(Hypertension. 1999;34:790-794.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
Modulation of Coronary Flow and Cardiomyocyte Size by Sensory Fibers
From the Department of Physical Education, Biosciences Institute (A.Z.), Paulista State University, Rio Claro (SP) and the Department of Pharmacology, Faculty of Medical Sciences (S.K.P.C., S.R.R., L.P.N., C.F. de O., E.A., G. De N.); and Department of Histology and Embryology, Biology Institute, State University of Campinas, (I.M.S. De L.), Campinas (SP), Brazil.
Correspondence to Angelina Zanesco, PhD, Department of Physical Education, Biosciences Institute, Paulista State University, Av 24A No. 1515, Bela Vista, CEP 13506-900, Rio Claro (SP), Brazil. E-mail azanesco{at}bestway.com.br
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Abstract |
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Abstract—Cardiac tissue is densely innervated by sensory neurons that are believed to play important modulatory roles in cardiac functions. In this study, pretreatment of neonate rats with capsaicin was performed. In adult rats, cardiomyocyte size and amount of fibrous tissue in left ventricles as well as in vitro coronary flow were evaluated. The chronotropic and inotropic responses to ß-adrenoceptor agonists (norepinephrine and isoproterenol), muscarinic agonists (carbachol and pilocarpine), and calcitonin gene–related peptide (CGRP) were also investigated with the use of the isolated right atria preparation. Capsaicin pretreatment significantly (P<0.05) reduced both basal coronary flow (18% reduction) and cardiomyocyte size (34% reduction) without affecting the amount of fibrous tissues in the left ventricles. The positive inotropic and chronotropic effects in response to norepinephrine in the isolated rat heart did not significantly differ between control and capsaicin-treated rats. Similarly, the positive chronotropic effects in response to norepinephrine, isoproterenol, and CGRP as well as the negative chronotropic responses to carbachol and pilocarpine in the isolated right atria were not affected by capsaicin pretreatment. Our data are consistent with the suggestion that reductions of both basal coronary flow and cardiomyocyte size seen in hearts from capsaicin-pretreated rats may be consequences of CGRP depletion. The cardiomyocyte size reduction produced by capsaicin treatment may be related to a modulatory role of CGRP as a growth factor.
Key Words: capsaicin • receptors, muscarinic • receptors, adrenergic, beta • neuropeptides • peptides
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Introduction |
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Sensory fibers have been associated with the control of smooth muscle tone, autonomic ganglia transmission, immunologic processes, tissue growth,1 and heart functions.2 3 4 5 A number of substances, including the peptides substance P and calcitonin gene–related peptide (CGRP), are released by the sensory nerve endings.1 6 Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is a highly selective neurotoxin that, on systemic administration, causes CGRP and substance P depletion from sensory neurons7 8 9 and thus has largely been used to study the involvement of sensory fibers in different pathophysiological functions.10
In the heart, capsaicin increases contractile force and spontaneous heart rate3 as well as evokes coronary vasodilation through CGRP release.11 12 Previous studies reported the presence of CGRP in the heart, predominantly in the right atria, followed by the left atria and right and left ventricles,3 13 where it causes concentration-dependent and long-lasting positive inotropic and chronotropic effects in several species,9 14 15 16 17 including humans.18 Although CGRP has also been described as a potent hypertrophic factor for cardiomyocytes,19 no study has been performed to investigate the effect of CGRP depletion on cardiomyocyte size. The existence of interactions of sympathetic and parasympathetic nerves with sensory fibers in in vitro20 21 and in vivo22 preparations has been reported.23 In this study, pretreatment of neonate rats with capsaicin was performed and the rats were used at adult ages. We then evaluated the following parameters in the heart: (1) cardiomyocyte size, (2) basal and stimulated coronary flow in vitro, and (3) chronotropic and inotropic responses of ß-adrenoceptors and muscarinic agonists as well as CGRP, with both whole rat isolated heart and isolated right atria.
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Methods |
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Capsaicin Treatment
The experiments were performed in Wistar rats of both sexes bred in the department of Animal Care of the Faculty of Medical Sciences, State University of Campinas (São Paulo, Brazil). In total, 102 neonatal rats were pretreated subcutaneously on the second day of life with capsaicin (50 mg/kg; Sigma Chemical Co) or the corresponding volume (100 µL) of capsaicin-vehicle (10% ethanol and 10% Tween 80, in 0.9% [wt/vol] NaCl solution) under ether anesthesia.8 Rats were used 12 to 14 weeks after capsaicin pretreatment, at which time they weighed 200 to 300 g. All procedures were designed in accordance with the guidelines of the State University of Campinas for animal care.
