The relation of atrial natrluretic factor to isolated atrial amyloid

EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

52,266278

The Relation of Atrial Natriuretic Atrial Amyloid

(I!?&))

Factor to Isolated

BJORNJOHANSSONAND PER~ESTERMARK Department of Pathology, University of Uppsala, Uppsala, Sweden, and Department of Pathology, University of Linkiiping, Linkiiping S-581 85, Sweden Received August 3, 1989, and in revised form January 31, 1990 Isolated atrial amyloid (IAA) is a very common age-related amyloid form which is seen only in the atria of the heart. Chemical characterization has indicated that the major subunit protein is atrial natriuretic factor (ANF). In this ultrastructural study we show that the fibrils in IAA most frequently are located extracellularly especially along the cell membranes of the myocytes, but that small deposits also seem to be present intracelhdarly. No obvious relation was noted between the tibrils and the endocrine granules. Antiserum to a low molecular fraction of IAA labeled amyloid fibrils and granules in the same way as a commercial antiserum to ANF, but no other structures in the myocyte. Finally we show that ANF can polymerize to tibrils with an amyloid appearance. The study thus supports the fact that ANF is an important and integrated part in the IAA fib&. o 1990ACZUI~C PISS, IIK.

INTRODUCTION Amyloid is characterized by the amyloid tibril which is a polymer of usually low molecular weight subunit proteins. Amyloid deposits in the aging heart are a common finding at autopsy (Cornwell et al., 1983; Hodkinson and Pomerance, 1977; Steiner, 1987; Westermark et al., 1979; Wright and Calkins, 1975). In the majority of cases it is an age-related cardiac amyloidosis. The systemic primary, secondary, p,-microglobulin-derived, or familial amyloidoses which also can affect the heart are more rare. The age-related cardiac amyloidoses can be separated into at least two groups. One is a systemic form now designated senile systemic amyloidosis (Pitkanen et al., 1984) which is seen at autopsy in about 15% of persons 70 years and older (Cornwell et al., 1983; Westermark et al., 1979). Here, the fibril protein, derived from transthyretin (TTR; previously called prealbumin) (Sletten et al., 1980), is designated protein ATTR.’ In a majority of persons 60 years and older there is an amyloid restricted to the atria of the heart (Cornwell et al., 1983; Westermark et al., 1979). This amyloid is designated isolated atria1 amyloid (IAA) (Westermark et al., 1979). The subunit fibril protein (protein AANF’) has recently been shown to be derived from atria1 natriuretic factor (ANF) (Johansson et al., 1987). ANF is’s potent natriuretic and diuretic hormone which also has vasorelaxing properties (Andersson and Bloom, 1986). In man it is produced mainly in the atria of the heart as 151 amino acid preprohormone (Oikawa et al., 1984). The C-terminal 28 amino acid fragment of the 126 amino acid prohormone which is stored in the endocrine granules of the atrial myocytes (Thibault et al., 1987) is considered to be the active circulating form (Flynn et al., 1983; Kangawa and Matsuo, 1984). The main stimulus for ANF release seems to be stretching of the atria1 wall (Bilder et af., 1986). Increased levels of ANF both ’ Suggestions for nomenclature: First International Symposium on Familial Amyloidotic ropathy and other transthyretin related disorders, Granja, Portugal, September 1989. 266 0014-4800/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All tights of reproduction in any form reserved.

Polyneu-

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in the heart and in the plasma are seen in patients with heart disease, especially congestive heart failure (Cody et al., 1986; Fyhrquist and Tikkanen, 1988; Nozuki et al., 1986; Riegger et al., 1986; Saito et al., 1989). The clinical importance of IAA is still a matter of discussion. There are only a few reports of a correlation between IAA and heart disease (Cornwell et al., 1983). Theoretically, when heavily deposited, IAA could have a deleterious influence on the function of the atria1 myocytes. IAA may also indicate an impaired ANF processing or release. The study of the amyloid deposits and their relation to the endocrine granules and other cell structures can bring some light to the matter of where the amyloid fibrils are formed and how they could influence atria1 function. MATERIALS

