International Journal of Cardiology, 34 (1992) 237-247 Q 1992 Elsevier Science Publishers B.V. All rights reserved
CARDJO
0167-5273/92/$05.00
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Localization of brain and atria1 natriuretic peptide in human and porcine heart Angela Pucci lT2,John Wharton r, Eloisa Arbustini ‘, Maurizia Grass0 2, Marta Diegoli Philip Needleman 4, Mario Viganb 3, Gonzalo Moscoso 5 and Julia M. Polak ’
2,
’ Department of Histochemistry, Royal Postgraduate Medical School, London, U.K.; ’ lstituto di Anatomia Patologica and .’ Ditisione di Cardiochirurgia, Istituto di Ricotsero e Cura a Carattere Scientific0 (IRCCS), Policlinico “S. Matteo’: Par,ia. Italy; ‘Monsanto Corporation, St Louis, Missouri. U.S.A.; ‘Department of Morbid Anatomy, Kings College Hospital. London U.K. (Received
22 July 1991: accepted
3 October
1991)
Pucci A, Wharton J, Arbustini E, Grass0 M, Diegoli M, Needleman P, Vigano M, Moscoso G, Polak JM. Localization of brain and atria1 natriuretic peptide in human and porcine heart. Int J Cardiol 1992;34:237-247. We have compared the localization of brain and atrial natriuretic peptide-like immunoreactivity in human and porcine hearts, using immunohistochemical techniques at both the light and ultrastructural level and specific antisera to amino-(cardiodilatin) and carboxy-terminal regions of the atrial natriuretic precursor molecule and to brain natriuretic peptide. Atrial myocardial cells in human fetal, normal adult and failing explanted hearts, displayed immunoreactivity for both brain and atrial natriuretic peptide-like sequences. At the subcellular level, brain natriuretic peptide-, cardiodilatinand alpha-atria1 natriuretic peptide-like immunoreactivity were co-localized to secretory granules in atrial myocardial cells. Immunoreactivity was also detected in the left (64%) and right ventricular free walls (23%) of 22 failing explanted hearts, but not in donor cardiac tissues. A gradient of natriuretic peptide immunostaining was observed across ventricular free walls and immunoreactivity for both natriuretic peptide sequences co-localized to secretory granules in a subpopulation of myocardial cells, concentrated in subendocardial regions of the ventricular walls. Brain and atrial natriuretic peptide-like immunoreactivity were also demonstrated in porcine atrial myocardium and cells of the ventricular conduction system. The parallel distribution of cardiac brain and atrial natriuretic peptide-like immunoreactivity suggests a dual regulation and co-storage of the natriuretic peptides in human and porcine hearts.
Key words: Atrial natriuretic peptide; chemistry; Conduction system
Brain natriuretic
Correspondence to: Dr. J. Wharton, Dept. of Histochemistry, Royal Postgraduate Medical School, Du Cane Road, London W12 ONN, U.K. A. Pucci was supported by a grant from the “Collegio Nuovo” Pavia and grant “Trapianto Cardiaco” from the Health Ministry to IRCCS Policlinico “S. Matteo”, Pavia 27100, Italy. The work was supported by the Council for Tobacco Research, U.S.A.
peptide;
Electron
microscopy;
Immunohisto-
Introduction
A family of homologous natriuretic peptides have now been identified in the mammalian heart and nervous system and implicated in regulating body fluid and electrolyte balance, comprising atria1 natriuretic peptide, brain natriuretic pep-
238
tide and C-type natriuretic peptide [1,2]. Although they are derived from distinct genes, brain and atria1 natriuretic peptides have a homologous structure and display a similar, but species dependent, range of effects [3,4]. Although originally identified in the brain [l] and shown to have a distinct distribution to atria1 natriuretic peptide in the nervous system [5-71, a number of studies have now reported the cloning of complementary deoxyribonucleic acid sequences encoding the brain natriuretic peptide precursor in the heart [g-lo]. Whilst several endogenous molecular forms of the peptide have also been isolated from mammalian cardiac tissues [7-9,11-131, the cellular localization of cardiac brain natriuretic peptide has not been established. Atria1 natriuretic peptide is produced and stored as a high molecular weight precursor in atria1 secretory granules [14] and proteolytic cleavage, during or soon after release, gives rise to active carboxy- and amino-terminal peptide sequences [15,16]. The processing pathway of the porcine brain natriuretic peptide precursor is similar to that of the atria1 peptide in mammalian hearts, whereas in rat and human cardiac tissues smaller molecular forms predominate, with brain natriuretic peptide 1-32 being the main storage and circulating form in man [7,11,12]. The concentration of human brain natriuretic peptide-like immunoreactivity is relatively low in the atria, ranging from 0.2-3.6% of atria1 natriuretic peptide, and the levels found in the ventricles are only l-7% of those occurring in atria1 extracts [13,17,181. A similar differential distribution and relative concentration of brain natriuretic peptide immunoreactivity has also been described in porcine heart [19]. Mature brain and atria1 natriuretic peptides have been shown to be co-secreted from cultured porcine atria1 myocytes [20], but recent studies in the rat indicate that autonomic and sensory neurones may represent a further source of natriuretic peptides in the heart [6,21]. In the present study we have used immunohistochemical techniques and specific antisera raised to amino- and carboxy-terminal regions of the atria1 natriuretic peptide precursor and to brain natriuretic peptide l-26 in order to compare the
cellular and ultrastructural localization of both peptides in the human and porcine heart. Materials and Methods Tissues Samples of human atria and ventricles were obtained from the failing explanted hearts of 22 consecutive patients (age 12-63 years) undergoing primary cardiac transplantation. These patients comprised 20 males and 2 females, 9 of whom suffered from ischaemic heart disease, 8 from dilated cardiomyopathy, 3 from valvular disease and 2 from anthracyclin-induced cardiomyopathy. All the patients were in New York Heart Association functional class IV, with left ventricular ejection fractions of less than 30%. Left and right residual atria1 cuffs, ventricular free walls and apical regions of the right ventricular septum were sampled immediately after excision. Right ventricular endomyocardial biopsies (l-3 per case) were obtained using a bioptome inserted through the exposed right atrium, from 97 donors (age 4-50 years) with apparently normal hearts which were considered suitable for transplantation. Further samples were also taken from a donor heart which was found to be unsuitable for transplantation as it belonged to a chronic hepatitis B “s” antigen carrier. Human fetal hearts (n = 4; 9-14 weeks gestation) were obtained following legal abortion, for reasons other than suspected cardiac abnormality. Additional tissues were also taken from the atria and ventricles of neonatal (n = 3) and adult pigs (n = 3) immediately after death. All the tissues were collected following the ethical standards of the institutions in which they were obtained. Immunohistochemistry Surgical samples and endomyocardial biopsies were fixed by immersion in a 10% buffered formalin solution for 12-24 hours, dehydrated and embedded in wax. Serial 4-5 pm thick sections were subsequently taken for haematoxylin-eosin, Masson’s trichrome and immunohistochemical staining. Sections for immunostaining were de-
