ARTICLE |
Correspondence to: Dina Lewinson, Dept. of Anatomy and Cell Biology, The Bruce Rappaport Faculty of Medicine, TechnionIsrael Inst. of Technology, POB 9649, 31096 Haifa, Israel. E-mail: dinal@tx.technion.ac.il
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The natriuretic peptides are believed to play an important role in the pathophysiology of congestive heart failure (CHF). We utilized a quantitative cytomorphometric method, using double immunocytochemical labeling, to assess the characteristics of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in atrial granules in an experimental model of rats with CHF induced by aortocaval fistula. Rats with CHF were further divided into decompensated (sodium-retaining) and compensated (sodium-excreting) subgroups and compared with a sham-operated control group. A total of 947 granules in myocytes in the right atrium were analyzed, using electron microscopy and a computerized analysis system. Decompensated CHF was associated with alterations in the modal nature of granule content packing, as depicted by moving bin analysis, and in the granule density of both peptides. In control rats, the mean density of gold particles attached to both peptides was 347.0 ± 103.6 and 306.3 ± 89.9 gold particles/µm2 for ANP and BNP, respectively. Similar mean density was revealed in the compensated rats (390.6 ± 81.0 and 351.3 ± 62.1 gold particles/µm2 for ANP and BNP, respectively). However, in rats with decompensated CHF, a significant decrease in the mean density of gold particles was observed (141.6 ± 67.3 and 158.0 ± 71.2 gold particles/µm2 for ANP and BNP, respectively; p<0.05 compared with compensated rats, for both ANP and BNP). The ANP:BNP ratio did not differ between groups. These findings indicate that the development of decompensated CHF in rats with aortocaval fistula is associated with a marked decrease in the density of both peptides in atrial granules, as well as in alterations in the quantal nature of granule formation. The data further suggest that both peptides, ANP and BNP, may be regulated in the atrium by a common secretory mechanism in CHF. (J Histochem Cytochem 49:12931300, 2001)
Key Words: ANP, BNP, heart failure, immunocytochemistry, quantitative microscopy, atrium, rat
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE NATRIURETIC PEPTIDE FAMILY includes three peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), of which the first two, ANP and BNP, are expressed in the heart (
Congestive heart failure (CHF) is a syndrome characterized by structural and functional impairment of the myocardium and a severe disturbance of body fluid balance. A variety of neurohormonal systems are activated in CHF (
Most studies conducted on experimental models of CHF have focused on ANP, whereas less attention has been given to the role of BNP in the pathogenesis of CHF. BNP is considered to be a more reliable prognostic marker than ANP in patients with CHF (
In recent years, the actions of the natriuretic peptides in CHF have been investigated in our laboratory in an experimental model of CHF in the rat. In this model, CHF is induced by an arteriovenous (AV) fistula between the abdominal aorta and the inferior vena cava (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Studies were performed in male Wistar rats (250300 g; Harlan Laboratories, Jerusalem, Israel). The animals were kept in individual metabolic cages and were maintained on normal rat diet containing 0.5% NaCl and tapwater ad libitum. All animal handling was according to the institutional guidelines.
CHF was induced by surgical creation of an AV fistula between the abdominal aorta and the inferior vena cava, according to the method of
Immunocytochemistry
On the seventh postoperative day, control and CHF rats were anesthetized and the right internal jugular vein and left internal carotid artery were cannulated with polyethylene tubes (PE-50). The aorta was severed proximal to the shunt and a perfusion of 3% glutaraldehyde in 0.1 mM sodium cacodylate, pH 7.4, was introduced simultaneously through the internal jugular vein and the internal carotid artery. The perfusion lasted until the effluent fluid of the abdominal aorta became clear. After perfusion, small specimens of anterior right atrial tissue, including the auricle, were immersed and fixed overnight in a solution of 3% glutaraldehyde in 0.1 mM sodium cacodylate. After a wash in 10% sucrose in 0.1 mM cacodylate buffer, pH 7.4, the samples were postfixed with 1% osmium tetroxide, dehydrated in graded ethanols, cleared in propylene oxide, and embedded in Epon. Three blocks from the right atrium of each rat were selected randomly for sectioning (LKB; Nova Ultratome, Bromma, Sweden).