AND METHODS

Tissues Right auricles from five patients, 71-75 years old undergoing heart surgery, were collected. Four patients suffered from angina pectoris and were provided with an aortocoronary bypass. One of the four also had a stenosis of the aortic valve and one patient had only an aortic stenosis. These two patients received an artificial valve. Small pieces were immediately fixed in a solution of 2% paraformaldehyde and 1% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2, containing 0.15 M NaCl. Part of the material was embedded in paraffin and sections were stained with Congo red and studied in polarized light. Green birefringent deposits along the muscle fibers and in small vessels as described before (Steiner, 1987; Westermark er al., 1979) were taken as evidence that the amyloid was of the atria1 type. This was also confirmed immunohistochemically as described below. The rest of the material was embedded in Epon for electron microscopy. For immunohistochemistry, sections of amyloid-containing tissues from patients with verified amyloid of AL, AA, ATTR, and AANF types were used as controls. Proteins Amyloid tibrils were isolated as described (Johansson ef al., 1987) from the auricles of two hearts rich in IAA, taken at autopsy, (patients 613 and 352). The tissue was repeatedly homogenized in 0.15 M NaCl followed by water and then treated with collagenase. After solubilization in concentrated formic acid and drying, the material was redissolved in 6 M guanidine HCl solution containing 0.1 M dithiothreitol, gel filtered through a 1.6 x 30-cm Sephadex G-50 fine column, and eluted with 5 M guanidine HCl in distilled water. This yielded a typical elution pattern (Johansson et al., 1987) with one retarded peak containing protein AANF. This material was dialyzed against saturated ammonium sulfate followed by deionized water and lyophihzed. The protein AANF material from patient 613 was used for immunization and that from patient 352 for absorption experiments. Human amyloid P-component was purified from saline washes of systemic amyloids as described (Pepsys et ul., 1977). Human ANF was purchased from Boehringer (Mannheim, West Germany). Antisera Antiserum

to protein AANF was raised in a rabbit by bimonthly

immunization

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with the protein AANF containing fraction from patient 613 using standard techniques. This antiserum (AA 78), obtained after 3 months, was absorbed with homogenized and lyophilized normal human heart ventricle powder (6 mg/ml undiluted serum) and purified human amyloid P-component (3 mg/ml undiluted serum). The peroxidase-antiperoxidase method was performed as described (Stemberger, 1979) on amyloid-containing sections from patients with amyloid of AL, AA, ATTR, and AANF types. AA 78 was used in dilutions 1:400 and 1:SOO. Rabbit antiserum to human ANF was purchased from Milab (Malmo, Sweden) and used in dilutions I:300 and 1:600. Immune

Electron

Microscopic

Studies

For immunogold studies the antisera were diluted 1:25 and I:50 in 0.05 M Tris-HCl buffer containing 0.5 M NaCl, 1% bovine serum albumin, and 0.02% Tween 20 (Merck, Mtinchen, Germany). AA 78 was used with and without absorption which was performed at room temperature for 8 hr, either with ANF (5 mg/ml undiluted antiserum) or the partially purified protein AANF of patient 352 (10 mg/ml undiluted antiserum). The antiserum to ANF was also used with and without absorption with the same antigens. Thin sections mounted on Formvarcoated nickel grids were etched with saturated sodium metaperiodate, blocked with 5% bovine serum albumin in 0.05 M Tris-HCl buffer, pH 7.4, and incubated for 2 hr with the primary antiserum. After rinsing, the sections were treated with protein A-gold conjugate (Auroprobe, 15-nm particles, Janssen, Beerse, Belgium) for 30 min, rinsed, and contrasted with uranyl acetate and lead citrate. The sections were studied in a JEOL 100 SX electron microscope. Fibril

Formation

Human ANF (50 pg) was dissolved in 5 pl 10% acetic acid and kept at room temperature for 15 hr. This procedure has been used at our laboratory to produce tibrils from fragments of islet amyloid polypeptide (IAPP) (manuscript in preparation). Before electron microscopy, 20 pJ distilled water was added and one drop of the suspension was put on a Formvar-coated copper grid and negatively contrasted with 2% uranyl acetate in distilled water. RESULTS Light Microscopy

Polarization microscopy following Congo red staining of the five auricles obtained at surgery revealed amyloid with an appearance typical of IAA in three cases. When the peroxidaseantiperoxidase-labeled sections from the different types of amyloid were studied in the light microscope only AA 78 showed reaction with amyloid of the isolated atrial type. No reaction was obtained with amyloid of AL, AA, or ATTR type. Electron