waxed, rehydrated through a graded series of ethanol concentrations and endogenous peroxidase activity inhibited by incubation in a solution of 0.3% hydrogen peroxide in 0.15 M Tris buffered saline (pH 7.4) for 30 minutes at room temperature. After rinsing in buffer, the sections were overlayed with normal goat serum (diluted 1 : 30) for 30 minutes and then incubated, overnight at 4°C with diluted primary antisera. Sections were subsequently overlayed with biotinylated goat anti-rabbit immunoglobulin (Vector Laboratories, Peterborough, U.K.) and then with avidin-biotin-peroxidase complex (Vector Laboratories) for 1 hour each at room temperature. Peroxidase activity was localised using a glucose oxidase-nickel enhancement method [22], with 3,3’-diaminobenzidine-tetrahydrochloride (Sigma Chemical Co., St. Louis) as chromogen in 0.1 M acetate buffer, pH 6.0. Human fetal hearts and pig cardiac tissues were fixed by immersion in a modified Bouin’s solution for 16-24 hours at 4°C rinsed in several changes of phosphate buffered saline (0.01 M, pH 7.2) containing 15% sucrose and processed for indirect immunofluorescence staining [231. Briefly, cryostat sections (15 Frn thick) were cut at - 25°C mounted on Vectabond treated slides (Vector Laboratories) and air-dried for 1 hour at room temperature. They were then immersed in buffer containing 0.2% Triton X-100 for 30 minutes and stained with pontamine sky blue (BDH Chemicals, U.K.). The sections were subsequently overlayed with diluted primary antisera, overnight at 4°C and then with fluorescein isothiocyanate labelled sheep anti-rabbit immunoglobulin (diluted 1: 100; Tago Inc., U.S.A.), for 1 hour at room temperature, and mounted in glycerol mixed in equal parts with phosphate buffered saline. Electron
microscopy
Small fragments of atria and ventricle tissues from donor hearts, with an ischaemic duration of less than 100 minutes, were fixed in a solution of 2% paraformaldehyde and 1% glutaraldehyde in 0.1 M cacodylate buffer for 1 hour at 4°C. After rinsing overnight in cacodylate buffer, some tissue fragments were post-fixed in 1.5% osmium
tetroxide in 0.1 M cacodylate buffer for 1 hour at 4°C. All the tissue samples were subsequently dehydrated in a graded series of ethanol concentrations and infiltrated with LR White resin or cleared in propylene oxide and infiltrated with Epon. Ultrathin sections were collected on uncoated or formvar carbon-coated 200 mesh nickel grids. For the ultrastructural localisation of peptide immunoreactivity, grid-mounted ultrathin sections were immunostained using either single, double or triple immunogold staining techniques as previously described [24-271. Single immunogold labelling was performed using either goldlabelled protein A or goat anti-mouse immunoglobulin. The immunogold labelling of two antigens in the same section was achieved with four different methods, comprising the double face technique of Bendayan [24]; sequential double immunostaining with 20 and 10 nm labelled goat anti-rabbit immunoglobulin and exposure to paraformaldehyde vapour in order to denature free binding sites [2.5]; applying a formvar membrane coating to the first immunostained surface prior to labelling the opposite side of the ultrathin section [26] and finally by simultaneous labelling with two primary antisera raised in different species [27]. Triple immunostaining was achieved by denaturing free binding sites with paraformaldehyde vapour after the localisation of both the first and second antigens. At least 5 grids were immunostained for each sample. Sections of osmicated tissue were treated with 5% (w/v) sodium metaperiodate for 30 minutes prior to immunostaining [28]. Gold-labelled protein A (10 and 20 nm>, goat anti-rabbit immunoglobulin (5, 10 and 20 nm) and goat anti-mouse immunoglobulin (10 nm> were used at a dilution of 1 : 30 (Bioclinical Services Ltd., Cardiff, U.K., and Bio-Rad Laboratories Ltd., Hemel Hempstead, U.K.). All sections were counterstained with uranyl acetate and lead citrate and examined using a Zeiss transmission electron microscope (EM 10). Antisera
Region-specific polyclonal antisera were raised in rabbits against pure synthetic human atria1
240
natriuretic peptide 1-16 (cardiodilatin, code XA6), atria1 natriuretic peptide Tyr41 l-40 (cardiodilatin, code BE3) precursor sequences, and to alpha-atria1 natriuretic peptide l-28 (code 16321, as previously described [23,291. A brain natriuretic peptide selective antiserum (Code BNP33) was raised by immunising rabbits with porcine brain natriuretic peptide l-26 conjugated to bovine thyroglobulin in Freund’s adjuvant [6,21]. This antiserum displays at least 500 times greater affinity for brain than atria1 natriuretic peptide [21] and showed no detectable cross-reactivity with human cardiodilatin or alpha-atria1 natriuretic peptide sequences, in either liquid phase absorption or solid phase enzyme-linked immunoassay experiments. An additional purified mouse monoclonal antibody to atria1 natriuretic peptide 4-28 (code CBL 66, Cymbus Bioscience Ltd., Southampton, U.K.) was also employed. All the primary antisera were used at a dilution of 1 : 400-l : 800. Controls included the substitution of primary antisera with pre-immune serum, replacement of gold-labelled probes with inappropriate gold conjugates and the application of primary antisera preabsorbed (liquid phase) with natriuretic peptide sequences (0.1-10 PM; Peninsula Laboratories Europe, Ltd., St. Helens, U.K.). Results Immunohistochemistry
Human. Brain natriuretic peptide-like immunoreactivity was localized to myocardial cells in samples of right and left atria, from both failing explanted and donor hearts (Fig. 1). Whilst the immunostaining appeared less intense and extensive than that obtained using antisera raised to either human alpha-atria1 natriuretic peptide or cardiodilatin sequences, it displayed a similar distribution pattern and a characteristic perinuclear localization in myocardial cells. Brain natriuretic peptide-, alpha-atria1 natriuretic peptideand cardiodilatin-like immunoreactivity were not detected in any of the ventricle samples taken from donor hearts; however, they were observed
Fig. 1. Brain natriuretic peptide-like dial cells in the right atrium of a diomyopathy, immunostained using technique. Bar =
immunoreactive myocarpatient with dilated carthe avidin-biotin complex 50 pm.
in myocardial cells in the left (64%) and right ventricular free walls (23%) of failing explanted hearts, 4 of which displayed immunostaining in both chambers. The immunoreactivity for all three peptide sequences exhibited a differential distribution pattern, with immunoreactive myocardial cells concentrated in the subendocardial region of the ventricles. No immunoreactivity was localized to vascular endothelial, smooth muscle, endocardial or epicardial cells and cardiac nerves were not immunostained using antisera raised to natriuretic peptides. As in adult tissues, brain natriuretic peptide-like immunostaining of myocardial cells in the fetal heart appeared to be less intense than that observed with antisera to alpha-atria1 natriuretic peptide or cardiodilatin. A similar distribution pattern of immunostaining was also observed for all three sequences, with immunoreactivity concentrated in atria1 myocardium (Fig. 2). Atria1 natriuretic peptide and cardiodilatin immunoreactivity displayed a concentration gradient across the myocardium of both ventricle walls and were also localized to cells in the left and right bundles of the ventricular conduction system. Brain natriuretic peptide-like immunoreactivity was not, however, detected in human fetal ventricular myocardium (Fig. 2). Sections incubated without primary antisera, with
241
pre-immune sera or diluted antisera preabsorbed with corresponding peptide sequences (2 X 10e5 to lo-’ M) showed no detectable immunostaining (Fig. 3).
septum (Fig. 4). No immunostaining was detected in either porcine ventricular myocardial cells or in the cardiac innervation. Electron
Brain natriuretic peptide-like immunoreactivity also exhibited a similar distribution pattern to that of alpha-atria1 natriuretic peptide and cardiodilatin in both adult and neonatal porcine tissues, albeit at an apparently lower concentration. Immunostaining for all three sequences occurred throughout the atria1 myocardium and in cells of the ventricular conduction system, which traversed the interventricular
microscopy
Porcine.
