than 150 bpm fall) in
response to light onset was observed from in oculo
atria but not from the host rat, verifying that the electrograms recorded from in oculo heart leads originated
from the transplanted heart and not the host heart. The
light stimulus was repeated once during the baseline
period and during subsequent pharmacological treatments. Failure to observe light-induced bradycardia
after methylatropine treatment confirmed successful
blockade of muscarinic receptors. Drugs were administered via a subcutaneous catheter. The intrinsic heart
rate recording protocol comprised sequential pharmacological blockade of parasympathetic influence with
atropine methyl nitrate (10 pig/kg) and sympathetic
influence with atenolol (1.0 mg/kg). Dose-response
curves were generated for each drug during preliminary studies; these doses were selected as the lowest
which produced a maximal blockade to challenge with
agonists.
Maximal heart rate response to /3-adrenergic receptor stimulation was determined by administering isoproterenol sulfate (1.0 ^tg/kg). Determination of maximal heart rate was separated from the intrinsic heart
rate protocol by 2-3 days to assure clearance of the
receptor blockers from the host rat.
CATECHOLAMINE CONTENT OF IMPLANTED ATRIA. TO
confirm prevention of sympathetic innervation of atria
implanted into sympathetically denervated anterior eye
chambers, norepinephrine (NE) and epinephrine (E)
content of implanted atria were measured by high pressure liquid chromatography with electrochemical detection. Host rats were killed by decapitation and the
eyes rapidly removed. The implanted atria were dissected from the anterior eye chamber in ice cold saline,
the iris was trimmed from around the graft, and the
537
Tucker and Gist Innervation of Rat Atria in Oculo
Implanted
Atria
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0.5 sec
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Baseline
Light on
Light off
implanted atria were placed in a microcentrifuge tube
and frozen in liquid nitrogen. Implants from both eyes
were dissected simultaneously.
Tissue samples were weighed on a Metier H54 AR
analytical balance and placed in a microhomogenizer
(Kontes Glassware, Vineland, N.J.). The tissue was
homogenized in 400 fil of 0.1 M perchloric acid containing 0.5 mM glutathione as a antioxidant and 5
ng/ml 3,4,-dihydroxybenzylamine (DHBA; Sigma
Chemical, St. Louis, Mo.) as an internal standard.
Protein determination was by a modification of the
FIGURE 1. Panel A shows a line drawing of heart
tissue grafted into the anterior eye chamber. Angiogenesis is evident in the iris and implant. Panel B
presents ECG tracings recorded simultaneously from
the host rat (4-25 R2) and from atria grafted into his
right and left eyes. The heart rate of the host was 360
bpm and the rates of the transplants were 240 and 260
bpm, respectively. These recordings were made 2 min
after TV injection of 1.0 fug/kg methylatropine. Panel
C illustrates the heart rate change observed in atria
implanted in oculo with changes in ambient light (Rat
SD-9). The dark-adapted baseline of the implanted
atria was 264 bpm; when the chamber light was
turned on it dropped to 96 bpm, returning to baseline
when the light was turned off. Pharmacological interventions indicate that the "light-on" response is
largely parasympathetic while the "light-off' response has both sympathetic and parasympathetic
components.
1 me
method of Bradford35 from 100 fil of the homogenate.
The remaining homogenate was centrifuged at 12,000
rpm for 10 minutes in an Eppendorf 5414 centrifuge.
Supernatant (200 /xl) was added to 1 ml of 0.1 M
phosphate buffer (pH 7.0) and 50 mg of washed alumina (BioAnalytical Systems, West Lafayette, Ind.).
Tris buffer (1 ml of 1.5 M, pH 8.6) was added, the
tubes were capped and shaken by hand for 10 minutes.
After the alumina had settled, the liquid was aspirated
off and the alumina was washed with distilled water
three times. The alumina was transferred to micro-
Table 1. Protocols for Determining Intrinsic and Maximal Heart Rate
Heart rate
Intrinsic
I. Dark-adapted baseline heart rate
II.