Microscopy

When studied in the electron microscope, all five atrial specimens exhibited fibrils of amyloid appearance (Fig. 1). The tibrils had a diameter of about 10 nm

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FIG. 1. Extracellular amyloid fibrils (A) close to a myocyte (M). In one area (arrows), parallel amyloid fibrils run perpendicular to the sarcolemma which in part is not clearly demonstrable. x5o.ooo.

and were of varying length. The amyloid tibrils were unevenly distributed in the auricles and the amount was largest extracellularly near the cell membrane (sarcolemma) of the myocyte. Small deposits sometimes seemed to occur intracellularly in muscle cells (Fig. 2). Such deposits were not limited by any membrane. As

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AND WESTERMARK

FIG. 2. Amyloid fibrils (A) interposed between atrial myocytes (M). Possible intracellular is indicated by arrows. ~25,000.

amyloid

a rule the fibrils seemed to be unorganized. Near the muscle cells, however, parallel fibrils could be seen, directed to the sarcolemma which here was often not clearly demonstrable (Fig. 1). No real invaginations of the sarcolemma with amyloid fibrils were seen.

271 The amyloid fibrils had no obvious relation to the endocrine granules. Most frequently, granules were seen near the fib&, but usually not in direct contact with them (Figs. 3 and 4). There were also areas with large extracellular deposits of amyloid fibrils without granules in the adjacent muscle cells. RELATION

OF ANF TO IAA

FIG. 3. Immunogold labeling of amyloid fibrils (A) and myocytic granules (arrows) using antiserum to ANF. ~22,000.

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FIG. 4. Immunogold labeling of amyloid fibrils (A) and myocytic granules (arrows) using antiserum to a subunit protein (protein AANF) of isolated atrial amyioid. x22,000.

Immune Electron Microscopy

Immunogold labeling confirmed that the amyloid was of the IAA type. Thus both the antisera to ANF and to protein AANF (AA 78) labeled amyloid fibrils and granules strongly and in a similar fashion. No other structures exhibited any

RELATION

OF ANF TO IAA

labeling above a slight background. This labeling auricles obtained at surgery (Figs. 3 and 4). Absorption

pattern

273 was seen in all five

of Antisera

Strong labeling of amyloid tibrils and endocrine granules was still seen when AA 78 was absorbed with the partially purified protein AANF from patient 352. On the other hand, when the antiserum to ANF was absorbed with ANF no labeling was seen at all. When AA 78 was absorbed with ANF the reaction with amyloid fibrils was completely abolished, but a strong reaction with the endocrine granules still remained (Fig. 5). This labeling pattern (a strong reaction with the granules and no reaction with the fibrils) was also seen when the antiserum to ANF was absorbed with the partially purified protein AANF from patient 352. Fibril Formation The negatively contrasted material, studied in the electron microscope, appeared as bundles of fibrils. The fibrils were even, nonbranching, and slightly wavy. They had a diameter of about 10 nm and were of varying length. Sometimes a composition of two thinner subunit fibrils, twisted around each other, seemed to constitute the lo-nm fibrils (Fig. 6).

FIG. 5. Atrial section treated with antiserum to a protein AANF, absorbed with ANF. The reaction with amyloid fibrils (A) is abolished, but the reaction with myocytic granules (arrows) persists. x42.000.

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JOHANSSON

FIG. 6. Fibrils formed

in

AND WESTERMARK

vitro by human ANF dissolved in 10% acetic acid. x 120,000.

DISCUSSION The immune electron microscopic finding clearly showed that the antisera to ANF and to the purified IAA protein AANF labeled exactly the same atrial