Characteristic membrane-bound secretory granules were identified in all the atria specimens examined. No granules were found in donor ventricle tissues, but they were detected in left (32%) and right (9%) ventricular myocardial cells of failing explanted hearts (Table l), located in both perinuclear and peripheral regions of the sarcoplasm. The number of granules appeared to be
Fig. 2. Immunofluorescence micrographs showing cardiodilatin(A, C) and brain natriuretic peptide-like immunoreactivity (B, D) in serial sections of the right atrium (A, B) and subendocardial region of left ventricular free wall (C. D) of a human fetal heart at 12 weeks gestation. Bar = 50 pm.
atria1 and ventricular myocardial cells (Fig. 5). l-5% of secretory granules exhibited immunogold labelling which was indistinguishable from background levels and a further l-3% of granules displayed no labelling. There was no apparent difference in the immunogold labelling density using either of the double immunostaining techniques. No crossreactivity was found between antisera to cardiodilatin and alpha-atria1 natriuretic peptide. Brain natriuretic peptide-like immunostaining was abolished following preabsorption of diluted antiserum with brain natriuretic peptide l-26 (0.1-1.0 PM), but was unchanged by preabsorption with either amino- or carboxyterminal atria1 natriuretic peptide precursor sequences (20 FM).
Fig. 3. Control preparation demonstrating the abolition of immunofluorescence staining of human fetal atrial myocardium following absorption of the diluted brain’natriuretic peptide antiserum (BNP33) with lo-’ M brain natriuretic peptide l-26. Bar = 50 Fm.
Discussion
greater in both atria1 and ventricular myocardial cells in failing hearts compared to normal donors (Table 1). Atria1 secretory granules were immunolabelled with antisera raised to each of the three natriuretic peptide sequences, in all of the cases where suitable tissue was available, but in contrast to alpha-atria1 natriuretic peptide and cardiodilatin, brain natriuretic peptide-like immunoreactivity was only detected in non-osmicated tissues (Table 2). Double and triple immunogold labelling demonstrated that alpha-atria1 natriuretic peptide-, cardiodilatin- and brain natriuretic peptide-like immunoreactivity were colocalized to the same secretory granules in both
The cellular and ultrastructural localization of brain and atria1 natriuretic peptide-like immunoreactivity suggests that, in contrast to the central nervous system, there is dual expression of brain and atria1 natriuretic peptide genes in the human and porcine heart and that translated peptides are co-stored in the same secretory granules. The distribution and relative amount of natriuretic peptide immunoreactivity in the heart, demonstrated by immunohistochemical means, is in accord with radioimmunoassay data obtained for both human and porcine tissue extracts, indicating that immunoreactivity is concentrated in the atria and the amount of brain natriuretic
TABLE 1 Incidence of secretory granules in human atrial and ventricular myocardial cells Right atrium
Left atrium
n
n
%
No.
Right ventricle *
Left ventricle n
%
No.
n
%
No.
%
No.
Normal heart
100
5.5 f 0.8
1
100
ND
98
0
0
Failing heart 100 22
35.2 +- 2.8
22
100
23.1 f 2.2
22
9
2.6 + 0.6
1
n = number of cases examined; % = percentage
1
0
22
32
0 2.4 f 0.03
of granule-containing myocardial cells; ND = not determined; * = right ventricle samples comprised of tissues from 1 unused donor heart and endomyocardial biopsies from a further 97 transplanted donor hearts; No. = number of granules identified in granule-containing cells expressed as the mean f SEM. A minimum of 50 (usually 80-90) myocardial cells were examined in each sample of atrium and ventricle from every case.
243
peptide-like immunoreactivity is relatively low in the normal heart [13,17,19]. Despite the much lower affinity of the brain natriuretic peptide antiserum for alpha-atria’ natriuretic peptide 1-28 compared to brain natriuretic peptide l-26 [21] and the apparent lack of cross-reactivity with other natriuretic peptide sequences, demonstrated in both preabsorption and enzyme linked immunoassay experiments, the precise structure of the brain natriuretic peptide-like material identified in the human and porcine heart is uncertain and has therefore been referred to as “brain natriuretic peptide-like immunoreactivity”.
The results of initial immunoblotting experiments (data not shown) indicate, however, that it is distinct from the 13 kDa molecular form of atria1 natriuretic peptide precursor found in extracts of human atria [30]. Previous experiments to characterise the specificity of the brain natriuretic peptide antiserum also indicate that it does not recognise the 5 kDa cardiac peptide which occurs in extracts of the rat heart and corresponds to the carboxy-terminal 45 amino acid sequence of the rat brain natriuretic peptide precursor [ 11,211. The major molecular forms of brain natriuretic peptide in the human heart are thought to be the
Fig. 4. Immunofluorescence micrographs demonstrating atria1 natriuretic peptide-(A, C) and brain natriuretic peptide-like immunoreactivity (C, D) in serial sections of neonatal porcine left atrium (A, B) and interventricular septum (C, D). Characteristic perinuclear immunostaining occurs in both atria1 myocardium (A, B) and cells of the ventricular conduction system (C, D. arrows). Bar = 50 urn.
244
brain natriuretic precursor l-108 and the carboxy-terminal 77-108 amino acid (brain natriuretic peptide l-32) sequence [ill, whereas in the porcine heart brain natriuretic peptide-like immunoreactivity exists mainly as the larger precursor molecule [12]. The parallel distribution of brain and atria1 natriuretic peptide-like immunoreactivity in atria1 myocardium is consistent with studies indicating that immunoreactive forms of both peptides are co-released from myocardial cells. The mechanism of cardiac brain natriuretic peptide release is not known, but the main secretory and circulating forms of brain natriuretic peptide, in both pig and man, appear to be small molecular weight sequences analogous to alphaatria1 natriuretic peptide l-28. ,,rain natriuretic peptide-like immunoreactivity has been identified in both porcine plasma and the perfusate of isolated neonatal hearts [193 and is co-secreted with
TABLE
2
Ultrastructural Case
1 2 3 4 5 6 7 8 9 10 11 12 13 14 1.5 16 17 18 19 20 21 22
atria1 natriuretic peptide immunoreactivity from cultured neonatal porcine atria1 myocytes as low molecular weight brain and atria1 natriuretic peptide sequences [20]. A low molecular weight form of brain natriuretic peptide-like immunoreactivity also occurs in human plasma [31] and like the atria1 natriuretic peptide, the heart seems to be the major source of circulating BNP-like immunoreactivity [ 181. The present findings confirm those of previous studies, demonstrating that failing human ventricles express ANP [23,30,32-351 and indicate that both brain and atria1 natriuretic peptide-like immunoreactivity are localized to secretory granules in a subpopulation of ventricular myocardial cells. It has been suggested that, in contrast to the atria, the production of ventricular atria1 natriuretic peptide is a constitutive process, with minimal peptide storage in myocardial secretory gran-
distribution
of natriuretic
peptide-like
immunoreactivity
LA
RA Card
ANP
+ + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + +
BNP
+ + -
+ + + + + + + + + + + +
in explanted
human
hearts LV
RV
Card
ANP
BNP
Card
ANP
+ + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + +
_ + _ + _
0
0
0
0
0
0
0
0
0
0
0 0 0 0
0
+
+ + + _ + + + + -t + + + +
BNP
Card
ANP
0
0 +
0
+ + + +
0 + + + + +
0
0
0
0
+
+ 0
_
+
0 0
0
0
0
0
0 0
0
0
0
0
+
+
+
+
0 _
0
0
0
0
0
0
0 0 0 0 0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0
0 0
0
RA = right atrium; LA = left atrium; RV = right ventricle; LV = left ventricle; Card = cardiodilatin; ANP = alpha-atria1 peptide; BNP = brain natriuretic peptide; + = immunogold labelling of secretory granules; 0 = no immunoreactive granules detected; - = not determined due to absence of non-osmicated tissue samples.