Sections (80100 nm) were mounted on nickel grids and were immunostained by a double-labeling immunocytochemical method modified from -ANP (1:500) (Phoenix Pharmaceuticals; Mountain View, CA) that has no crossreactivity with BNP as determined in an RIA system (personal communication) was administered to the grid and incubated overnight in a humidified chamber at 4C. This was followed by 10-nm colloidal gold-tagged goat anti-rabbit second antibody (1:10) (Zymed Laboratories; San Francisco, CA) and a thorough wash in 0.1% BSA in PBS. Next, a polyclonal antibody against rat BNP (1:500) (Peninsula Laboratories; Belmont, CA), which has no crossreactivity with ANP as determined in an RIA system (personal communication), was administered to the other side of the grid, following the same procedure, with 15-nm gold-tagged goat anti-rabbit second antibody (1:10) (Zymed).
The double-labeled sections were then scanned without further staining in a JEOL 100 SX transmission electron microscope (Tokyo, Japan), and the most technically adequate and clearly stained sections were selected. Secretory granules were photographed randomly at a magnification of x30,000 and were printed at a final magnification of x60,000 from at least five different cells from each block (i.e., at least 50 prints/treatment group). Both types of gold particles in each individual granular profile (10- and 15-nm) were counted separately, directly from the final printed micrographs. Using an HP-9111A graphic tablet connected to a Power Macintosh 7100/60 AV, the profile area of each granule was measured by a program developed by /3)(Ai/
)3/2. The resulting volume equivalents were plotted as a histogram. The multimodal histogram was analyzed by the moving-bin technique to reveal true peaks, as explained elsewhere in detail (
The data were correlated with the respective mean granule equivalent volume [A = Ai/25, equivalent volume = (4
/3)(A/
)3/2].
Statistical Analysis
Data are presented as mean ± SE. Statistical significance was evaluated by the nonparametric Kolmogorov-Smirnov test (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Representative electron micrographs of granules in the right atrium in each of the three groups (compensated and decompensated rats with CHF and controls), double-labeled by the two different gold particle sizes (10 and 15 nm), are shown in Fig 1. A total of 947 granules were counted, of which 226 were from the control group, 370 from the decompensated CHF subgroup, and 351 from the compensated CHF subgroup. As shown, most of the labeling was concentrated within the limits of the granules, with minimal background activity. Moreover, the density of both sizes of gold particles was higher in the granules of normal and compensated rats (Fig 1A and Fig 1B) compared with granules of decompensated rats (Fig 1C). In control rats, the mean densities of gold particles were 347.0 ± 103.6 and 306.3 ± 89.9 gold particles/µm2 for ANP and BNP, respectively. Similar mean densities were revealed in the compensated rats (390.6 ± 81.0 and 351.3 ± 62.1 gold particles/µm2 for ANP and BNP, respectively). However, in rats with decompensated CHF, a significant decrease in the mean density of gold particles was observed (141.6 ± 67.3 and 158.0 ± 71.2 gold particles/µm2 for ANP and BNP, respectively; p<0.05 for both ANP and BNP vs controls and compensated rats). When non-immune rabbit serum replaced both primary antibodies, no gold particles of any size were observed in the atrial granules (Fig 1D).
|
To evaluate the characteristics of granule formation and release, we applied the technique of moving-bin analysis (
|
|
|
An important observation in this study was the significant difference between the compensated group and the decompensated group in the density of gold particles attached to both ANP and BNP (Fig 4B and Fig 4C). On the other hand, there was no significant difference between the control group and the compensated rats (Fig 4A and Fig 4B). However, despite a significant reduction in the absolute values of ANP and BNP in rats with decompensated CHF, the ANP:BNP ratio was not significantly different in this subgroup. Of note is the finding that when the mean densities of gold particles were plotted relative to the mean granule equivalent volumes in the three groups (Fig 4), a similar ratio was obtained irrespective of the granule size.
There was a significant difference between the compensated and control rats compared with the decompensated subgroup. Because peaks were not evident in all three groups, we took data from Fig 2 and labeled the graphs of Fig 4 at the location of the quanta of the calculated granules. In Fig 4A and Fig 4B, in both cases, the modes of the graphs are approximately near the indicated arrows, whereas in the decompensated subgroup the quantal nature is significantly less obvious. However, it should be noted that because peaks in this graph were not evident in all three cases, the modes might have been due to noise (
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our study provides important insight into the regulation of both ANP and BNP in the atria of rats with AV fistula, an experimental model of CHF. Using a quantitative morphometric immunocytochemical method, we confirmed previous observations that both ANP and BNP are stored in the same granules in cardiac atriocytes. Moreover, rats with severe decompensated CHF display a significant decrease in the density of both peptides in the atrial granules, irrespective of granule size, as well as alterations in the quantal nature of atrial granule formation. The results further suggest that the generation of ANP and BNP in the atria shares a common regulatory mechanism, both under normal conditions and in animals with CHF.
The finding that atrial granules contain both ANP and BNP is in agreement with previous reports in the literature.
