Myocardial glycoproteins in diabetes: Type VI collagen is a major PAS-reactive extracellular matrix protein

J Mol Cell Cardiol

Myocardial

24, 397-410 (1992)

Glycoproteins PAS-Reactive Mary

Jane

Spiro,

in Diabetes: Extracellular B. R. Rajesh

Type VI Collagen Matrix Protein

Kumar

and Thomas

is a Major

J. Crowley

Department of Medicine and Biological Chemistry and Molecular Pharmacology, Harvard Medical School anld theJoslin Diabetes Center, Boston, MA 02215, USA (Received 5 September 1991; accepted in revisedform 27 November 1991.) M. J. SPIRO, B. R. RAJESH KUMAR, AND T. J. CROWLEY. Myocardial Glycoproteins in Diabetes: Type VI Collagen is a Major PAS-Reactive Extracellular Matrix Protein. Journal ofMolecular and Cellular Cardiology (1992) 24, 397-410. An investigation of myocardial glycoproteins was undertaken to elucidate the molecules of the increased extracellular matrix of diabetic cardioresponsible for the periodic acid-Schiff (PAS) reactivity myopathy. Perfusion with radiolabeled mannose indicated an enhanced formation of matrix components in the diabetic compared to the normal rat heart. Electrophoretic separation of radiolabeled extracts demonstrated the presence of glycoproteins with Mr values of 205, 142 and 90 kDa which could be separated by Bio-Gel A-5 rn filtration. Fractionation of non-perfused hearts resulted in the isolation of only the 205 and 142 kDa components, which were shown by amino acid analyses and collagenase digestion to belong to the collagen family of proteins and by immunoblotting to represent type VI collagen. The carbohydrate content of these rat myocardial type VI collagen subunits, determined from monosaccharide analyses, was 11 and 12 % , respectively, and N-glycanase digestion of the 142 kDa chain resulted in a decrease in size of approximately 14 kDa, indicating the presence of asparagine-linked units. Examination of normal and diabetic rat heart sections indicated that the latter contained abundant PAS-positive strands and nodules which corresponded to the distribution of anti type VI collagen reactivity. Moreover, immunoblots showed higher levels of Type VI collagen in diabetic than in normal heart extracts. Type VI collagen therefore appears to represent a major glycoprotein of myocardial extracellular matrix and to be implicated in diabetic cardiomyopathy. KEY WORDS: Diabetic cardiomyopathy; Extracellular matrix; Carbohydrate units;

PAS; Glycoproteins; Heart perfusion.

Introduction Although little information is available about glycoproteins present in the heart, interest in this area has been stimulated by the finding in diabetic cardiomyopathy of periodic acidSchiff (PAS)’ reactive deposits in the myocardium [ 11. This cardiopathy is a complication of diabetes mellitus in which heart failure is thought to be due not to coronary atherosclerosis but rather to an expansion in the myocardial extracellular matrix both between the muscle cells and in contact with the capillary walls [l-5]. Although a diffuse fibrosis of the myocardium also occurs as a consequence of other conditions, such as pressure overload hypertrophy, the changes observed in these situations appear to be due *Correspondence ‘The following bovine serum

to: Mary

Jane Spiro, Joslin

Diabetes

+ 14 $03.00/O

VI

collagen;

Type

IV

collagen;

to increased formation of the main con stituents of the myocardial extracellular matrix, types I and II collagens [6-lo]. It ifs however unlikely that these collagens, which are poorly glycosylated, can be responsible for the PAS reactive carbohydrate containing deposits seen in the diabetic heart. In order to identify the molecules involved in the diabetic myocardial changes, we undertook a search for constituents of normal and diabetic rat hearts which could be detected with the PAS stain after electrophoretic separation and which could be metabolically radiolabeled with [3H]mannose during perfusion. After fractionation of the myocardial constituents by means of a sequential extrac tion procedure, PAS-reactive molecules were found associated primarily with the cellular Center,

abbreviations are used: PAS, periodic acid-Schiff, albumin; PBS, phosphate buffered saline.