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OF ANF

TO IAA

275

structures, i.e., secretory granules within muscle cells and amyloid fibrils. This finding further supports previous immunological (Kaye et al., 1986) and biochemical (Johansson et al., 1987; Linke et al., 1988) findings which have indicated that ANF is a constituent of the IAA tibrils. Whether ANF is the only constituent of the fibrils is a subject of further studies. However, to date copolymerization between unrelated proteins have not been proved in any amyloid form. Furthermore, the present study shows that intact ANF alone is capable of forming tibrils with amyloid appearance. In amyloid in medullary carcinomas and in the islets of Langerhans both the mature hormone and the prohormone or fragments thereof seem to constitute the fibrils (Sletten et al., 1976; Westermark et al., 1989; unpublished results). In previous studies of the low molecular weight component of IAA we obtained no evidence of a prohormone constituent (Johansson et al., 1987). However, it has not been possible to rule out that pro-ANF (or parts thereof) occurs in IAA fibrils. IAA is a localized form of amyloid with a unique chemical composition (Westermark et al., 1979; Johansson et al., 1987). It belongs to the hormone-derived (endocrine) amyloid forms which probably constitute a fairly large group of amyloids. Other hormones which have been proven to give rise to amyloid fibrils are calcitonin/procalcitonin in medullary carcinoma of the thyroid (Sletten et al., 1976) IAPP in insulinomas and in islets of Langerhans in type 2 diabetes (Westermark et al., 1986, 1987; Cooper et al., 1987), and insulin in rare cases of iatrogenic-localized amyloid deposits (Dische et al., 1988). The reasons why some polypeptide hormones form amyloid fibrils are unclear. In the pathogenesis of all amyloid fibrils, a high degree of p-pleated sheet structure in the subunit proteins is believed to be of basic importance (Eanes and Glenner, 1968; Glenner et al., 1974). A partial degradation of precursor proteins also seems to take place in the amyloidogenesis (Glenner, 1980) and it is possible that this can open up molecules and expose sequences prone to polymerize. It is also possible that an abnormally high concentration of a polypeptide hormone is of importance for the formation of amyloid. Thus, amyloid is commonly found in some polypeptide hormone producing tumors (Westermark et al., 1977). Islet amyloid formation by IAPP in the glucose intolerant cat occurs in islets with an abnormally strong P-cell IAPP immunoreactivity (Johnson et al., 1989). A similar mechanism might occur in the formation of fibrils in IAA since there is obviously an abnormally high atria1 concentration of ANF in patients with cardiac insufficiency (Akimoto et al., 1988) which is a state with a high incidence of pronounced IAA (unpublished observation) . The exact location of the formation of amyloid fibrils has not been determined in any type of amyloid. Parallel fibrils running perpendicularly into pockets of the cell membrane have been taken as evidence for formation of fibrils on or in these cells (Shirahama and Cohen, 1973). In experimental (AA-) amyloidosis, fibril formation has been proposed to occur in lysosomes (Shirahama and Cohen, 1975) or extracellularly on the plasma membrane (Lavie et al., 1978). It was not possible to point out the exact location of amyloid fibril formation in the present study. However, in some areas amyloid fibrils close to myocytes were found more or less perpendicular to the sarcolemma. Here the sarcolemma often showed signs of degeneration. Most evidence favors proteolytic cleavage of pro-ANF (1-126) to ANF (99-126) in or in close contact with the myocyte (Shields ef al., 1988). It is possible that polymerization to amyloid tibrils takes place directly after the re-

276

JOHANSSON

AND

WESTERMARR

lease of ANF. On the other hand, the present study and previous studies (Kaye et al., 1986; Westermark et al., 1980) indicate that some amyloid fibrils may also be deposited intracellularly in IAA. The results from the absorption experiments are difficult to interpret. In the studies with antiprotein AANF, it was only possible to abolish the reaction with the amyloid fibrils and only with ANF (with the concentrations of the antigens used). Likewise, the reaction of anti-ANF with granules, but not with amyloid fibrils, persisted after absorption with the purified protein AANF. At present, there is no explanation for these phenomena, but the tertiary structure of ANF in the protein AANF form may be different compared to that of nonflbrillar ANF. Antigenic epitopes might also be hidden within the amyloid librils. Amyloid proteins have been shown to posses antigenic sites different from those of the native precursor. Thus, in double immunodiffusion, antiserum to TTR does not react with protein ATTR although the latter is derived from TTR. In the same way, antiserum to protein ATTR does not react with TTR (Cornwell et al., 1981). It is also possible that partial degradation of ANF in the librillogenesis alters the antigenic behavior. Finally due to very small amounts of material, the purified protein AANF used for absorption and for immunization was from two different patients and it is possible that the composition varied slightly between individuals. ACKNOWLEDGMENTS Thanks are due to Marie-Louise Eskilsson, Linkoping, for expert help with the immunogold labeling and to Dr. Leif Nilsson, Uppsala, for supplying us with the biopsy material. Supported by the Swedish Medical Research Council (Project 5941) and the Research Fund of King Gustaf V.

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