JOURNAL OF PATHOLOGY, VOL.

165: 235-241 (1991)

ATRIAL AMYLOID DEPOSITS IN THE FAILING HUMAN HEART DISPLAY BOTH ATRIAL AND BRAIN NATRIURETIC PEPTIDE-LIKE IMMUNOREACTIVITY ANGELA PUCCI*T, JOHN WHARTON*, ELOISA ARBUSTINIT, MAURIZIA GRASSO?, MARTA DIEGOLIT, PHILIP NEEDLEMAN$, MARIO V I G A N O ~AND JULIA M. POLAK*

*Department of Histochernistry, RoyaI Postgraduate Medical School, London, U.K.; TIstituto di Anatornia Patologica and SDivisione di Cardiochirurgia, Istituto di Ricovero e Cura a Carattere Scientifico ( I R C C S ) Policilinico 'S.Matteo ', Pavia, Italy; IMonsanto Corporation , St Louis, Missouri, U.S.A. Received 7 March, 1991 Accepted 29 April, 1991

SUMMARY Atrial amyloid deposits are common in the ageing human heart and contain a-atrial natriuretic peptide (proANP,,-,,,) immunoreactivity. However, atrial myocytes secrete both amino and carboxy terminal fragments of the A N P prohormone (proANP, , 2 6 ) and also express an homologous, but separate brain natriuretic peptide (BNP). Characteristic amyloid deposits were identified in the atria of 9/22 patients (26-63 years of age) with end-stage heart failure. Amyloid fibrils displayed immunoreactivity for both amino and carboxy terminal fragments of proANP,-,,, and for the distinct BNP sequence. As in other endocrine organs, both mature and precursor peptide sequences appear to be constituents of amyloid fibrils. Whilst immunoreactivity for cardiac peptide hormones is co-localized in atrial amyloid deposits, it is uncertain whether the increase in natriuretic peptide expression which accompanies cardiac failure contributes to the incidence of isolated atrial amyloidosis. KEY

WORDS-Natriuretic

peptides, ANP, BNP, cardiodilatin, amyloid, heart failure, immunoelectron microscopy

INTRODUCTION Cardiac amyloidosis is a relatively common finding in elderly patients, 70-80 years or more of age, and consists of two types, involving either the ventricles as well as the atria (senile cardiac amyloid) or more frequently the atria alone (isolated atrial amyloid). Unlike senile cardiac amyloid, which is derived from prealbumin (tran~thyretin),~ immunohistochemical and biochemical studies indicate that mature atrial natriuretic peptide (ANP,,-,,, or aANPI-,,) is a major immunoreactive subunit of isolated atrial amyloid fibrils."' It is now established that cardiac myocytes synthesize and store ANP as a high molecular weight precursor sequence (proANP,-,,,) which undergoes proteolytic cleavage soon after release, giving rise to the mature carboxy Addressee for correspondence:Dr J. Wharton, Department of Histochemistry, Royal Postgraduate Medical School, Du Cane Road, London W12ONN, U.K. 0022-3417/91/110235-07 $05.00 0 1991 by John Wiley & Sons, Ltd.

terminal peptide a-ANP,-,, and amino terminal proANP,_,, (cardiodilatin) sequences. Immunoreactive a-ANP,-,, and cardiodilatin are secreted together in normal individuals and in patients with congestive heart failure. In the latter, plasma peptide levels correlate directly with the severity of the A distinct brain natriuretic peptide (BNP) is expressed in the human heart, albeit at a lower level than ANP, and also appears to be synthesized and stored as a prohormone (BNP,_,,J which gives rise to a mature carboxy terminal sequence BNP,,-,,, 'I Immunoreactive BNP circulates in (BNf)21-32). man and in cases of heart failure exhibits a proportionally greater increase in plasma concentration than u-ANP,-~,.'~. Several polypeptide hormones have been identified in amyloid fibril proteins, as both mature peptides and prohormone sequences, including procalcitonin in medullary carcinoma of the t h y r ~ i d , ' ~

236

A. PUCCl ET A L .

islet amyloid polypeptide or amylin in pancreatic islets in type 2 diabetes" and insulin in insulindependent type 1 diabetes.16 Studies of atrial amyloidosis have, however, focused on the incorporation of u-ANP,-,,, rather than proANP,-,, sequences, in fibril proteins and have not examined the possible presence of BNP immunoreactivity.6 In the present immunoelectron microscopic study, we have used region-specific polyclonal antisera to investigate the presence of both amino and carboxy terminal proANP sequences, as well as BNP-like immunoreactivity, in isolated atrial amyloid deposits of failing hearts from patients undergoing primary cardiac transplantation.