0022-2828/92/040397

Type

One Joslin

Place,

SDS, sodium

Boston,

dodecylsulfate;

MA

02215, USA. kDa, kilodaltons;

0 1992 Academic

BSA,

Press Limited

398

M.J. Spiro

membranes and extracellular matrix. In the diabetic heart, the relative amount of monosaccharide incorporated into the extracellular matrix components increased and examination of these molecules indicated the primary PAS-reactive molecule to be type VI collagen, a carbohydrate-containing protein which has been noted to be increased in the glomerular mesangium of human diabetic kidneys [U]. In this report, isolation of and characterization of myocardial type VI collagen is described, together with studies showing its increase in the diabetic heart both by immunohistochemical and immunoblotting techniques.

Methods Animals

and tissues

Male albino rats (CD strain) were purchased from Charles River Laboratories. Diabetes was induced by the intrafemoral injection of alloxan monohydrate (37mglkg of body weight) after an overnight fast. The average length of the diabetes was 4 weeks; blood ‘glucose values for normal and diabetic rats were 120 + 9 and 460 + 27 mg/dl, respectively, while body weights were 419 f 34 and 285 + 43 g. Ventricle weights (g/100 g body weight) were 0.32 f 0.05 for normal rats and 0.36 + 0.03 for diabetics. Insulin was administered only as necessary to avoid death of the animal. Rat hearts (quick frozen) obtained from Rockland Inc. (Gilbertsville, PA) were used in the large scale fractionations, For radiolabeling by the Langendorff procedure [12], hearts of rats anesthetized with Nembutal (Abbot Laboratories, N. Chicago, IL, USA) were treated in situ by injection of 1OOU of Panheprin (Abbott) and then removed and the aorta cannulated. Radiolabeling was performed for 60min at 70mmHg with 67&i/ml of [2-3H]mannose (27.2CYmmol) or 17pCi/ml of [4,5-3H] leucine (56 Ci/mmol); both preperfusion and radiolabeling were carried out in oxygenated (O,:CO,, 95:5) glucose or leucine-free Krebs-Henseleit buffer containing Eagle’s amino acids and 2 mM pyruvate. The preperfusion (20 min) medium contained 0.34 while 1.5% fatty units/ml of heparin, acid-free bovine serum albumin (BSA) pretreated sodium oleate (0.5 mM final concentra-

et al.

tion, both from Sigma) was added during radiolabeling; at the end of the incubation, the heart was perfused by syringe with cold 0.15 M NaCl after which the ventricles were separated and frozen. All hearts were stored at - 80°C until treatment by a sequential extraction procedure previously described [13] which employed 50 mM Tris-acetate, pH 7.4; 4 M NaCl; 0.6M KCl; 1% Triton X-100; 4M guanidine hydrochloride; 4M guanidine hydrochloride with 20 mM dithiothreitol; and 2% SDS-5% 2-mercaptoethanol. In some experiments, SDS-mercaptoethanol extraction alone was used after the guanidine extraction step.

Chromatographic

separation of PAS-reactive components

For isolation of glycoproteins present in the extracellular matrix, extraction was performed either on [3H]mannose-labeled normal and diabetic hearts or on combined labeled and unlabeled normal hearts. Fractionation of the guanidine-dithiothreitol extract was accomplished on a 2.1 x 76 cm column of BioGel A-5m (200-400 mesh) equilibrated with 1% SDS in 50 mM Tris acetate, pH 7.0, containing 1 mM dithiothreitol and 2 mM EDTA. The dialyzed sample was lyophilized and dissolved by heating at 100’ in 10 ml of 1% SDS-5 % 2-mercaptoethanol. Elution of components was monitored by radioactivity and electrophoretic mobility.

Immunoassays The sources of antisera (all rabbit) were as follows: antihuman type VI collagen [II], was kindly provided by Dr W. G. Carter (Fred Hutchinson Cancer Research Center, Seattle, WA, USA) and also obtained from Telios Pharmaceuticals (San Diego, CA, USA); antiserum against rat Type IV collagen was prepared in this laboratory as previously described [15]; anti rat heart type VI collagen (142 kDa component purified as described in this report) was prepared in rabbits by multiple intradermal injections [16]. For solidphase immunoassays, diluted antigens were allowed to absorb to immunowells prior to their reaction with antiserum followed by lz51labeled Protein A [15].