BNP
0
natriuretic secretory
ules [36]. Characteristic secretory granules have, however, been identified displaying atria1 natriuretic peptide immunoreactivity in both cultured neonatal rat ventricular myocytes [37] and ventricular myocardial cells in cardiomyopathic hamsters [38]. Although relatively few in number, the immunogold labelling of secretory granules in human ventricles, suggests that the synthesis, storage and release of natriuretic peptides in failing ventricular myocardium may occur, in part at least, via a regulated uathway similar to that found in the atria. Hypertrophied rat ventricular myocytes also appear to possess the cellular mechanisms required for the regulated secretion of natriuretic peptides [39]. As in previous studies
of ventricular atria1 natriuretic peptide expression in failing hearts with dilated cardiomyopathy, valvular disease and myocardial infarction [23,30,32-351, myocardial cells in the ventricles of failing hearts, but not in normal donors, displayed both brain and atria1 natriuretic peptidelike immunoreactivity. A subendocardial to epicardial gradient was observed in the distribution of ventricular natriuretic peptide immunoreactivity, with the preferential localization of brain and atria1 natriuretic peptide-like immunoreactive myocytes in subendocardial regions of the left ventricle. As this region of the ventricular wall is thought to be subject to the highest levels of stress 1391it is possible that common factors such
Fig. 5. Double immunogold labelling of left atrium from a patient with ischaemic heart disease (A) showing cardiodilatin(20 nm gold particles) and brain natriuretic peptide-like immunoreactivity (10 nm gold particles) localized to several secretory granules. Cardiodilatin(B) and brain natriuretic peptide-like immunoreactivity (C) arc also localized (10 nm gold particles) to single secretory granules in the left ventricle of a patient with ischaemic heart disease (B) and the right ventricle of a patient with anthracyclin-induced cardiomyopathy (C). Bar = 500 nm.
246
as wall tension regulate the ventricular expression of both peptides. Tissue and plasma levels of atria1 natriuretic peptide correlate with indices of the severity of heart failure [40-431 and in accordance with the proposed co-storage and release of natriuretic peptides from myocardial cells, plasma brain natriuretic peptide levels have also been found to rise in heart failure [183. The augmentation of ventricular natriuretic peptide immunoreactivity seems to be independent of the underlying pathological condition leading to heart failure and there appears to be a differential effect on atria1 and brain natriuretic peptide expression and secretion. Thus, compared to atria1 natriuretic peptide, a proportionally greater increase in ventricular and plasma brain natriuretic peptide-like immunoreactivity was found in patients with heart failure [18]. The mechanism and significance of this differential response is not known, but may reflect differences in gene regulation, transcription, peptide processing and the clearance rates of mature circulating peptides. Northern blot analysis indicates that the ventricles represent a major site of cardiac brain natriuretic expression in the failing heart and the increase in plasma levels is thought to reflect the augmented expression, and release of brain natriuretic peptide from the ventricles 1181. The relative significance of cardiac brain and atria1 natriuretic peptide expression remains to be established, however, the apparent co-storage of the two peptides in atria1 and ventricular myocardial cells, during fetal, neonatal and adult stages of deveIopment, suggests that similar factors may regulate the storage and release of both peptides in the human and porcine heart.
3
4
5
6
7
8
9
10
11
12
13
14
Acknowledgements The authors wish to thank Patricia Harley for excellent technical assistance. References 1 Sudoh T. Kangawa K, Minamino N, Matsuo H. A new natriuretic peptide in porcine brain. Nature 1988;332:7881. 2 Sudoh T, Minamino N, Kangawa K, Matsuo H. C-type
15
16
natriuretic peptide (CNP): a new member of natriuretic peptide family identified in porcine brain. Biochem Biophys Res Commun 1990;168:863-870. McGregor A, Richards M, Espiner E, Yandle T, Ikram H. Brain natriuretic peptide administered to man: actions and metabolism. J Clin Endocrinol Metab 1990,70:1103-1107. Kambayashi Y. Nakao K, Kimura H et al. Biological characterization of human brain natriuretic peptide (BNP) and rat BNP: species-specific actions of BNP. Biochem Biophys Res Commun 1990;173:599-605. Ueda S, Minamino N. Sudoh T, Kangawa K, Matsuo H. Regional distribution of immunoreactive brain natriuretic peptide in porcine brain and spinal cord. Biochem Biophys Res Commun 1988;155:733-739. Saper CB, Hurley KM, Moga MM et al. Brain natriuretic peptides: differential localisation of a new family of neuropeptides. Neurosci Lett 1989;96:29-34. Nakao K, ltoh H. Kambayashi Y et al. Rat brain natriuretic peptide. Isolation from rat heart and tissue distribution. Hypertension 1990;15:774-778. Maekawa K, Sudoh T, Furusawa M et al. Cloning and sequence analysis of cDNA encoding a precursor for porcine brain natriuretic peptide. Biochem Biophys Res Commun 1988;157:410-416. Aburaya M, Hino J, Minamino N, Kangawa K, Matsuo H. Isolation and identification of rat brain natriuretic peptides in cardiac atrium. Biochem Biophys Res Commun 1989;163:226-232. Sudoh T, Maekawa K, Kojima M, Minamino N, Kangawa K, Matsuo H. Cloning and sequence analysis of cDNA encoding a precursor for human brain natriuretic peptide. Biochem Biophys Res Commun 1989;159:1427-1434. Hino J, Tateyama H, Minamino N, Kangawa K, Matsuo H. Isolation and identification of human natriuretic peptides in cardiac atrium. Biochem Biophys Res Commun 1990;167:693-700. Minamino N. Kangawa K, Matsuo H. Isolation and identification of a high molecular weight brain natriuretic peptide in porcine cardiac atrium. Biochem Biophys Res Commun 1989;157:402-409. Kambayashi Y, Nakao K, Mukoyama M et al. Isolation and sequence of human brain natriuretic.peptide in human atrium. FEBS Lett 1990;259:341-345. Thibault G, Haile-Meskel H, Wrobel-Konrad E et al. Processing of the atrial natriuretic factor propeptide by atrial cardiocytes as revealed by immunocryoultramicrotomy. Endocrinology 1989;124:3109-3116. Meleagros L, Ghatei MA, Gibbs JSR, Bloom SR. Pro-atria1 natriuretic peptide (l-98): The circulating cardiodilatin in man. Peptides 1989;10;545-550. Buckley MG, Sagnella GA, Markandu ND, Singer DRJ, MacGregor GA. Immunoreactive N-terminal pro-atria1 natriuretic peptide in human plasma: plasma levels and comparisons with alpha human atria1 natriuretic peptide in normal subjects, patients with essential hypertension, cardiac transplant and chronic renal failure. Clinical Science 1989:77:573-579.