'

MATERIALS AND METHODS

Tissue sampling Tissue samples of the right and left atrial cuffs and the lateral walls of both ventricles were taken from the explanted failing hearts of 22 consecutive patients undergoing primary cardiac transplantation. The patients ranged in age from 12 to 63 years and comprised 20 males and 2 females. Nine patients suffered from ischaemic heart disease, eight from dilated cardiomyopathy, three from valve disease and two from anthracyclin-induced cardiomyopathy (Fig. 1). All the patients were in New York Heart Association (NYHA) functional class IV, with left ventricular ejection fractions of less than 30 per cent. None of the patients was referred because of systemic or cardiac amyloidosis and right ventricular endomyocardial biopsies from patients with dilated cardiomyopathy did not show amyloid storage. Light microscopy Samples for light microscopy were fixed by immersion in 10 per cent buffered formalin or Zamboni's solution" for 6-24 h, washed in Trisbuffered saline solution ( O . ~ M , pH 7.4) for 24h, dehydrated and embedded in paraffin. Serial sections (4-5 pm thick) were stained with haematoxylin-eosin, Masson's trichrome and Congo red. Electron microscopy Small fragments of atrial and ventricular tissue were immediately fixed in a solution of 2 per cent paraformaldehyde and 1 per cent glutaraldehyde in

0.1 M cacodylate buffer for 1 h at 4°C. After rinsing

overnight in cacodylate buffer, some tissue fragments were post-fixed in 1.5 per cent osmium tetroxide in 0.1 M cacodylate buffer for 1 h 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 and counterstained with uranyl acetate and lead citrate for conventional ultrastructural examination. Inimunoelectron microscopy For the ultrastructural localization of peptide immunoreactivity, grid-mounted ultrathin sections were immunostained using either single or double immunogold staining techniques as previously described." Sections of osmicated tissue were treated with 5 per cent (w/v) sodium metaperiodate prior to immunostaining. Gold-labelled protein A (1 0 and 20 nm), goat anti-rabbit IgG (10 and 20 nm) and goat antimouse IgG (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 EM 10 transmission electron microscope. Antisera Polyclonal antisera were raised in rabbits against pure synthetic human proANP,-,6 (cardiodilatin, code XA6), proANP Tyr4', 4o (cardiodilatin, code BE3), proANP,,-,,, (a-ANP, zx, code 1632) and porcine BNP,_,, Their specificity has been previously d e ~ c r i b e d . ' ~An , ' ~additional purified mouse monoclonal antibody to ANP,,, (code CBL 66, Cymbus Bioscience Ltd., Southampton, U.K.) and a polyclonal antiserum to islet amyloid polypeptide or amylin (code BT7) were also employed. All the primary antisera were used at a dilution of I :400-1:800. 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 (0.1-1 0 p ~ ;Peninsula Laboratories Europe, Ltd., St Helens, U.K.). Antisera specificity for natriuretic peptide sequences was also analysed using an enzyme-linked immunoassay system.

237

ANP AND BNP IN ATRIAL AMYLOID DEPOSITS

T a b l e I-Atrial

amyloid deposits and natriuretic peptide immunoreactivity Amyloid

Diagnosis

No.

RA

Ischaemic h e a r t disease Dilated c a r d i o m y o p a t h y Valve disease

619 1jS 213

-

-

Immunoreactivi ty

LA

Both

ANP

Card

BNP

1

2

216 oi 1 212

4/4*

-

3 1 2

516

-

l/l 212

ND 212

Ultrastructural evidence of amyloid in either the right atrium (RA), left atrium (LA) or both atria. ANP, alpha-atrial natriuretic peptide; Card, cardiodilatin; BNP, brain natriuretic peptide. *Non-osmicated samples available in only 4/6 cases in which amyloid deposits were identified. ND, not determined due to absence of non-osmicated samples.