Myocardial

Type

VI

Polyaqlamide gel electrophoresis and immunoblotting Samples for electrophoresis were dried and removal of guanidine or urea as well as salts was accomplished by extraction with 80% ethanol prior to suspension in 4% sodium dodecyl sulfate (SDS)-5 % 2-mercaptoethanol in 80mM Tris/chloride, pH6.8, and boiling for 3 min. After electrophoresis in SDS on vertical slab gels (4- 12 % acrylamide gradient, 1.5 mm thick) by the procedure of Laemmli [17J, the proteins were either stained with the PAS and Coomassie-blue reagents [18] or were transferred to nitrocellulose [19] using a Trans-Blot Cell (Bio-Rad, Richmond, CA, USA) for immunoassay. The nitrocellulose sheets were pretreated with phosphatecontaining 0.1% BSA buffered saline (PBS-BSA) followed by diluted goat serum and detection of antigens was accomplished by sequential treatment with appropriate antiserum and lz51-labeled Protein A followed by radioautography at - 70°C using Kodak XOmat AR film. For dilution of sera and washing of the nitrocellulose between immunoreagents, 0.05 % Tween 20 was added to the PBS-BSA.

Treatment with collagenase and N-glycanase Samples of the guanidine-dithiothreitol extract of pooled rat hearts were dried by lyophilization and extracted 3 times with 80% ethanol to remove the guanidine. Treatment with or without collagenase (10 fig of Sigma type VII) was carried out for 2 h at 37’C in 100~1 of 0.1 M Tris acetate, pH 7.4, containing 5 mM calcium acetate. Aliquots of the 142 kDa component of rat heart type VI collagen purified by Bio-Gel A-5m filtration were dried, extracted with ethanol as above to remove urea and salts and solubilized by boiling in 20mM sodium phosphate. Incubation with or without Nglycanase (0.2 units, Oxford Glycosystems, Rosedale, NY, USA) was performed in the phosphate buffer in the presence of 0.07% SDS and mercaptoethanol, 0.35% octylglucoside and 35mM EDTA for 18 hr at 37’C. At the end of the incubations, samples were boiled in the presence of 2.5 % SDS and 5 % 2-mercaptoethanol in preparation for electrophoresis.

Collagen

39!3

in Diabetes

Analytical procedures Protein was determined by the Peterson [20] procedure using bovine serum albumin as standard; radioactivity was measured by scintillation counting with Monofluor (National Diagnostics, Manville, NJ, USA); amino acids and hexosamines were determined as their phenylthiocarbamyl derivatives [21] using a Pica-Tag column and HPLC equipment for Waters (Milford, MA, USA). After formation of their glycamines [22] neutral sugars were also analyzed by HPLC as phenylthiocarbamyl derivatives; sialic acild was determined by the thiobarbituric acild reaction [23].

Treatment of tissue sections For histochemical studies, rat heart slices were prepared with a cryostat, affixed to a slide anid treated with methanol at - 2O’C. Prior t’o immunostaining they were treated (pH 5.3, room temperature, 60 min) with hyaluronidase [24], 1 mg/ml (Sigma, St. Louis, MO, 295 units/mg) to enhance penetration of the antibodies; after blocking with normal goart serum (Sigma) for 60min at room tempera.ture, sections were reacted overnight with antibody or preimmune serum and were then treated with fluorescently labeled goat antirabbit IgG (Cappel Laboratories, Richmondi, VA, USA). All sera were diluted with phosphate buffered saline pH 7.4 containing 1 mg/ml BSA (PBS-BSA) and washing of sections was also performed with this reagent. The heart sections exhibited an intrinsic fluorescence which could be substantially quenched [25] by treatment with 0.5% Eriochrome black (Sigma). Prior to treatment with the PAS stain, sections were digested for 1 h at 37OC with (Yamylase [26], 1 mg/ml in PBS containinig 2 mM phenylmethyl sulfonyl fluoride. Slices were then washed with PBS and reacted witlh 0.5 % periodic acid followed by the Schiff fuchsin reagent [26].