Langendorff Preparation
Male Wistar rats (weight, 250 to 350 g) were
anesthetized with sodium pentobarbital (Sagatal; 50 mg/kg IP)
and given heparin (500 IU/kg IP) 5 minutes before thoracotomy. The
hearts were rapidly excised and mounted on a Langendorff
apparatus and perfused at constant pressure (65
mm Hg) with oxygenated (95% O2/5%
CO2) Krebs-Henseleit solution containing (in
mmol/L) NaCl 118, KCl 4.7,
KH2PO4 1.2,
MgSO4 1.2, CaCl2 2.5,
NaHCO3 25, and glucose 11, pH 7.4, at 37°C.
Left ventricular developed pressure (LVDP) (mm Hg) and
heart rate (HR) (bpm) were recorded via a latex balloon inserted
into the left ventricle (basal end-diastolic pressure,
5 mm Hg). A polyethylene cannula was then connected to a
pressure transducer (model PRC 21/3, Ugo Basile) and a 2-channel
recorder (Gemini 7070, Ugo Basile). The preparations were allowed
to stabilize for 30 minutes. Coronary flow (mL/min) was
measured manually by 15-second timed collections of the
coronary effluent.
Functional Assays With Isolated Right Atria
Animals were anesthetized with halothane, and the hearts
were rapidly removed. The right atria were isolated and mounted in a
water-jacketed tissue chamber (10-mL volume) containing Krebs-Henseleit
solution, pH 7.3 to 7.5, at 37°C and gassed with 95%
O2/5% CO2. The composition
of the Krebs-Henseleit solution was as follows (mmol/L): NaCl 124, KCl
4.75, MgSO4 1.30, CaCl2
2.25, NaHCO3 25.0,
NaH2PO4 0.6, dextrose 10.0,
sodium ascorbate 0.3, and disodium EDTA 0.03. Ascorbate and EDTA were
added to inhibit the oxidation of
catecholamines.24 To block the neuronal and
extraneuronal uptake of catecholamines, both
phenoxybenzamine (10 µmol/L; Sigma Chemical Co) and
17ß-hydroxyestradiol (5 µmol/L; Sigma Chemical Co) were added
to the tissue bath.25
Construction and Analyses of Concentration-Response
Curves
Concentration-response curves for the positive and negative
chronotropic actions of isoproterenol, norepinephrine, rat
CGRP, carbachol, and pilocarpine (all Sigma Chemical Co) were
constructed by the cumulative variation of agonist concentration at
one-half log unit increments.26
All concentration-response data were evaluated for a fit to a logistic
function in the following form:
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is the response observed in the
absence of added agonist. Nonlinear regression analyses to
determine the parameters Emax, log
EC50, and n were performed with GraphPad Prism
(GraphPad Software) with the constraint that =zero.
Stereological Procedures
Stereological analysis was performed according to the
method described by Aherne.27 Formalin-fixed left
ventricle and septum were cut into 5 equidistant rings perpendicular to
the long axis of the ventricle. The rings were then embedded in
paraffin, and 5-µm sections were stained with Masson's trichrome.
Analysis of the slides was performed in blinded fashion on a
light microscope (Zeiss), and the relative volume occupied by each
element of the ventricle (myocardial fibers and fibrous tissue) was
measured with a special ocular apparatus containing a
25-point reticulum (5 parallel lines with 5 points each, kpl x8,
Zeiss). To determine cardiomyocyte size, 15 cells
randomly selected from the subepicardial, midmyocardial, and
subendocardial regions were measured for each animal from the different
experimental groups. For counting, 50 microscopic fields were
evaluated, and the relative volume (Ppi) occupied by each component was
calculated as follows: Ppi=p/P-R, where p is the number of
reticular points hitting each cardiac element, P is the total number of
reticular points, and R is the number of points hitting artifactual
retraction areas.
Blood Pressure Measurement
The systolic blood pressure was measured by a modified
tail-cuff method in awake animals.28 The measurements were
performed 24 hours before the animals were killed.
Statistical Analysis
All values are expressed as mean±SEM. The program InStat
(GraphPad Software) was used for statistical analyses. When
appropriate, 1-way ANOVA followed by a Bonferroni multiple comparisons
post hoc test was performed to determine whether the treatments had an
effect. In some cases, a paired or unpaired Student's t
test was used. P<0.05 was accepted as significant.
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Results |
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Body Weight, Stereological Analysis, and Systolic Blood Pressure
Body weight and systolic blood pressure were not significantly affected by the capsaicin pretreatment (n=12) compared with the control group (n=5; Table 1). Additionally, stereological analysis of the left ventricle revealed a 34% reduction (P<0.05) of cardiomyocyte size in the capsaicin-pretreated rats without change in the amount of fibrous tissues (Table 1).
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Whole Heart Preparations
Bolus injection of norepinephrine (0.6 nmol) caused
similar increases in LVDP and HR in both control and capsaicin-treated
animals (Table 2).
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Capsaicin pretreatment significantly reduced basal coronary
flow by 18% (P<0.05; Table 2). Bolus
injection of norepinephrine (1.0 nmol) caused a significant
decrease in coronary flow in the control group at 0.25 minutes
after injection (35% reduction), whereas in capsaicin-treated rats the
reduction in coronary flow by norepinephrine was
attenuated (18.7% reduction; Table 2).