7 0-

60-

;

50-

8 -

0 0

h

oe

0)

L-l

m

0

40-

a,

30-

0 0

20-

0

v

a,

n

Q

h

2

+

10-

___

50.9t 3.1

31 .5*4.1

41.3

I

I

I

IHD

DCM

VHD

(619)

(118)

(21’3)

13 1

A-CM (012)

Fig. I-Summary of patient population, relating age with the presence ( 0 )and absense (0)of detectable atrial amyloid deposits in cases of ischaemic heart disease (IHD), dilated cardiomyopathy (DCM), valvular heart disease (VHD) and anthracyclin-induced cardiomyopathy (A-CM), Values are expressed as the mean age (yr) k SEM

RESULTS Light microscopy Congo red staining, viewed with polarization microscopy, revealed typical apple-green birefringence in sections of atrial tissue from 5/22 cases.

Electron microscopy Ultrastructural analysis revealed the presence of typical amyloid fibrils in atrial samples from 9/22

cases (Table 1, Fig. I), this being four more than was distinguished by Congo red staining. Focal amyloid deposits were identified in the atria as early as 26 years of age, but the incidence of amyloidosis was found to increase with age (Fig. 1). The incidence of amyloidosis was greater in ischaemic heart disease patients (6/9) compared to those with dilated cardiomyopathy ( 1 /8), however, the mean ages of the two groups were significantly different (Fig. 1). Amyloid-like fibrils were approximately 10 nm in

238

A. PUCCI E r AL.

Fig. 2-Amyloid fibrils displaying a-ANP immunogold labelling in the atrial myocardium of patients with dilated cardiomyopathy (A) and ischaemic heart disease (B, C). Immunoreactive amyloid fibrils occur in both interstitial (A) and cellular deposits, with (B) and without (C) an apparent limiting membrane. Scale bar = 500 nm

diameter and occurred in linear, non-branching, aggregated deposits. Isolated, focal deposits of amyloid were found distributed between myocytes and around intramyocardial blood vessels. Occasionally, deposits also appeared to occur inside myocytes. These were generally considered to represent pseudo-inclusions, possessing a surrounding membrane. Some cellular deposits apparently lacked a limiting membrane. There was no obvious relationship between amyloid fibrils and either myocyte secretory granules or the sarcolemmal membrane. Finally, amyloid fibrils were never identified in ventricle samples.

Immunoelectron microscopy Antisera raised to the amino (cardiodilatin) and carboxy terminals (a-ANP,-,, or a-ANP,,,) of

proANP,_,, immunolabelled atrial amyloid fibrils in the cases in which amyloid deposits were identified by either Congo red staining or conventional electron microscopy. Extracellular amyloid deposits displayed specific high density gold labelling, with very low background labelling of the parenchyma (Fig. 2A). ANP immunoreactivity was also localized to pseudo-inclusions, with or without a limiting membrane (Fig. 2B and C). Immunoreactivity for amino terminal (cardiodilatin), as well as carboxy terminal proANP,_,,, sequences was localized to amyloid deposits (Fig. 3A). Whilst u-ANP,_,* and cardiodilatin immunoreactivity were detected in either osmicated ar nonosmicated tissue, BNP-like immunoreactivity could only be detected in the latter. Antiserum to BNP immunolabelled amyloid fibrils (Fig. 3B) in six of

A N P AND B N P IN ATRIAL AMYLOID DEPOSITS

239

Fig. 3-Cardiodilatin (A, C) and BNP (B, C) immunogold labelling localized to amyloid fibrils in the atria of patients with ischaemic heart disease (A, C) and valvular heart disease (B). Double immunogold labelling demonstrates the colocalization of cardiodilatin (10-nm diameter gold particles, single arrows) and BNP immunoreactivity to amyloid fibrils (20-nm diameter gold particles, double arrows). Arrowhead, BNP-like immunoreactivity localized to an atrial secretory granule (B). Scale bar = 500 nm

the cases displaying a-ANP,-,, and/or cardiodilatin immunoreactivity and in one case in which no ANP immunoreactivity was found (Table 1). Double immunogold labelling revealed the dual localization ofcardiodilatin and BNP immunoreactivity in atrial amyloid deposits (Fig. 3C). While only 9/22 cases were found to contain amyloid deposits, all the atrial tissues examined possessed myocardial secretory granules containing atrial and brain natriuretic peptide immunoreactivity. Amyloid polypeptide (amylin) immunoreactivity was not found in iso-

lated atrial amyloid deposits and no immunolabelling was detected when primary antisera were either replaced with preimmune serum or preabsorbed with appropriate peptide sequences (0.1-1.0 p ~ ) No cross-reactivity was found between amino terminal cardiodilatin and a-ANP,~,, or u-ANP,~, antisera. BNP-like immunostaining of amyloid deposits was abolished following preabsorption of thediluted antiserum with BNP,-,, (0.1-1-0 p ~ )but , was unchanged by preabsorption with either amino (ANP Tyr4’,40) or carboxy terminal (a-ANP,-,,)

.