Light stimulus (5-second pulse,
repeated once)
Muscarinic receptor blockade
(10 /xg/kg methylatropine s.c.)
Light stimulus
HI. /3-adrenergic receptor blockade
(1.0 mg/kg atenolol s.c.)
Light stimulus
Maximal
I. Dark-adapted baseline
II.
Isoproterenol
(1.0
Interpretation
Rate under conditions of high
sympathetic tone
Functional innervation
Tonic parasympathetic control
Infer parasympathetic component
of response to light stimulus
Tonic sympathetic control and
intrinsic heart rate
Infer sympathetic component of
response to light stimulus
Rate under conditions of high
sympathetic tone
Maximum heart rate to direct
/3-adrenergic receptor stimulation
Circulation Research
538
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filters (BioAnalytical Systems) and centrifuged to remove the water. A new recovery cap was placed on the
filter and 200 /xl of 0.1 M perchloric acid added to the
alumina and vortexed. The acid/alumina slurry was
allowed to stand for 5 minutes, vortexed again and
centrifuged at 3000 rpm for 5 minutes. A 50 \x\ sample
of the acidic extract was injected into the HPLC system. The HPLC system consisted of an LDC minipump (Milton Roy, Rivera Beach, Fla.), an IBM C-18
column (IBM Instruments, Danbury, Conn.), LC4B
electrochemical detector (BioAnalytic Systems) and a
Hewlett Packard HP3390A integrator (Hewlett-Packard Instruments, Atlanta, Ga.). Mobile phase conditions were 0.02 M citrate/0.02 M Na2H2PO4/acetonitrile (650:300:40) pH 3.75 with 1.5 mM TEA and
octyle sodium sulfate 115 mg/L. All chemicals were
analytical grade (Sigma Chemical, St. Louis, Mo.).
DATA ANALYSIS. Developmental changes in the
transplant size and rate were analyzed using multivariate profile analysis. Univariate analyses of variance
were used to follow up significant multivariate results.
The dark-adapted heart rate, light response and response to drug treatments of sympathetically innervated and noninnervated atria were compared by analysis
of variance. Transplant weight and catecholamine content were compared using Student's t test. Follow-up
comparisons were made using Newman-Kuel's tests.
Data are reported throughout as mean ± standard error
(SEM).
Results
OBSERVATIONS OF FETAL ATRIAL TISSUE IN OCULO.
Within 24 hours after implantation, sinuses of blood
were evident on implanted atria, with the tissue surface
becoming well vascularized within 1 week. In oculo,
fetal heart tissue does not grow to the size it would
achieve in situ and does not retain distinct chambers.
Instead blood-filled sinusoids are observed. The coronary arterial system does not develop in oculo. Blood
vessels grow in from the ground plexus of the iris and
are relatively uniform in their distribution.
CATECHOLAMINE CONTENT OF ATRIAL GRAFTS. Fig-
ure 2 presents norepinephrine content of atria cultured
in sympathetically innervated and noninnervated anterior eye chambers. Measurement of norepinephrine
content of atria implanted into sympathetically denervated eye chambers confirmed nondetectable to very
low catecholamine levels (range, 0-0.550 ng/implant,
mean = 0.17 ± 0.07 ng/implant compared to range
1.4-11.9 ng/implant, mean = 5.7 ± 1.1 ng/implant
for innervated implants). As care was taken to remove
the iris not directly adherent to the atrial graft, the
norepinephrine content of atria maturing in sympathetically innervated eye chambers suggests that the
expected ingrowth of sympathetic fibers into the grafted atria did, in fact, occur. Measurable epinephrine
content was found in 2 noninnervated and one innervated implant (1.6 and 1.4 ng/implant in noninnervated and 7.8 ng/implant in innervated implants). The
very low to nondetectable catecholamine content of
noninnervated atrial implants suggests that noninner-
Vol 59, No 5, November 1986
Catecholamine Content of jn_ Oculo Atria
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FIGURE 2. Norepinephrine (NE) content of fetal rat atrial
grafts cultured in intact and in sympathetically denervated anterior eye chambers is compared. Data are reported inpicograms
of norepinephrine per implant (H = 8 implants per group). The
relatively high NE content of atrial grafts into sympathetically
innervated and denervated eye chambers suggests that sympathetic innervation of the implants did occur, while the very low
to nondetectable NE levels in grafts into denervated eye chambers indicates that the sympathetic denervation was permanent
[t (14) = 5.0, p < 0.001 for differences in NE content].