Results

Electrophoretic

examination

of heart extracts

In the sequential procedure employed, after treatment with dilute Tris buffer (Fig. 1, lane

M. J. Spiro et al.

400

FIGURE 1. Identification of glycoproteins in heart extracts by PAS staining. Fractions prepared from rat ventricles by sequential treatment with 50 mM Tris acetate, pH 7.4 (l), 4 M N&l(2), 1% Triton X-100 (3), 4 M guamdine/ mM tris acetate, pH 7.4 (4) and 2% SDS containing 5% 2-mercaptoethanol(5) were examined by SDS-polyacrylamide gel electrophoresis (4-12s gradient). Bands which stained with the PAS reagent were marked with India ink (indicated by arrows) and counterstaining was performed with Coomassie-blue to detect proteins not containing carbohydrate. Molecular weight standards included in the electrophoresis were myosin (205kDa), P-galactosidase (llGkDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (29 kDa). In order to demonstrate the glycoproteins, all lanes contained approximately 1 mg of protein except for the Triton extract (3) for which 250 pg were used. TABLE

1.

Glycoconjugate

Synthesis

by

Normal

and

Diabetic

Rat

Hearts” Extract

Substrate

Animal

Tris

Blood Glucose

NaCl

mg/dl [3H]Man

[3H]Leu

N (5)b D (6) D/N P value N (2) D (3) D/N

and

Triton

Guanidine SDS-ME

KC1 % of total

122 522

11.7 (4.2) 15.0 (2.4) 1.28

d

92 499

incorporated

23.3 (2.7) 26.5 (3.4)

radioactivity

43.8 (7.3) 25.9

1.1

21.2 (1.5) 32.5 (3.6)

(2.9)

0.59

1.5

0.05

0.02

4.4 4.0

76.9 79.6

1.04

and

18.8 15.8 0.84

0.91

=Heart perfusion was performed for 1 hr as described in the Methods section with 67 &i/ml [2-3H]mannose or 17pCilml [4,5-3H]leucine after which the tissue was sequentially extracted with the reagents listed. The ratio of total radioactivity incorporated per g by diabetic hearts to that by normal hearts was 0.55 from mannose and 1.22 from leucine; data for each fraction is expressed as percentage of total incorporation. bThe number of normal (N) and diabetic normal values. CFigures in these parentheses represent dAfter these perfusions, NaCl extraction

(D)

animals

is given

in parentheses;

D/N

the standard error of the mean; P values was performed without prior treatment

represents

the ratio

of diabetic

are based on the Student’s with the Tris acetate buffer.

t test.

to

Myocardial

Type

VI

Collagen

4’0 1

in Diabetes

(b) Peak

'2

50

100 Elution

150 200 250 volume

3

4

5'

300

(ml)

FIGURE 2. Fractionation of [3H]mannose-labeled myocardial proteins. After perfusion of rat hearts with radiolabeled mannose, sequential extracts were prepared as shown in Figure 1 and the guanidine-dithiothreitol extract (used instead of SDS-mercaptoethanol) representing 3.5 g of ventricles was fractionated by Bio-Gel A-5 m filtration (A), Radioactive peaks were examined by SDS-polyacrylamide electrophoresis and stained with the PAS reagent (B). Monitoring with anti-human fibroblast type VI collagen (C) of fractions after preparative gel filtration of pooled extractsof perfused radiolabeled plus nonperfused rat hearts (representing 10 g of ventricles) on the same column used in Figure Z(a). Molecular weight standards were as in Figure 1.

1) the large amount of myosin present in the heart muscle cells was preferentially removed by 4 M NaCl (lane 2, 205 kDa band). Subsequent extraction with Triton X-100 (lane 3) was used to release constituents associated with the cellular membranes, while 4M guanidine resulted in the solubilization of proteoglycans [.23] as well as in the release of some additional myosin (lane 4). Other extracellular matrix components were solubilized either by treatment of the remaining tissue directly with 2 % SDS containing 5 % 2-mercaptoethanol by boiling for 5 min (lane 5) as in this experiment or by treatment first with 4 M guanidine containing 20 mM dithiothreitol. Examination of the electrophoretically separated myocardial constituents with the PAS reagent indicated the presence of only a small number of reactive bands (shown by the arrows in Fig. 1) which were detected in the Triton, guanidine and SDS-mercaptoethanol extracts (or guanidine-dithiothreitol extract if this preceded the SDS treatment); upon staining the same gel with Coomassie-blue to

visualize other protein constituents (Fig. I), PAS-reactive bands were still detectable since they had assumed a purple hue.