240

A. P U C C I ET A L .

proANP sequences (10 p ~ )BNP . antiserum showed no cross-reactivity with u-ANP,-,,, cardiodilatin or rat BNP,-,, in an enzyme-linked immunoassay system. DISCUSSION

patients was also significantly greater ( P < 0.02) than those with other forms of heart disease. Tissue and plasma ANP levels correlate with indices of the severity ofheart failure, varying directly with NYHA functional class and inversely with left ventricular ejection f r a c t i ~ n . ~ ”As ~ ’all ~ ~the ’ ~ patients ~ in the present study exhibited severe impairment ofcardiac function prior to transplantation, it would appear that age-related factors, rather than increased natriuretic peptide expression, are a contributory component influencing the incidence of atrial amyloidosis.

The immunoelectron microscopic demonstration of immunoreactivity for both mature carboxy proANP,,,,, (u-ANP,_,,) and amino terminal proANP,-,, sequences in atrial amyloid deposits confirms and extends the findings of previous studies indicating that u-ANP,-,, is a major subunit of ACKNOWLEDGEMENTS atrial amyloid fibrils.“’ As in other forms of endocrine amyloidosis, such as medullary carcinoma of the thyroid14and in pancreatic islet^,'^ both mature A. Pucci was supported by a grant from ’Collegio and prohormone sequences or peptide fragments Nuovo’, Pavia and grant ‘Trapianto Cardiaco’ from are present in atrial amyloid deposits. The co- the Health Ministry to IRCCS Policlinico ‘S. localization of ANP- and BNP-like immunoreact- Matteo’, Pavia 27100, Italy. The work was supivity also suggests that two distinct cardiac peptide ported in part by the British Heart Foundation and hormones may be constituents of atrial amyloid the Council for Tobacco Research. U.S.A. fibrils. It is unclear why some peptide hormones give rise REFERENCES to amyloid fibrils, but the presence of abnormally highlevels ofpeptidemaybeacontributoryfactor, as I . Hodkinson H M , Pomerance A. The clinical significance of senile endocrine-type amyloid has been found in situations cardiac amyloidosis: a prospective clinico-pathological study. Q J where there are high peptide levels, including some Med 1977;46 381-387. 2. Westermark P, Johansson B, Natvig JB. Senile cardiac amyloidosis: peptide-producing turn our^'^ and the site of repeated evidence of two different amyloid substances in the ageing heart. insulin injections. I 6 Heart failure is accompanied by Scand J Immunol1979; 1 0 303-308. an increase in cardiac ANP expression, raised tissue 3. Cornwell G J S 111, Murdoch WL, Kyle RA, Westermark P, Pitkanen P. Frequency and distribution ofsenile cardiovascular amyloid. Am J and plasma levels of natriuretic peptides and altered Med 1983; 15:618-623. processing of the ANP p r e c u r s ~ r , ~but ~ ~further ~ , ~ ~ , 4~ ~Steiner 1.Theprevelanceof isoldtedatrialamyloid.JPathoI 1987;153 studies will be required to determine whether there is 395-398. 5. Sletten K, Westermark P, Natvig JB. Senile cardiac amyloid is related an association between tissue peptide levels and to prealbumin. Scand J rmmunol1980; 1 2 503-506. amyloid deposition in the atria. 6. Kaye GC, Butler MG, d’Ardenne AJ, Edmondson SJ, Camm AJ, Slavin G . Isolated atrial amyloid contains atrial natriuretic peptide: a Previous studies of isolated atrial amyloid have report of six cases. Br Hearl J 1986; 56: 317--320. used cardiac tissues obtained post-mortem from 7. Johansson B, Wernstedt C , Westermark P. Atrial natriuretic peptide eldery patients and have shown an age-related deposited as atrial amyloid fibrils. Biochem Biophys Res Commun 1987; 148: 1087-1092. increase in the incidence of amyloid deposits.’“ In a 8. Linke RP, Voigt C, Storkel FS, Eulitr M. N-Terminal amino acid study more comparable to our own, Kaye and cosequence analysis indicates that isolated atrial amyloid is derived from workers6 examined right atrial appendage samples atrial natriuretic peptide. Virchows Arch [ B ] 1988;5 5 125-127. 9. Itoh H, Nakao K, Mukoyama M, er al. Secretion of N-terminal fragfrom patients (19-73 years of age) undergoing ment of human atrial natriuretic polypeptide. Hypertension 1988; 11: coronary artery bypass graft operations and ident(Suppl I); 152-156. ified ultrastructural evidence of amyloidosis in 8/22 10. Meleagros L, Gibs JSR, Ghatei MA, Bloom SR. Increase in plasma concentrations of cardiodilatin (amino terminal pro-atrial natriuretic (36 per cent) of the cases. Four of these were found to peptide) in cardiac failure and during recumbency. Br Heart J 1988; display ANP-immunoreactive amyloid fibrils. Our 60: 39-44. study population had a similar range ( 1 2-63 years of 11. Hino J, Tateyama H, Minamino N, Kangawa K. Matsuo H. Isolation and identification of human natriuretic peptides in Cardiac atrium. age) and exhibited approximately the same incidence Biochem Biophys Res Commun 1990; 167: 693-700. of amyloidosis (41 per cent). Whilst the proportion 12. Togashi K, Hirata Y, Ando K, Takei Y, Kawakimi M , Marumo F. Brain natriuretic peptide-like immunoreactivity is present in human of cases (67 per cent) displaying immunoreactive plasma. FEBS Lerr 1989; 250: 235-237. amyloid deposits was greater in patients with is- 13. Mukoyama M , Nakao K, Saito Y . et a/. Human brain natriuretic peptide, a novel cardiac hormone. Lancet 1990;335: 801. chaemic heart disease, the mean age of this group of