247 Tateyama H. Hino J, Minamino N. Kangawa K. Ogihara T, Matsuo H. Characterisation of immunoreactive brain natriuretic peptide in human cardiac atrium. Biochem Biophys Res Commun 1990:166:1080-1087. Mukoyama M. Nakao K, Hosoda K et al. Brain natriuretic peptide as a novel cardiac hormone in humans. Evidence for an exquisite dual natriuretic peptide system, atria1 natriuretic peptide and brain natriuretic peptide. J Clin Invest lY91:87:1402-1412. Saito Y, Nakao K, Itoh H et al. Brain natriuretic peptide is a novel cardiac hormone. Biochem Biophys Res Commun lY89:158:360-36X. 20 lida T. Hirata Y. Takemura N. Togashi K, Nakagawa S, Marumo F. Brain natriuretic peptide is cosecreted with atrial natriuretic peptide from porcine cardiocytes. FEBS Lett lY90;26O:YX-100. 21 Saper CB. Kibbe MR. Hurley KM et al. Brain natriuretic peptide-like immunoreactive innervation of the cardiovascular and cerebrovascular systems in the rat. Circ Res 1990:67:1345-1354. 22 Shu S. Ju G. Fan L. The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system. Neurosci Lett lY88;85:169-171. RH. Springall D et al. Localisation 73 Wharton J. Anderson of atrial natriuretic peptide immunoreactivity in the ventricular myocardium and conduction system of the human fetal and adult heart. Br Heart J 1988;60:?67-274. M. Double immunocytochemical labelling ap24 Bendayan plying the protein A-gold technique. J Histochem Cytochem 198?;30:81-85. LI. Simultaneous demonstration of 2s Wang BL. Larsson multiple antigens>by indirect immunofluorescence or immunogold staining. Histochemistry 1985;83:47-56. 2h Merighi A. Polak JM. Fumagalli G. Theodosis DT. Ultrastructural localisation of neuropeptides and GABA in rat dorsal horn: a comparison of different immunogold labelling techniques. J Histochem Cytochem 1989;37:529540. IM, Polak JM. Double immunostaining proce‘I Varndell dures: techniques and applications. In: Polak JM, Varndell IM. eds. Immunolabelling for electron microscopy. Amsterdam: Elsevier/North-Holland. 1984;155-177. 28 Bendayan M. Zollinger M. Ultrastructural localisation of antigenic sites on osmium-fixed tissues applying the protein A-gold technique. J Histochem Cytochem 1983;31: 101-100. 29 Meleagros L, Ghatei MA, Anderson JV et al. The presence and molecular forms of cardiodilatin immunoreactivity in the human and rat right atrium. Clin Chim Acta 1988:172:199-210. 30 Arbustini E, Pucci A. Grass0 M et al. Expression of natriuretic peptide in ventricular myocardium of failing human hearts and its correlation with the severity of clinical and haemodynamic impairment. Am J Cardiol lY90:66:973-980.
31 Togahsi K, Hirata Y. Ando K, Takei Y. Kawakimi M. Marumo F. Brain natriuretic peptide-like immunoreactivity is present in human plasma. FEBS Lett lY89:250:235237. 32 Edwards BS. Ackermann DM, Lee ME, Reeder GS, Wold LE. Burnett JC Jr. Identification of atrial natriuretic factor within ventricular tissue in hamsters and humans with congestive heart failure. J Clin Invest lYXX;Xl:82-86. 33 Tsuchimochi H. Kurimoto F. Ieki K et al. Atrial natriuretic peptide distribution in fetal and failed adult hearts, Circulation 1988;78:9?0-927. 34 Saito Y. Nakao K. Arai H et al. Augmented expression of atrial natriuretic polypeptide gene in ventricle of human failing heart. J Clin Invest 198Y:83:298-305. G. Fujiwara H, Horike K et al. Ventricular 35 Takemura expression of atria1 natriuretic polypeptide and its relations with hemodynamics and histology in dilated human hearts - immunohistochemical study of the endomyocardial biopsy specimens. Circulation 198Y:80: 1137-I 147. 36 Bloch KD. Seidman JG, Naftilan JD. Fallon JT, Seidman CE. Neonatal atria and ventricles secrete atrial natriuretic factor via tissue-specific pathways. Cell lVXh:47:6Y5-707. S et al. Ventricular 37 Hassall CJS, Wharton J, Gulbenkian and atrial myocytes of newborn rats synthesist: and secrete atria1 natriuretic peptide in culture: light- and rlectron-microscopic localisation and chromatographic examination of stored and secreted molecular forms. Cell Tiss Res 198X:25l:lhl-169. G. Gutkowska J et al. Cardiac and 38 Ding J. Thibault plasma atria1 natriuretic factor in experimental congestive heart failure. Endocrinology lYX7;121:248-257. 39 Kinnunen P, Taskinen T, Jarvinen M. Ruskoaho H. Effect of phorbol ester on the release of atrial natriuretic peptide from the hypertrophied rat myocardium. Br J Pharmacol lYYl:lO2:4S3-461. L, Gibs JSR. Ghatei MA. Bloom SK. Increase 40 Meleayros in plasma concentrations of cardiodilatin (amino terminal pro-atria1 natriuretic peptide) in cardiac failure and during recumbency. Br Heart J 1988;60:3Y-44, H. Minamino N. Kangawa K. Matsuo 41 Hino J. Tateyama H. Isolation and identification of human natriuretic peptides in cardiac atrium. Biochrm Biophys Rrs Commun 1990: 167:693-700. A. Nakao K, Morii N et al. Synthesis of atria1 42 Sugawara natriuretic polypeptide in human failing hearts. Evidence of altered processing of atria1 natriuretic polypeptide precursor and augmented synthesis of P-human ANP. J Clin Res lY88;81:1962-1970. H. Umeda Y, Nishikawa M et al. Heart with 43 Matsubara circulatory ,failure secretes and processes atrial natriuretic peptide in a manner different from normal heart. Clin Cardiol 19X8:11: 197-203.
CARDJO
0167-5273/92/$05.00
01394
Localization of brain and atria1 natriuretic peptide in human and porcine heart Angela Pucci lT2,John Wharton r, Eloisa Arbustini ‘, Maurizia Grass0 2, Marta Diegoli Philip Needleman 4, Mario Viganb 3, Gonzalo Moscoso 5 and Julia M. Polak ’
2,
’ Department of Histochemistry, Royal Postgraduate Medical School, London, U.K.; ’ lstituto di Anatomia Patologica and .’ Ditisione di Cardiochirurgia, Istituto di Ricotsero e Cura a Carattere Scientific0 (IRCCS), Policlinico “S. Matteo’: Par,ia. Italy; ‘Monsanto Corporation, St Louis, Missouri. U.S.A.; ‘Department of Morbid Anatomy, Kings College Hospital. London U.K. (Received
22 July 1991: accepted
3 October
1991)
Pucci A, Wharton J, Arbustini E, Grass0 M, Diegoli M, Needleman P, Vigano M, Moscoso G, Polak JM. Localization of brain and atria1 natriuretic peptide in human and porcine heart. Int J Cardiol 1992;34:237-247. We have compared the localization of brain and atrial natriuretic peptide-like immunoreactivity in human and porcine hearts, using immunohistochemical techniques at both the light and ultrastructural level and specific antisera to amino-(cardiodilatin) and carboxy-terminal regions of the atrial natriuretic precursor molecule and to brain natriuretic peptide. Atrial myocardial cells in human fetal, normal adult and failing explanted hearts, displayed immunoreactivity for both brain and atrial natriuretic peptide-like sequences. At the subcellular level, brain natriuretic peptide-, cardiodilatinand alpha-atria1 natriuretic peptide-like immunoreactivity were co-localized to secretory granules in atrial myocardial cells. Immunoreactivity was also detected in the left (64%) and right ventricular free walls (23%) of 22 failing explanted hearts, but not in donor cardiac tissues. A gradient of natriuretic peptide immunostaining was observed across ventricular free walls and immunoreactivity for both natriuretic peptide sequences co-localized to secretory granules in a subpopulation of myocardial cells, concentrated in subendocardial regions of the ventricular walls. Brain and atrial natriuretic peptide-like immunoreactivity were also demonstrated in porcine atrial myocardium and cells of the ventricular conduction system. The parallel distribution of cardiac brain and atrial natriuretic peptide-like immunoreactivity suggests a dual regulation and co-storage of the natriuretic peptides in human and porcine hearts.