vated implants do not sequester catecholamines from
the circulation. This finding is consistent with Potter's
report3* that the capability of denervated myocardium
to take up and store catecholamines is minimal (6% of
capacity of innervated myocardium).
GROWTH OF ATRIA MATURING IN OCULO. Sympathetically innervated grafts showed significantly greater
growth than grafts into denervated eye chambers [F (3,
22) = 5.25, p < 0.007]. Subsequent analyses indicated differential growth between weeks 2 and 4 and
between weeks 4 and 6. By 8 weeks after implant, atria
maturing in sympathetically innervated anterior eye
chambers were 86% larger than atria maturing in sympathetically denervated eye chambers (2.22 ± 0.29
vs. 1.19 ± 0.13 mm2). Figures 3 and 4 show the
differential growth of atria in sympathetically innervated and noninnervated eye chambers. Because the
implants are three dimensional structures, surface dimensions are likely to underestimate actual differences
in growth as a function of experimental manipulations.
To examine this possibility, implanted atria from 8
host rats were weighed. Sympathetically innervated
atrial grafts weighed three times more than noninnervated atria (2.35 ± 0.75 mg vs. 0.76 ± 0.21 mg).
When viewed through the surgical microscope, sympathetically innervated transplants were dark red in
color and appeared to be thicker than the more ambercolored noninnervated transplants.
RATE OF ATRIA MATURING IN OCULO. Atria maturing
in sympathetically noninnervated eye chambers maintained a higher rate than innervated transplants when
measurements were made with the host rats anesthetized with ether and using the white light source on the
surgical microscope (see Figure 5). These measurement conditions produce high parasympathetic tone to
the anterior chamber (white light) and high levels of
539
Tucker and Gist Innervation of Rat Atria in Oculo
Growth of Atria In Oculo
Effect of Sympathetic Innervation
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FIGURE 3. Effects of sympathetic innervation on growth of
fetal rat atria transplanted into the anterior chamber of the eye.
Atria placed into anterior chambers denervated by removal of
the ipsilateral superior cervical ganglion did not differ in size at
the time of implantation (i.e., they were from the same litter of
fetuses and implanted at the same time), yet by 6 weeks in oculo
atrial grafts into sympathetically innervated anterior eye chambers had more than doubled in size, while atria in denervated
eyes failed to grow (t (55) = 2.3, p < 0.05). Data are mean ±
SEM.
circulating catecholamines (ether anesthesia). Thus,
the higher basal rates in sympathetically noninnervated
atria could reflect an increased intrinsic heart rate,
reduced parasympathetic tone and/or an increased sensitivity to circulating catecholamines in sympathetically noninnervated transplants.
After two months in oculo, intrinsic heart rate and
autonomic controls were estimated from ether-anesthetized hosts using white microscope light (Figure 6).
Parasympathetic tone was estimated by the heart rate
increase after intraperitoneal injection of atropine
methyl nitrate (10 pig/kg). Sympathetic tone was estimated by the heart rate decrease after injection of aten-
olol (1.0 mg/kg). Intrinsic heart rate was estimated
after combined muscarinic and /3-adrenergic receptor
blockade. There was no evidence of reduced functional parasympathetic control in sympathetically noninnervated transplants. Instead, the intrinsic heart rate of
transplants not receiving sympathetic innervation was
increased (167 ± 6 vs. 111 ± 18 bpm, p < 0.01). A
possible enhanced sensitivity to circulating catecholamines was suggested by the significant fall in heart rate
after blockade of /3-adrenergic receptors with atenolol
in both sympathetically innervated and denervated
transplants ( —30 ± 13 and - 5 8 ± 10 bpm, respectively).