The densities of ANP and BNP cannot be used as a quantitative estimate of the absolute content of ANP and BNP in the granule, because the antibodies may differ in their affinities for the peptides. However, the BNP:ANP density ratio can provide valuable information about the alterations in the granule content of each hormone during the evolution of CHF from a compensated to a decompensated state, thus hinting at either common or separate regulation of the two hormones in this disease state. Indeed, contradictory data are available on the regulation of ANP and BNP in the atria during the development of heart failure. Our findings support previous studies by
However, other investigators have shown differences in the ANP:BNP ratio in the atria.
An additional interesting feature in the present study was the loss of the modes with the deterioration of CHF into a decompensated state (Fig 4). The secretory granule equivalent volume in the decompensated subgroup was multimodal and periodic. However, the scattergram analysis correlating the immunohistochemical data with granule size was not multimodal as in the other two groups. In a previous study from our laboratory, using morphometric analysis of the granules in atriocytes of rats with decompensated CHF, we demonstrated changes compatible with accelerated generation and release of the granules in this severe form of CHF (
In summary, in the present study we used a double-labeling cytomorphometric approach to follow the alterations in the ANP- and BNP-containing granules in atria of rats with experimental CHF. The data demonstrate that the progression of CHF from a compensated to a decompensated state is associated with marked alterations in the density of both peptides in the atrial granules and in the quantal nature of granule formation. The findings further suggest that both ANP and BNP in atrial granules share a common regulatory mechanism in heart failure.
![]() |
Acknowledgments |
---|
We thank Ms Pesia Shentzer and Ms Irena Reiter for excellent technical assistance and Ms Ruth Singer for editing the manuscript.
Received for publication November 29, 2000; accepted May 16, 2001.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abassi Z, Burnett JC, Jr, Grushka E, Hoffman A, Haramati A, Winaver J (1991) Atrial natriuretic peptide and renal cGMP in rats with experimental heart failure. Am J Physiol 261:R858-864
Asano K, Masuda K, Okumura M, Kadosawa T, Fujinaga T (1999) Plasma atrial and brain natriuretic peptide levels in dogs with congestive heart failure. J Vet Med Sci 61:523-529[Medline]
Atlas SA, Cody RJ, Laragh JH (1992) Atrial natriuretic peptide in heart failure. In Braunwald E, ed. Heart Disease Update. New York, WB Saunders, 19-30
Avramovitch N, Hoffman A, Winaver J, Haramati A, Lewinson D (1995) Morphometric analysis of atrial natriuretic peptide-containing granules in atriocytes of rats with experimental congestive heart failure. Cell Tissue Res 279:575-583[Medline]
Brodsky S, Gurbanov K, Abassi Z, Hoffman A, Ruffolo RR, Jr, Feuerstein GZ, Winaver J (1998) Effects of eprosartan on renal function and cardiac hypertrophy in rats with experimental heart failure. Hypertension 32:746-752
Doyama K, Fukumoto M, Takemura G, Tanaka M, Oda T, Hasegawa K, Inada T, Ohtani S, Fujiwara T, Itoh H, Nakao K, Sasayama S, Fujiwara H (1998) Expression and distribution of brain natriuretic peptide in human right atria. J Am Coll Cardiol 32:1832-1838[Medline]
Friedl W, Mair J, Thomas S, Pichler M, Puschendorf B (1999) Relationship between natriuretic peptides and hemodynamics in patients with heart failure at rest and after ergometric exercise. Clin Chim Acta 281:121-126[Medline]
Hammel I, Lagunoff D, Bauza M, Chi E (1983) Periodic, multimodal distribution of granule volumes in mast cells. Cell Tissue Res 228:51-59[Medline]
Hammel I, Lagunoff D, Krüger P-G (1988) Studies on the growth of mast cells in rats. Changes in granule size between 1 and 6 months. Lab Invest 59:549-554[Medline]
Hammel I, Lagunoff D, Krüger PG (1989) Recovery of rat mast cells after secretion: a morphometric study. Exp Cell Res 184:518-523[Medline]