Key words: Atrial natriuretic peptide; chemistry; Conduction system
Brain natriuretic
Correspondence to: Dr. J. Wharton, Dept. of Histochemistry, Royal Postgraduate Medical School, Du Cane Road, London W12 ONN, U.K. A. Pucci was supported by a grant from the “Collegio Nuovo” Pavia and grant “Trapianto Cardiaco” from the Health Ministry to IRCCS Policlinico “S. Matteo”, Pavia 27100, Italy. The work was supported by the Council for Tobacco Research, U.S.A.
peptide;
Electron
microscopy;
Immunohisto-
Introduction
A family of homologous natriuretic peptides have now been identified in the mammalian heart and nervous system and implicated in regulating body fluid and electrolyte balance, comprising atria1 natriuretic peptide, brain natriuretic pep-
238
tide and C-type natriuretic peptide [1,2]. Although they are derived from distinct genes, brain and atria1 natriuretic peptides have a homologous structure and display a similar, but species dependent, range of effects [3,4]. Although originally identified in the brain [l] and shown to have a distinct distribution to atria1 natriuretic peptide in the nervous system [5-71, a number of studies have now reported the cloning of complementary deoxyribonucleic acid sequences encoding the brain natriuretic peptide precursor in the heart [g-lo]. Whilst several endogenous molecular forms of the peptide have also been isolated from mammalian cardiac tissues [7-9,11-131, the cellular localization of cardiac brain natriuretic peptide has not been established. Atria1 natriuretic peptide is produced and stored as a high molecular weight precursor in atria1 secretory granules [14] and proteolytic cleavage, during or soon after release, gives rise to active carboxy- and amino-terminal peptide sequences [15,16]. The processing pathway of the porcine brain natriuretic peptide precursor is similar to that of the atria1 peptide in mammalian hearts, whereas in rat and human cardiac tissues smaller molecular forms predominate, with brain natriuretic peptide 1-32 being the main storage and circulating form in man [7,11,12]. The concentration of human brain natriuretic peptide-like immunoreactivity is relatively low in the atria, ranging from 0.2-3.6% of atria1 natriuretic peptide, and the levels found in the ventricles are only l-7% of those occurring in atria1 extracts [13,17,181. A similar differential distribution and relative concentration of brain natriuretic peptide immunoreactivity has also been described in porcine heart [19]. Mature brain and atria1 natriuretic peptides have been shown to be co-secreted from cultured porcine atria1 myocytes [20], but recent studies in the rat indicate that autonomic and sensory neurones may represent a further source of natriuretic peptides in the heart [6,21]. In the present study we have used immunohistochemical techniques and specific antisera raised to amino- and carboxy-terminal regions of the atria1 natriuretic peptide precursor and to brain natriuretic peptide l-26 in order to compare the
cellular and ultrastructural localization of both peptides in the human and porcine heart. Materials and Methods Tissues Samples of human atria and ventricles were obtained from the failing explanted hearts of 22 consecutive patients (age 12-63 years) undergoing primary cardiac transplantation. These patients comprised 20 males and 2 females, 9 of whom suffered from ischaemic heart disease, 8 from dilated cardiomyopathy, 3 from valvular disease and 2 from anthracyclin-induced cardiomyopathy. All the patients were in New York Heart Association functional class IV, with left ventricular ejection fractions of less than 30%. Left and right residual atria1 cuffs, ventricular free walls and apical regions of the right ventricular septum were sampled immediately after excision. Right ventricular endomyocardial biopsies (l-3 per case) were obtained using a bioptome inserted through the exposed right atrium, from 97 donors (age 4-50 years) with apparently normal hearts which were considered suitable for transplantation. Further samples were also taken from a donor heart which was found to be unsuitable for transplantation as it belonged to a chronic hepatitis B “s” antigen carrier. Human fetal hearts (n = 4; 9-14 weeks gestation) were obtained following legal abortion, for reasons other than suspected cardiac abnormality. Additional tissues were also taken from the atria and ventricles of neonatal (n = 3) and adult pigs (n = 3) immediately after death. All the tissues were collected following the ethical standards of the institutions in which they were obtained. Immunohistochemistry Surgical samples and endomyocardial biopsies were fixed by immersion in a 10% buffered formalin solution for 12-24 hours, dehydrated and embedded in wax. Serial 4-5 pm thick sections were subsequently taken for haematoxylin-eosin, Masson’s trichrome and immunohistochemical staining. Sections for immunostaining were de-
waxed, rehydrated through a graded series of ethanol concentrations and endogenous peroxidase activity inhibited by incubation in a solution of 0.3% hydrogen peroxide in 0.15 M Tris buffered saline (pH 7.4) for 30 minutes at room temperature. After rinsing in buffer, the sections were overlayed with normal goat serum (diluted 1 : 30) for 30 minutes and then incubated, overnight at 4°C with diluted primary antisera. Sections were subsequently overlayed with biotinylated goat anti-rabbit immunoglobulin (Vector Laboratories, Peterborough, U.K.) and then with avidin-biotin-peroxidase complex (Vector Laboratories) for 1 hour each at room temperature. Peroxidase activity was localised using a glucose oxidase-nickel enhancement method [22], with 3,3’-diaminobenzidine-tetrahydrochloride (Sigma Chemical Co., St. Louis) as chromogen in 0.1 M acetate buffer, pH 6.0. Human fetal hearts and pig cardiac tissues were fixed by immersion in a modified Bouin’s solution for 16-24 hours at 4°C rinsed in several changes of phosphate buffered saline (0.01 M, pH 7.2) containing 15% sucrose and processed for indirect immunofluorescence staining [231. Briefly, cryostat sections (15 Frn thick) were cut at - 25°C mounted on Vectabond treated slides (Vector Laboratories) and air-dried for 1 hour at room temperature. They were then immersed in buffer containing 0.2% Triton X-100 for 30 minutes and stained with pontamine sky blue (BDH Chemicals, U.K.). The sections were subsequently overlayed with diluted primary antisera, overnight at 4°C and then with fluorescein isothiocyanate labelled sheep anti-rabbit immunoglobulin (diluted 1: 100; Tago Inc., U.S.A.), for 1 hour at room temperature, and mounted in glycerol mixed in equal parts with phosphate buffered saline. Electron
microscopy
Small fragments of atria and ventricle tissues from donor hearts, with an ischaemic duration of less than 100 minutes, were fixed in a solution of 2% paraformaldehyde and 1% glutaraldehyde in 0.1 M cacodylate buffer for 1 hour at 4°C. After rinsing overnight in cacodylate buffer, some tissue fragments were post-fixed in 1.5% osmium
tetroxide in 0.1 M cacodylate buffer for 1 hour at 4°C. All the tissue samples were subsequently dehydrated in a graded series of ethanol concentrations and infiltrated with LR White resin or cleared in propylene oxide and infiltrated with Epon. Ultrathin sections were collected on uncoated or formvar carbon-coated 200 mesh nickel grids. For the ultrastructural localisation of peptide immunoreactivity, grid-mounted ultrathin sections were immunostained using either single, double or triple immunogold staining techniques as previously described [24-271. Single immunogold labelling was performed using either goldlabelled protein A or goat anti-mouse immunoglobulin. The immunogold labelling of two antigens in the same section was achieved with four different methods, comprising the double face technique of Bendayan [24]; sequential double immunostaining with 20 and 10 nm labelled goat anti-rabbit immunoglobulin and exposure to paraformaldehyde vapour in order to denature free binding sites [2.5]; applying a formvar membrane coating to the first immunostained surface prior to labelling the opposite side of the ultrathin section [26] and finally by simultaneous labelling with two primary antisera raised in different species [27]. Triple immunostaining was achieved by denaturing free binding sites with paraformaldehyde vapour after the localisation of both the first and second antigens. At least 5 grids were immunostained for each sample. Sections of osmicated tissue were treated with 5% (w/v) sodium metaperiodate for 30 minutes prior to immunostaining [28]. Gold-labelled protein A (10 and 20 nm>, goat anti-rabbit immunoglobulin (5, 10 and 20 nm) and goat anti-mouse immunoglobulin (10 nm> were used at a dilution of 1 : 30 (Bioclinical Services Ltd., Cardiff, U.K., and Bio-Rad Laboratories Ltd., Hemel Hempstead, U.K.). All sections were counterstained with uranyl acetate and lead citrate and examined using a Zeiss transmission electron microscope (EM 10). Antisera
Region-specific polyclonal antisera were raised in rabbits against pure synthetic human atria1
240
natriuretic peptide 1-16 (cardiodilatin, code XA6), atria1 natriuretic peptide Tyr41 l-40 (cardiodilatin, code BE3) precursor sequences, and to alpha-atria1 natriuretic peptide l-28 (code 16321, as previously described [23,291. A brain natriuretic peptide selective antiserum (Code BNP33) was raised by immunising rabbits with porcine brain natriuretic peptide l-26 conjugated to bovine thyroglobulin in Freund’s adjuvant [6,21]. This antiserum displays at least 500 times greater affinity for brain than atria1 natriuretic peptide [21] and showed no detectable cross-reactivity with human cardiodilatin or alpha-atria1 natriuretic peptide sequences, in either liquid phase absorption or solid phase enzyme-linked immunoassay experiments. An additional purified mouse monoclonal antibody to atria1 natriuretic peptide 4-28 (code CBL 66, Cymbus Bioscience Ltd., Southampton, U.K.) was also employed. All the primary antisera were used at a dilution of 1 : 400-l : 800. Controls included the substitution of primary antisera with pre-immune serum, replacement of gold-labelled probes with inappropriate gold conjugates and the application of primary antisera preabsorbed (liquid phase) with natriuretic peptide sequences (0.1-10 PM; Peninsula Laboratories Europe, Ltd., St. Helens, U.K.). Results Immunohistochemistry
Human. Brain natriuretic peptide-like immunoreactivity was localized to myocardial cells in samples of right and left atria, from both failing explanted and donor hearts (Fig. 1). Whilst the immunostaining appeared less intense and extensive than that obtained using antisera raised to either human alpha-atria1 natriuretic peptide or cardiodilatin sequences, it displayed a similar distribution pattern and a characteristic perinuclear localization in myocardial cells. Brain natriuretic peptide-, alpha-atria1 natriuretic peptideand cardiodilatin-like immunoreactivity were not detected in any of the ventricle samples taken from donor hearts; however, they were observed
Fig. 1. Brain natriuretic peptide-like dial cells in the right atrium of a diomyopathy, immunostained using technique. Bar =
immunoreactive myocarpatient with dilated carthe avidin-biotin complex 50 pm.