To obviate concerns about the effects of anesthesia
of the host and measurement under white light, recordings of heart rate were also made from chronic
recording electrodes. On the first day of testing, a
dark-adapted baseline heart rate was obtained simultaneously from atria implanted into sympathetically innervated and denervated eye chambers in each host.
Baseline heart rates of sympathetically innervated and
noninnervated atria were virtually identical (289 vs.
290, see Figure 7). Adaptation to a darkened chamber
produces high sympathetic and low parasympathetic
tone to the anterior eye chamber.
A profound bradycardia after presentation of a light
stimulus (— 205 ± 33 bpm for sympathetically innervated and — 207 ± 35 bpm for noninnervated transplants) confirmed that electrograms were from in oculo
atria rather than the host rat. No significant change in
rate of the in oculo atria was observed after atropine
treatment (see Figure 7). This result was expected,
given the low parasympathetic tone to the anterior
chamber when host rats are adapted to a darkened
environment. However, muscarinic blockade attenuated substantially the bradycardic response to light stimulation (to — 40 ± 16 for sympathetically innervated
and —38 ± 19 for noninnervated transplants). Substantial sympathetic control of in oculo atria when host
rats are in a darkened environment was confirmed by
B
FIGURE 4. Photographs of sympathetically innervated (Panel A) and noninnervated (Panel B) atrial graphs that illustrate the
difference in size observed as a function of sympathetic innervation.
Circulation Research
540
Vol 59, No 5, November 1986
Controls of i n Oculo Heart Rate
Heart Rate of In Oculo Atria
Ether Anesthetized Host
Freely Moving Host Rats
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Drug Treatment
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FIGURE 5. Effects of sympathetic innervation on rate (mean
± SEM) of atria maturing in sympathetically innervated (N =
34) and denervated (N = 34) anterior eye chambers. Measurements were made using a surgical microscope with host rats
anesthetized with ether. By 6 weeks in oculo atria in sympathetically denervated anterior eye chambers had a significantly
higher rate than innervated implants (t (36) = 3.1, p < 0.01).
the heart rate decrease after /3-adrenergic receptor
blockade with atenolol ( - 1 3 5 ± 16 and - 8 8 ± 15
bpm in innervated and noninnervated transplants). In
noninnervated atrial grafts, the bradycardia in response to atenolol treatment represents functional conIntrinsic Rate and Autonomlc Control
Host AnwtfwttzMl with Eltw
300r
I
220
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• S C O Intact
1 nn
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200SCQ Ramwad
r
3 ioo-
Atroptna
Drug Treatment
FIGURE 6. Effects of sympathetic innervation during development of atria in oculo on autonomic controls and intrinsic rate.
Rates were counted through a surgical microscope while hosts
were anesthetized with ether. The higher baseline heart rate of
sympathetically denervated atria was maintained after both
blockade ofparasympathetic control of in oculo heart rate with
atropine methyl nitrate (10 pg/kg i.p.; t (15) = 3.3, p < 0.005)
and combined blockade with atropine and atenolol (1.0 mglkg,
i.p.; t (17) = 3.1, p < 0.01). The heart rate change after
atropine and atenolol treatments did not differ between sympathetically innervated and noninnervated grafts. These data suggest that atria which are never innervated by the sympathetic
nervous system develop a higher intrinsic beating rate than
sympathetically innervated atria. Data are mean ± SEM.
FIGURE 7. Recordings were made from unanesthetized
Sprague-Dawley hosts adapted to a darkened chamber. (Rates
are mean ± SEM.) Parasympathetic tone was estimated after
treatment of host rats with atropine methyl nitrate (10 ng/kg
s.c.) and sympathetic tone was estimated after injection of atenolol (1.0 mglkg s.c). The intrinsic rate was measured after
combined fi-adrenergic and muscarinic receptor blockade.