Hasegawa K, Fujiwara H, Itoh H, Nakao K, Fujiwara T, Imura H, Kawai C (1991) Light and electron microscopic localization of brain natriuretic peptide in relation to atrial natriuretic peptide in porcine atrium. Immunohistocytochemical study using specific monoclonal antibodies. Circulation 84:1203-1209[Abstract]
Hoffman A, Burnett JC, Jr, Haramati A, Winaver J (1988) Effects of atrial natriuretic factor in rats with experimental high-output heart failure. Kidney Int 33:656-661[Medline]
Horisberger M (1981) Colloid gold: a cytochemical marker for light and fluorescent microscopy and for transmission and scanning electron microscopy. Scan Electron Microsc 2:9-31
Langenickel T, Pagel I, Hohnel K, Dietz R, Willenbrock R (2000) Differential regulation of cardiac ANP and BNP mRNA in different stages of experimental heart failure. Am J Physiol 278:H1500-1506
Ledsome JR, Wilson N, Courneya CA, Rankin AJ (1985) Release of atrial natriuretic peptide by atrial distension. Can J Physiol Pharmacol 63:739-742[Medline]
Magga J, Vuolteenaho O, Tokola H, Marttila M, Ruskoaho H (1997) Involvement of transcriptional and posttranscriptional mechanisms in cardiac overload-induced increase of B-type natriuretic peptide gene expression. Circ Res 81:694-702
Moe GW, Grima EA, Wong NLM, Howard RJ, Armstrong PW (1993) Dual natriuretic peptide system in experimental heart failure. J Am Coll Cardiol 22:891-898[Medline]
Moe GW, Grima EA, Wong NLM, Howard RJ, Armstrong PW (1996) Plasma and cardiac tissue atrial and brain natriuretic peptides in experimental heart failure. J Am Coll Cardiol 27:720-727[Medline]
Mukoyama M, Nakao K, Hosoda K, Suga S, Saito Y, Ogawa Y, Shirakami G, Jougasaki M, Obata K, Yasue H, Kambayashi Y, Inouye K, Imura H (1991) Brain natriuretic peptide as a novel cardiac hormone in humans. Evidence for an exquisite dual natriuretic peptide system, atrial natriuretic peptide and brain natriuretic peptide. J Clin Invest 87:1402-1412[Medline]
Nakamura S, Naruse M, Naruse K, Kawana M, Nishikawa T, Hosoda S, Tanaka I, Yoshimi T, Yoshihara I, Inagami T (1991) Atrial natriuretic peptide and brain natriuretic peptide coexist in the secretory granules of human cardiac myocytes. Am J Hypertens 4:909-912[Medline]
Pemberton CJ, Yandle TG, Charles CJ, Rademaker MT, Aitken GD, Espiner EA (1997) Ovine brain natriuretic peptide in cardiac tissues and plasma: effects of cardiac hypertrophy and heart failure on tissue concentration and molecular forms. J Endocrinol 155:541-550
Pettersson A, Hedner J, Hedner T (1989) Renal interaction between sympathetic activity and ANP in rats with chronic ischaemic heart failure. Acta Physiol Scand 125:487-492
Sokal RR, Rohlf FJ (1969) Biometry. San Francisco, WH Freeman
Stumpe KO, Sölle H, Klein H, Krück F (1973) Mechanism of sodium and water retention in rats with experimental heart failure. Kidney Int 4:309-317[Medline]
Takemura G, Takatsu Y, Doyama K, Itoh H, Saito Y, Koshiji M, Ando F, Fujiwara T, Nakao K, Fujiwara H (1998) Expression of atrial and brain natriuretic peptides and their genes in hearts of patients with cardiac amyloidosis. J Am Coll Cardiol 31:754-765[Medline]
Thibault G, Charbonneau C, Bilodeau J, Schiffrin EL, Garcia R (1992) Rat brain natriuretic peptide is localized in atrial granules and released into the circulation. Am J Physiol 263:R301-309
Wei C-M, Heublein DM, Perrella MA, Lerman A, Rodeheffer RJ, McGregor CGA, Edwards WD, Schaff HV, Burnett JC, Jr (1993) Natriuretic peptide system in human heart failure. Circulation 88:1004-1009[Abstract]
Winaver J, Hoffman A, Abassi Z, Haramati A (1995) Does the heart's hormone, ANP, help in congestive heart failure? News Physiol Sci 10:247-253
Winaver J, Hoffman A, Burnett JC, Jr, Haramati A (1988) Hormonal determinants of sodium excretion in rats with experimental high-output heart failure. Am J Physiol 254:R776-784
Yasue H, Yoshiruma M, Sumida H, Kikuta K, Kugiyama K, Jougasaki M, Ogawa H, Okumura K, Mukoyama M, Nakao K (1994) Localization and mechanism of secretion of B-type natriuretic peptide in comparison with those of A-type natriuretic peptide in normal subjects and patients with heart failure. Circulation 90:195-203[Abstract]
Yoshimura M, Yasue H, Okumura K, Ogawa H, Jougasaki M, Mukoyama M, Nakao K, Imura H (1993) Different secretion patterns of atrial natriuretic peptide and brain natriuretic peptide in patients with congestive heart failure. Circulation 87:464-469[Abstract]