in myocardial cells in the left (64%) and right ventricular free walls (23%) of failing explanted hearts, 4 of which displayed immunostaining in both chambers. The immunoreactivity for all three peptide sequences exhibited a differential distribution pattern, with immunoreactive myocardial cells concentrated in the subendocardial region of the ventricles. No immunoreactivity was localized to vascular endothelial, smooth muscle, endocardial or epicardial cells and cardiac nerves were not immunostained using antisera raised to natriuretic peptides. As in adult tissues, brain natriuretic peptide-like immunostaining of myocardial cells in the fetal heart appeared to be less intense than that observed with antisera to alpha-atria1 natriuretic peptide or cardiodilatin. A similar distribution pattern of immunostaining was also observed for all three sequences, with immunoreactivity concentrated in atria1 myocardium (Fig. 2). Atria1 natriuretic peptide and cardiodilatin immunoreactivity displayed a concentration gradient across the myocardium of both ventricle walls and were also localized to cells in the left and right bundles of the ventricular conduction system. Brain natriuretic peptide-like immunoreactivity was not, however, detected in human fetal ventricular myocardium (Fig. 2). Sections incubated without primary antisera, with
241
pre-immune sera or diluted antisera preabsorbed with corresponding peptide sequences (2 X 10e5 to lo-’ M) showed no detectable immunostaining (Fig. 3).
septum (Fig. 4). No immunostaining was detected in either porcine ventricular myocardial cells or in the cardiac innervation. Electron
Brain natriuretic peptide-like immunoreactivity also exhibited a similar distribution pattern to that of alpha-atria1 natriuretic peptide and cardiodilatin in both adult and neonatal porcine tissues, albeit at an apparently lower concentration. Immunostaining for all three sequences occurred throughout the atria1 myocardium and in cells of the ventricular conduction system, which traversed the interventricular
microscopy
Porcine.
Characteristic membrane-bound secretory granules were identified in all the atria specimens examined. No granules were found in donor ventricle tissues, but they were detected in left (32%) and right (9%) ventricular myocardial cells of failing explanted hearts (Table l), located in both perinuclear and peripheral regions of the sarcoplasm. The number of granules appeared to be
Fig. 2. Immunofluorescence micrographs showing cardiodilatin(A, C) and brain natriuretic peptide-like immunoreactivity (B, D) in serial sections of the right atrium (A, B) and subendocardial region of left ventricular free wall (C. D) of a human fetal heart at 12 weeks gestation. Bar = 50 pm.
atria1 and ventricular myocardial cells (Fig. 5). l-5% of secretory granules exhibited immunogold labelling which was indistinguishable from background levels and a further l-3% of granules displayed no labelling. There was no apparent difference in the immunogold labelling density using either of the double immunostaining techniques. No crossreactivity was found between antisera to cardiodilatin and alpha-atria1 natriuretic peptide. Brain natriuretic peptide-like immunostaining was abolished following preabsorption of diluted antiserum with brain natriuretic peptide l-26 (0.1-1.0 PM), but was unchanged by preabsorption with either amino- or carboxyterminal atria1 natriuretic peptide precursor sequences (20 FM).
Fig. 3. Control preparation demonstrating the abolition of immunofluorescence staining of human fetal atrial myocardium following absorption of the diluted brain’natriuretic peptide antiserum (BNP33) with lo-’ M brain natriuretic peptide l-26. Bar = 50 Fm.
Discussion
greater in both atria1 and ventricular myocardial cells in failing hearts compared to normal donors (Table 1). Atria1 secretory granules were immunolabelled with antisera raised to each of the three natriuretic peptide sequences, in all of the cases where suitable tissue was available, but in contrast to alpha-atria1 natriuretic peptide and cardiodilatin, brain natriuretic peptide-like immunoreactivity was only detected in non-osmicated tissues (Table 2). Double and triple immunogold labelling demonstrated that alpha-atria1 natriuretic peptide-, cardiodilatin- and brain natriuretic peptide-like immunoreactivity were colocalized to the same secretory granules in both
The cellular and ultrastructural localization of brain and atria1 natriuretic peptide-like immunoreactivity suggests that, in contrast to the central nervous system, there is dual expression of brain and atria1 natriuretic peptide genes in the human and porcine heart and that translated peptides are co-stored in the same secretory granules. The distribution and relative amount of natriuretic peptide immunoreactivity in the heart, demonstrated by immunohistochemical means, is in accord with radioimmunoassay data obtained for both human and porcine tissue extracts, indicating that immunoreactivity is concentrated in the atria and the amount of brain natriuretic
TABLE 1 Incidence of secretory granules in human atrial and ventricular myocardial cells Right atrium
Left atrium
n
n
%
No.
Right ventricle *
Left ventricle n
%
No.
n
%
No.
%
No.
Normal heart
100
5.5 f 0.8
1
100
ND
98
0
0
Failing heart 100 22
35.2 +- 2.8
22
100
23.1 f 2.2
22
9
2.6 + 0.6
1
n = number of cases examined; % = percentage
1
0
22
32
0 2.4 f 0.03
of granule-containing myocardial cells; ND = not determined; * = right ventricle samples comprised of tissues from 1 unused donor heart and endomyocardial biopsies from a further 97 transplanted donor hearts; No. = number of granules identified in granule-containing cells expressed as the mean f SEM. A minimum of 50 (usually 80-90) myocardial cells were examined in each sample of atrium and ventricle from every case.