While baseline heart rates did not differ between sympathetically innervated and noninnervated transplants, the heart rate
measured after combined blockade was higher in noninnervated
than in sympathetically innervated atria (F (2, 54) = 3.9, p <
0.03).
trol of in oculo heart rate by circulating catecholamines. In contrast, both circulating catecholamines
and high sympathetic neural tone to the anterior eye
chamber contribute to adrenergic stimulation of atria in
sympathetically innervated eye chambers.
The intrinsic heart rates estimated after combined /?adrenergic and muscarinic receptor blockade were 134
± 25 for sympathetically innervated atria and 213 ±
12 for noninnervated atria (p < 0.05; see Figures 7 and
8). This difference suggests that innervation by the
sympathetic nervous system can affect the intrinsic
pacemaker rate established by developing myocardium.
Maximal heart rates estimated after injection of 1
^tg/kg isoproterenol did not differ between sympathetically innervated and noninnervated atrial grafts (i.e.,
317 ± 18 vs. 334 ± 17 bpm; see Figure 8).
Discussion
Sympathetic innervation exerted a trophic effect on
the growth of fetal rat atria cultured in the anterior
chamber of the rat eye and depressed the intrinsic rate
at which the atrial grafts beat. These findings suggest
that differences in sympathetic stimulation during development may contribute to variability among individuals in heart size and rate.
ADVANTAGES OF IN OCULO CULTURE FOR STUDYING
THE EFFECTS OF SYMPATHETIC INNERVATION ON CARDIAC DEVELOPMENT. In oculo, atria from fetal rat hearts
grow, become vascularized, and are functionally innervated.30"33-37 In contrast, atrial strips from adult
Tucker and Gist Innervation of Rat Atria In Oculo
Intrinsic and Maximal In Oculo HR
400
-p 350
Effects of Sympathetic Innervation
Q SCO Intact
ED SCQ Rtmortd
T
J
Q. 300
Q
5250
m 200
QC
•C 150
a
© 100
50
Intrinsic HR
Maximum HR
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FIOURE 8. Intrinsic and maximal heart rates (mean ± SEM)
of atria cultured in sympathetically innervated and denervated
anterior eye chambers. Recordings were made after atria had
been in oculo for 3 months from chronically implanted electrodes. Intrinsic rate was determined after combined blockade
with atenolol and methylatropine. Maximal heart rate was determined after injection of 10 ng/kg isoproterenol into a subcutaneously implanted catheter. Maximal heart rate did not differ
between sympathetically innervated and noninnervated grafts
(p < 0.05).
heart are reported not to grow, with a loss in myocytes
during the first 2 months after implantation.38
The influence of innervation on cardiac development can also be studied in other model systems. Fetal
hearts placed in organ culture fail to grow, remaining
viable for only 2-3 weeks.27 Cells dispersed from fetal
and newborn rat hearts can be grown in culture. Their
environment can be precisely specified but is very different from their milieu in situ (e.g., myocytes are
isolated from rather than in close contact with other
heart cells). Culture of fetal heart in oculo is a pacemaker-driven model of heart development33 that preserves many aspects of heart tissue organization yet
allows the neural and hormonal environment to be
manipulated. Different questions about heart development are best examined in cell culture, in organ culture, in oculo, and in intact animals. Within these
model systems degree of experimental control is balanced against disruption of normal conditions for cardiac development.
Atria cultured in oculo become innervated at approximately the same time after conception as do atria
in situ. Olson and colleagues30" reported innervation
of fetal rat hearts cultured in the anterior eye chamber
to occur during the second and third weeks after implantation. For atria grafted at 12 days postconception
in our study, this would correspond to innervation in
oculo between 20 and 26 days after conception (E-20
to P-5), the period during which sympathetic innervation of the heart undergoes final maturation in situ.23
Prior to growth of catecholamine fibers into the grafted
atria, catecholamine stimulation would come both
from circulating catecholamines and, in sympathetically innervated eye chambers, by diffusion of norepinephrine released from the sympathetic nerves innervating the iris. The consequences of this early
541
catecholamine stimulation of hearts developing in
oculo remain to be determined.