A N P A N D BNP I N A T R I A L A M Y L O I D DEPOSITS 14. Sletten K, Westermark P, Natvig JB. Characterization of amyloid

fibrils proteins from medullary carcinoma of the thyroid. J E.rp Mrd 1976; 143 993-998. IS. Westermark P, Wernatedt C , Wilander E, Hayden DW, OBrien TD, Johnson KH. Aniyloid fibrils in human insulinomas and islet of Langerhans of the diabetic cat are derived from a novel neuropeptidelike protein also present in normal islet cells Proc Nut/ Acud Sci U S A 1987; 84: 3881-3885. 16. Dische FE, Wernstedt C , Westermark GT, et a / . Insulin as an amyloid-fibril protein at site of repeated insulin injections in a diabetic patient. Diuhrtologiu 1988: 31: 158-161 17. Wharton J, Anderson RH, Springall D, er ti/. Localization of atrial natriuretic peptide immunoreactivity in the ventricular myocardium and conduction system of the human fetal and adult heart. Br Heurr J 1988; M): 267-274.

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18. Varndell IM, Polak JM. Double immunostaining procedures: techniques and applications. In: Polak J M , Varndell IM, eds. Immunolabelling for Electron Microscopy. Amsterdam: Elsevier, 19x4; I5s- I 77 19. Saper CB, Hurley K M , Moga MM. e r a/. Brain natriuretic peptides: differential localization of a new family of neuropeptides. Nrurmci Lett 1989; 9 6 29-34. 20. Sugawara A, Nakao K, Morii N, c f ul. Synthesis of atrial natriuretic polypeptide in human failing hearts. Evidence of altered processing of atrial natriuretic polypeptide precursor and augmented sythesis of /l-human ANP. J. C'lin Rec 1988;81: 1962-1970. 21. Matsubara H, Umeda Y , Nishikawa M, et ul. Heart with circulatory failure secretes and processes atrial natriuretic peptide in a manner different from normal heart. Clin Curdid 1988; 11: 197-203.

Atrial amyloid deposits in the failing human heart display both atrial and brain natriuretic peptide-like immunoreactivity.

Atrial amyloid deposits are common in the ageing human heart and contain alpha-atrial natriuretic peptide (proANP99-126) immunoreactivity. However, at...
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