243
peptide-like immunoreactivity is relatively low in the normal heart [13,17,19]. Despite the much lower affinity of the brain natriuretic peptide antiserum for alpha-atria’ natriuretic peptide 1-28 compared to brain natriuretic peptide l-26 [21] and the apparent lack of cross-reactivity with other natriuretic peptide sequences, demonstrated in both preabsorption and enzyme linked immunoassay experiments, the precise structure of the brain natriuretic peptide-like material identified in the human and porcine heart is uncertain and has therefore been referred to as “brain natriuretic peptide-like immunoreactivity”.
The results of initial immunoblotting experiments (data not shown) indicate, however, that it is distinct from the 13 kDa molecular form of atria1 natriuretic peptide precursor found in extracts of human atria [30]. Previous experiments to characterise the specificity of the brain natriuretic peptide antiserum also indicate that it does not recognise the 5 kDa cardiac peptide which occurs in extracts of the rat heart and corresponds to the carboxy-terminal 45 amino acid sequence of the rat brain natriuretic peptide precursor [ 11,211. The major molecular forms of brain natriuretic peptide in the human heart are thought to be the
Fig. 4. Immunofluorescence micrographs demonstrating atria1 natriuretic peptide-(A, C) and brain natriuretic peptide-like immunoreactivity (C, D) in serial sections of neonatal porcine left atrium (A, B) and interventricular septum (C, D). Characteristic perinuclear immunostaining occurs in both atria1 myocardium (A, B) and cells of the ventricular conduction system (C, D. arrows). Bar = 50 urn.
244
brain natriuretic precursor l-108 and the carboxy-terminal 77-108 amino acid (brain natriuretic peptide l-32) sequence [ill, whereas in the porcine heart brain natriuretic peptide-like immunoreactivity exists mainly as the larger precursor molecule [12]. The parallel distribution of brain and atria1 natriuretic peptide-like immunoreactivity in atria1 myocardium is consistent with studies indicating that immunoreactive forms of both peptides are co-released from myocardial cells. The mechanism of cardiac brain natriuretic peptide release is not known, but the main secretory and circulating forms of brain natriuretic peptide, in both pig and man, appear to be small molecular weight sequences analogous to alphaatria1 natriuretic peptide l-28. ,,rain natriuretic peptide-like immunoreactivity has been identified in both porcine plasma and the perfusate of isolated neonatal hearts [193 and is co-secreted with
TABLE
2
Ultrastructural Case
1 2 3 4 5 6 7 8 9 10 11 12 13 14 1.5 16 17 18 19 20 21 22
atria1 natriuretic peptide immunoreactivity from cultured neonatal porcine atria1 myocytes as low molecular weight brain and atria1 natriuretic peptide sequences [20]. A low molecular weight form of brain natriuretic peptide-like immunoreactivity also occurs in human plasma [31] and like the atria1 natriuretic peptide, the heart seems to be the major source of circulating BNP-like immunoreactivity [ 181. The present findings confirm those of previous studies, demonstrating that failing human ventricles express ANP [23,30,32-351 and indicate that both brain and atria1 natriuretic peptide-like immunoreactivity are localized to secretory granules in a subpopulation of ventricular myocardial cells. It has been suggested that, in contrast to the atria, the production of ventricular atria1 natriuretic peptide is a constitutive process, with minimal peptide storage in myocardial secretory gran-
distribution
of natriuretic
peptide-like
immunoreactivity
LA
RA Card
ANP
+ + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + +
BNP
+ + -
+ + + + + + + + + + + +
in explanted
human
hearts LV
RV
Card
ANP
BNP
Card
ANP
+ + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + +
_ + _ + _
0
0
0
0
0
0
0
0
0
0
0 0 0 0
0
+
+ + + _ + + + + -t + + + +
BNP
Card
ANP
0
0 +
0
+ + + +
0 + + + + +
0
0
0
0
+
+ 0
_
+
0 0
0
0
0
0
0 0
0
0
0
0
+
+
+
+
0 _
0
0
0
0
0
0
0 0 0 0 0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0
0 0
0
RA = right atrium; LA = left atrium; RV = right ventricle; LV = left ventricle; Card = cardiodilatin; ANP = alpha-atria1 peptide; BNP = brain natriuretic peptide; + = immunogold labelling of secretory granules; 0 = no immunoreactive granules detected; - = not determined due to absence of non-osmicated tissue samples.
BNP
0
natriuretic secretory
ules [36]. Characteristic secretory granules have, however, been identified displaying atria1 natriuretic peptide immunoreactivity in both cultured neonatal rat ventricular myocytes [37] and ventricular myocardial cells in cardiomyopathic hamsters [38]. Although relatively few in number, the immunogold labelling of secretory granules in human ventricles, suggests that the synthesis, storage and release of natriuretic peptides in failing ventricular myocardium may occur, in part at least, via a regulated uathway similar to that found in the atria. Hypertrophied rat ventricular myocytes also appear to possess the cellular mechanisms required for the regulated secretion of natriuretic peptides [39]. As in previous studies
of ventricular atria1 natriuretic peptide expression in failing hearts with dilated cardiomyopathy, valvular disease and myocardial infarction [23,30,32-351, myocardial cells in the ventricles of failing hearts, but not in normal donors, displayed both brain and atria1 natriuretic peptidelike immunoreactivity. A subendocardial to epicardial gradient was observed in the distribution of ventricular natriuretic peptide immunoreactivity, with the preferential localization of brain and atria1 natriuretic peptide-like immunoreactive myocytes in subendocardial regions of the left ventricle. As this region of the ventricular wall is thought to be subject to the highest levels of stress 1391it is possible that common factors such
Fig. 5. Double immunogold labelling of left atrium from a patient with ischaemic heart disease (A) showing cardiodilatin(20 nm gold particles) and brain natriuretic peptide-like immunoreactivity (10 nm gold particles) localized to several secretory granules. Cardiodilatin(B) and brain natriuretic peptide-like immunoreactivity (C) arc also localized (10 nm gold particles) to single secretory granules in the left ventricle of a patient with ischaemic heart disease (B) and the right ventricle of a patient with anthracyclin-induced cardiomyopathy (C). Bar = 500 nm.
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as wall tension regulate the ventricular expression of both peptides. Tissue and plasma levels of atria1 natriuretic peptide correlate with indices of the severity of heart failure [40-431 and in accordance with the proposed co-storage and release of natriuretic peptides from myocardial cells, plasma brain natriuretic peptide levels have also been found to rise in heart failure [183. The augmentation of ventricular natriuretic peptide immunoreactivity seems to be independent of the underlying pathological condition leading to heart failure and there appears to be a differential effect on atria1 and brain natriuretic peptide expression and secretion. Thus, compared to atria1 natriuretic peptide, a proportionally greater increase in ventricular and plasma brain natriuretic peptide-like immunoreactivity was found in patients with heart failure [18]. The mechanism and significance of this differential response is not known, but may reflect differences in gene regulation, transcription, peptide processing and the clearance rates of mature circulating peptides. Northern blot analysis indicates that the ventricles represent a major site of cardiac brain natriuretic expression in the failing heart and the increase in plasma levels is thought to reflect the augmented expression, and release of brain natriuretic peptide from the ventricles 1181. The relative significance of cardiac brain and atria1 natriuretic peptide expression remains to be established, however, the apparent co-storage of the two peptides in atria1 and ventricular myocardial cells, during fetal, neonatal and adult stages of deveIopment, suggests that similar factors may regulate the storage and release of both peptides in the human and porcine heart.
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Acknowledgements The authors wish to thank Patricia Harley for excellent technical assistance. References 1 Sudoh T. Kangawa K, Minamino N, Matsuo H. A new natriuretic peptide in porcine brain. Nature 1988;332:7881. 2 Sudoh T, Minamino N, Kangawa K, Matsuo H. C-type
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