In oculo, sympathetically innervated and noninnervated atrial grafts are perfused by the same circulating
catecholamine and hormonal stimulation. By comparing sympathetically innervated and noninnervated
grafts, we can determine the effects of sympathetic
innervation per se on cardiac growth and rate. Growth
of sympathetically innervated atrial grafts may have
been enhanced because they received a greater quantity of norepinephrine stimulation than noninnervated
grafts or because they were exposed to some other
trophic substance released from sympathetic nerves. In
oculo, beating heart tissue does not pump against a
blood pressure gradient so the increased growth of
sympathetically innervated atrial grafts cannot be attributed to differential hemodynamic load.
TROPHIC INFLUENCES OF SYMPATHETIC INNERVATION
ON HEART GROWTH. This experiment
demonstrated that
sympathetic innervation promotes the growth of fetal
rat atria grafted into the anterior eye chamber. At the
time the atria were grafted, cell division was contributing to cardiac growth.22 Sympathetic innervation may
have increased graft size by promoting ongoing cell
division, increasing the size of individual myocytes, or
both. Claycomb's report4 that isoproterenol treatment
of newborn rats inhibited subsequent cell division argues against the possibility that sympathetic innervation of grafted fetal atria would promote cell division.
Since catecholamine stimulation is known to induce
hypertrophy of myocytes,6725 it seems likely that sympathetically innervated grafts had larger cells. However, determination of whether cell size or number or
both were increased by sympathetic innervation awaits
empirical verification.
Our findings are consistent with the report of Nayler
and colleagues8 that pharmacological blockade of sympathetic stimulation of young rabbits reduced heart
size and inconsistent with the reports of Kirby and
Stewart14 and of Higgins and Pappano13 that sympathectomy of the embryonic chick heart did not alter its
growth. In the present study, the surface dimensions of
sympathetically innervated atrial grafts were not significantly increased over noninnervated grafts for the
first 4 weeks in oculo. If Kirby and Stewart1-4 and
Higgins and Pappano15 had been able to follow their
sympathectomized chick hearts for a longer period of
time, growth differences might have been detected.
Alternatively, the immature sympathetic innervation
of the fetal heart may exert minimal control of cardiac
growth, with more substantial sympathetic influence
on heart size occuring after birth.
TROPHIC INFLUENCE OF SYMPATHETIC INNERVATION
ON OTHER CARDIOVASCULAR TARGET ORGANS. Sympathetic innervation has also been reported to exert a
trophic influence on developing blood vessels. Bevan
and Bevan3940 demonstrated that sympathetic innervation promotes the growth of blood vessels in the ear of
young rabbits using a strategy similar to that used in
the present experiment. They produced unilateral denervation by removing the superior cervical ganglion
542
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which supplies sympathetic innervation to the ipsilateral ear. In young rabbits (4 weeks of age), sympathetic denervation resulted in decreased vascular smooth
muscle proliferation, fewer vascular smooth muscle
cells and stiffer blood vessel walls.4°-42 Sympathetic
denervation of the arteries of older rabbits did not alter
the number of smooth muscle cells. However, in all
age groups, sympathetic denervation reduced force of
vascular contraction and produced postjunctional hypersensitivity to depolarizing stimuli.
Aprigliano and Hermsmeyer43 produced sympathetic denervation of the portal vein by 6-hydroxydopamine treatment of newborn rats. Postjunctional alterations were observed in the sympathectomized portal
veins which Aprigliano and Hermsmeyer43 attributed
to removal of a trophic influence of the sympathetic
nervous system. Specifically, a partial depolarization
was observed in denervated portal veins. They hypothesized that the associated ionic changes were components of the trophic interaction between sympathetic
nerves and blood vessels.
In spontaneously hypertensive rats (SHR), the hypertrophy of blood vessel walls and increased vascular
reactivity are at least partially dependent on sympathetic innervation. Unilateral surgical denervation of
cerebral arteries in 3-week-old stroke-prone SHR attenuated the wall thickening expected in intraparenchymal vessels 1 year later.44 Abel and Hermsmeyer45
demonstrated that sympathetic innervation may be
necessary for development of the membrane properties
which underly vascular hyperreactivity in SHR rats.