Radiolabeling

of normal

and diabetic

hearts

Perfusion of normal hearts by the Langendorff procedure with tritiated mannose for 1 hr led to incorporation of the largest portion of tlhe radioactivity into the fraction solubilized by Triton (Table l), which represents glycoproteins associated with cellular membranes and luminal contents. When a comparison was made between normal and diabetic hearts, it was noted that the relative amount of synthesis of these membrane-associated glycoproteins was reduced in diabetes while the formation of extracellular matrix constituents was substantially increased (Table 1). This enhanced synthesis was not observed when radiolabeled leucine was employed as the substrate (Table l), as might be anticipated from the observation that glycoproteins represent

402

M. J. Spiro

FIGURE 3. Preparation of PAS-reactive Fig. 2~) were examined by SDS-polyacrylamide Coomassie blue. Molecular weight standards

et al.

components. Peak gel electrophoresis, as in Figure 1.

only a small portion of myocardial constituents, judging from the small number of bands stained with the PAS reagent compared to those vitalized by Coomassie blue (Fig. 1).

Isolation of matrix glycoproteins Since the radiolabeling experiments indicated enhanced formation of extracellular matrix glycoproteins in diabetes, the search for PASreactive constituents characteristic of diabetic cardiomyopathy was focused on this fraction. Gel filtration on Bio-Be1 A-5 m of guanidinedithiothreitol-solubilized [3H]mannose-labeled glycoproteins formed by normal hearts yielded several discrete peaks [Fig. 2(a)]. SDSpolyacrylamide gel electrophoresis [Fig. 2(b)] followed by staining with the PAS reagent indicated the presence of bands with Mr values of 142 and 90 kDa in peaks 2 and 3, respectively, low molecular weight constituents in peak 4 and no detectable components

tubes from a preparative gel filtration (similar followed by staining first with PAS and then

to with

in peak 5, which probably represented residual substrate. A small amount of a PASreactive protein with Mr = ‘205 kDa was also present in Peak 1 (data not shown). Examination of mannose-labeled diabetic heart extracts yielded similar elution and electrophoretic patterns (data not shown). In order to obtain sufficient quantities of the observed matrix glycoproteins for characterization, mannose-labeled rat ventricles were added to a pool of unlabeled normal ventricles prior to extraction and gel filtration. Electrophoretic examination of the fractions obtained from the Bio-Gel A-5 m column indicated that the main PAS component present in these extracts had an Mr value of 142 kDa (Fig. 3); Coomassie-blue staining (Fig. 3) indicated as well a band at 205 kDa which reacted less strongly with the PAS reagent. Little of the 90 kDa glycoprotein was obtained from these extracts of the frozen hearts, suggesting that this component may have been formed durin?

Myocardial

FIGURE preparative Figure 3 (Pool B). the other collagen.

Type

VI

Collagen

in

Diabetes

403

4. Evaluation of presence. of types VI and IV collagen in Bio-Gel A-5m column pools. Tubes from a gel filtration column similar to that shown in Figure Z(c) were analyzed electrophoretically as shown in as well a fraction which contained bolth and pools made of the 205 (Pool A) and 142 (Pool C) kDa components, After electrophoresis of the pools in triplicate, one portion of the gel was stained with Coomassie-blue while two were electroblotted and reacted with either anti-human fibroblast type VI collagen or with anti rat type IV Molecular weight standards are as described in Figure 1.

perfusion by degradation of the strongly PAS positive 142kDa protein. Indeed, we have observed that the purified 142 kDa molecule, when frozen and thawed several times, tends to form smaller polypeptides including that observed at Mr = 90 kDa. In order to obtain sufficient amounts of these glycoproteins for analytical procedures as well as for antibody production, pools were made of the tubes containing the 205 [Fig. 4(a)] and 142 [Fig. 4(c)] kDa proteins from this and similar columns.