Isolated Right Atria
Addition of capsaicin (1 µmol/L) to the organ bath caused a
positive chronotropic response in isolated right atria of the control
group (44±4 bpm) that was attenuated by 45% (P<0.05)
in capsaicin-treated rats (20±2 bpm).
Chronotropic responses to both ß-adrenoceptor (isoproterenol and norepinephrine) and muscarinic (carbachol and pilocarpine) agonists are illustrated in the Figure. There was no significant shift of the concentration-response curves for norepinephrine between control and capsaicin groups (Figure, panel A). With respect to isoproterenol, although there was a tendency for a rightward shift in the concentration-response curves, the differences between both groups were not significant (Figure, panel B). The potency of muscarinic agonists was also unaffected by capsaicin pretreatment in isolated right atria (Figure, panels C and D).
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) and capsaicin-treated
(•) rats. Values are mean±SEM of 5 to 8 experiments.
Rat CGRP induced concentration-dependent positive chronotropic effects
of rat right atria that were 30% of maximum response for
isoproterenol and norepinephrine, as observed in either
control or capsaicin groups (Table 3).
Furthermore, there were no significant changes in the potency of rat
CGRP in isolated right atria after capsaicin pretreatment compared with
the control group (Figure, panel E). The maximum responses for
all studied agonists were similar in capsaicin-treated rats and the
control group (Table 3). Table 4
summarizes all studied agonists in isolated right atria from control
and capsaicin-treated rats.
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Discussion |
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In this study we clearly demonstrate that capsaicin pretreatment caused a significant reduction in both basal coronary flow and cardiomyocyte size. However, other cardiac parameters, such as the increase in LVDP in response to norepinephrine and chronotropic actions in response to norepinephrine, isoproterenol, carbachol, pilocarpine, and rat CGRP, were not affected by capsaicin pretreatment.
Many vascular tissues, including coronary vessels, are richly innervated by sensory fibers that, on stimulation, release substance P and CGRP.16 29 In the rat heart, substance P has no effect on vascular tone30 whereas CGRP exerts a potent vasodilatation.31 Capsaicin releases CGRP, leading to an enhancement of coronary flow.32 Maintenance of coronary vascular tone seems to be determined by endothelium-dependent and endothelium-independent mechanisms.33 Our findings showing a significant reduction of basal coronary flow in capsaicin-pretreated rats suggest that it is a consequence of CGRP depletion. In adult rats, systemic capsaicin treatment also reduces basal coronary flow.12
CGRP causes vasodilatation by a mechanism involving intracellular accumulation of cAMP34 that counteracts the norepinephrine-induced increase in inositol 1,4,5-trisphosphate levels.35 Thus, control of vascular tone in coronary vessels by CGRP may reflect a counterregulatory mechanism between these second messengers. Our results showing a decrease of basal coronary flow in the capsaicin-treated rats could be explained by a sustained decrease of cAMP levels. However, the lower responsiveness of coronary vessels to norepinephrine remains unclear.
CGRP has been described as a growth factor in the rat heart, where it exerts beneficial trophic actions on cardiomyocytes.1 Accordingly, our stereological studies have evidenced a decrease in cardiac muscle fiber size in capsaicin-treated rats. These results are therefore consistent with the suggestion that CGRP plays an important role in the maintenance of cardiomyocyte size under normal conditions by acting as a growth factor. Additionally, constant coronary flow as a consequence of continuous release of CGRP may also contribute to regulation of cardiac muscle fiber size. Reduction in coronary flow leads to an increase in the fibrous tissue in the heart as a result of ischemic processes.36 However, capsaicin-treated rats showed no alterations in the amount of fibrous tissues in the left ventricles, suggesting that the magnitude of the coronary flow reduction was insufficient to determine ischemic processes.
The positive chronotropic responses for isoproterenol, norepinephrine, and rat CGRP and the negative chronotropic responses for carbachol and pilocarpine were not affected in isolated right atria by capsaicin treatment. These results may be explained by the partial depletion (45%) of neuropeptides in our study. However, this is unlikely since previous work also found no alterations in the potency of both agonists, isoproterenol and rat CGRP, in right atria from guinea pigs and rats treated with capsaicin, where complete depletion of CGRP was detected.37 In a manner similar to that of chronotropism, capsaicin pretreatment had no effect on the inotropic responses to norepinephrine in isolated whole heart, indicating that cardiomyocytes may be able to retain their physiological responses, including contraction force, despite their size reduction. Taken together, these observations presumably indicate that sensory fibers do not play a role in the primary modulation of cardiac actions (chronotropism and inotropism) compared with the sympathetic nervous system, in which norepinephrine is the main neurotransmitter.
In summary, capsaicin treatment in neonatal rats causes a significant decrease of basal coronary flow, associated with diminishing cardiomyocyte size, as a consequence of CGRP depletion.
Received May 8, 1999; first decision June 22, 1999; accepted July 10, 1999.
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