They transplanted tail arteries from 2-week-old SHR
and WKY control pups into the anterior eye chamber
of SHR or WKY host rats. In sympathetically innervated eye chambers, the blood vessels underwent interconversion to membrane properties (resting potential
and NE sensitivity) characteristic of the host strain.
However, if cultured in sympathetically denervated
eye chambers, the arteries developed membrane properties characteristic of the donor strain.
Both increased sympathetic nervous system activity
and increased size of the heart and blood vessel walls
are observed in neonatal rats of the genetically hypertensive SHR strain. Because of technical difficulties in
selectively preventing sympathetic innervation of the
heart and blood vessels during fetal development, the
role of sympathetic stimulation in the early growth and
differentiation of cardiac and vascular smooth muscle
in situ has yet to be determined. By 4-5 days of age,
pharmacological blockade of sympathetic stimulation
produces a larger bradycardia1 and a greater fall in
blood pressure46 in SHR than in WKY pups. By 3
weeks of age, norepinephrine in plasma and dopamine
/3-hydroxylase in blood vessels are both increased in
SHR rats.4748 Measurements of heart weight, ventricular weight to body weight ratios, ventricular wall thickness and myocardial DNA and RNA content indicate
that the hearts of newborn SHR are larger than WKY,
contain more cells, and are synthesizing more proteins.21649-51 Similarly, the earliest measurements of
the wall to lumen ratios and medial wall thickness of
Circulation Research
Vol 59, No 5, November 1986
blood vessels in SHR indicate that hypertrophy begins
during the first 2 postnatal weeks.5253 Thus, there is
considerable circumstantial evidence linking enhanced
sympathetic activity with increased growth of cardiac
and vascular smooth muscle. However, the increased
hemodynamic demand placed on the heart and blood
vessels of young SHR by the higher mean arterial
pressure2'54-56 could also account for the cardiac and
vascular hypertrophy.
EFFECTS OF SYMPATHETIC INNERVATION ON INTRINSIC
In contrast to the tachycardic response
observed to phasic sympathetic stimulation, the results
of the present study indicate that tonic sympathetic
stimulation of the developing heart reduces the intrinsic rate of firing of its pacemaker. Little is understood
of how pacemaker rate is determined during development. Specialization of the sinoatrial region as pacemaker occurs very early in embryonic development.57-58 Electrical activity is initiated in the atrial
region of the embryonic chick heart.57"59 Rhythmic
electrical activity precedes mechanical activity and
histologic identification of pacemaker cells.5760 There
is a gradual increase in the rate at which the embryonic
heart beats during fetal and early postnatal development.'.10.".6i A n increased pacemaker rate in the maturing heart may result from changes inherent in the developing heart, from the changes in the fetal and
postnatal heart's neurohumoral milieu or from an interaction between intrinsic and neurohumoral factors.
The current data indicate that sympathetic innervation
can influence the pacemaker rate established by the
embryonic heart.
Individual differences in functional sympathetic
maturation and tonic activity exist early in development.1'46 Individual differences in sympathetic nervous
system function may be genetically based (e.g., SHR
rat146) or induced by environmental factors.62 The present results suggest that sympathetic innervation of developing cardiovascular target organs may be one effector system through which genetically determined
and environmentally induced differences influence
subsequent cardiovascular function.
HEART RATE.
Acknowledgments
Catecholamine assays were performed in the laboratory of S. Oparil by R. Gist; the authors thank Dr.
Oparil for her assistance. P. Alverson provided excellent technical assistance during data collection. The
comments of W. T. Woods on an earlier draft were
most helpful.
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KEY WORDS • fetal rat atria • in oculo culture
beating rate • sympathetic innervation
intrinsic
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Sympathetic innervation alters growth and intrinsic heart rate of fetal rat atria maturing in
oculo.
D C Tucker and R Gist
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Circ Res. 1986;59:534-544
doi: 10.1161/01.RES.59.5.534
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1986 American Heart Association, Inc. All rights reserved.
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