Immunocharacterization of matrix glycoproteins Since Mr values of the major glycoproteins detected in the heart matrix extracts (142 and 205 kDa) suggested the possibility that they might represent the CY~plus c+ and c~s chains, respectively, of type VI collagen, fractions from preparative gel filtration on Bio-Gel A-5 m of the guanidine-dithiothreitol extract of pooled perfused (radiolabeled) and nonperfused rat hearts were tested with antibody

raised against human fibroblast type VI collagen [Fig. 2(c)]. The results indicated that the mannose-labeled peaks also represented areas reactive with anti-type VI immunoglobulins, as detected by binding of [iz51]Protein A in the solid phase immunoassay [Fig. 2(c)]. The main peak of immunoreactivity occurred in the region of the 142 kDa component, corresponding to the major PAS-staining component (Fig. 3) obtained from this column. Immunoblotting of the Bio-Gel A-5 m pools (Fig. 4) with antiserum against type VI collagen indicated that while the strongest reaction occurred with the 142 kDa band, th’e 205 kDa component was also detected as we!11 as a band migrating slightly faster than thte 142 kDa glycoprotein (Fig. 4). A similar pattern was obtained when the entirle guanidine-dithiothreitol extract (Fig. 5, lante 2) was examined with the anti-type VI antibodies. A portion of the type VI collagen was also extracted by guanidine itself in the absence of reducing agent (Fig. 5, lane 1).

404

M.J.

Spiro

et al.

FIGURE 5. Types VI and IV collagen in rat heart extracts. Aproximately 400 gg of protein (lanes 1) and guanidine-dithiothreitol extracts (lanes 2) of normal rat hearts were electrophoresed staining sequentially with PAS and Coomassie-blue as well as immunoblotting with anti-human collagen and anti-rat type IV collagen. Molecular weight standards are in Figure 1.

FIGURE 6. Collagenax susceptibility of rat myocardial PAS-reactive components. extract (approximately 4OOag of protein) from normal rat ventricles was desalted and collagenase as described in the Methods section. After polyacrylamide gel electrophoresis stained directly with PAS and Coomassie blue or electroblotted for reaction with anti-rat weight standards are as in Figure 1.

from

the guanidine in triplicate for fibroblast type VI

The guanidine-dithiothreitol treated either with or without in duplicate, the gel was either type VI collagen. Molecular

Myocardial

Type

VI

Collagen

To evaluate whether any type IV collagen, which is also a glycoprotein and has chains of approximately 200 and 180kDa, might be after Rio-Gel A-5 m filtration, present immunoblotting was performed using antibody directed against rat glomerular basement membrane type IV collagen (Fig. 4); no evidence for this collagen in the purified fractions was obtained although it was detected in the entire guanidine-dithiothreitol extract (Fig. 5, lane 2). Antibody raised in rabbits against the isolated rat heart 142 kDa glycoprotein, when used in immunoblotting experiments, reacted with human fibroblast type VI collagen; moreover, when tested with either the isolated proteins or the heart guanidine-dithiothreitol extracts it gave a similar pattern to that obtained with the anti-human fibroblast type VI, delineating both the 142 and 205 kD bands (see Fig. 6).

Characterization

of heart vpe VI collagen

Several studies were undertaken to evaluate the identity of the matrix 205 and 142 kDa glycoproteins as chains to type VI collagen. The sensitivity of both bands to collagenase was demonstrated by their disappearance after treatment with this enzyme whether detected by PAS or Coomassie-blue staining or by reaction with anti-rat type VI collagen (Fig. 6). The amino acid analyses of the purified proteins (data not shown) indicated the presence of the specialized amino acids hydroxyproline characteristic and hydroxylysine of the collagen family of proteins. The monosaccharTABLE

2. Carbohydrate

composition

Sugara

Galactose Glucose Mannose Fucose Glucosamine Sialic acid Total Percentage

of Rat 142 kDa

Heart

in Diabetes

405

ide constituents detected (Table 2) included mannose, galactose, fucose, glucosamine and sialic acid as well as glucose which would be expected to be present in hydroxylysine-linked glucosylgalactose disaccharides characteristic of collagens [271; only trace amounts of galactosamine were found. Total carbohydrate was 12 % for the 142 kDa subunits and approximately 11 % for the 205 kDa chain. An important difference observed is the smaller quantity of sialic acid in the 205 kDa chain, probably explaining its lower PAS reactivity. Evaluation of the presence of asparaginelinked units with the use of the enzyme Nglycanase indicated a reduction of Mr value of 14kDa for the 142 kDa component (Fig. 7), accounting for removal of 83% of the carbohydrate determined from sugar analyses. Among the carbohydrate not removed by this enzyme treatment would be the glucosylgalactosyl hydroxylysine units. The decrease in Mr value observed with N-glycanase together with the ratios of the monosaccharides present would be consistent with a content of 5 to 6 N-linked biantennary complex units per 142 kDa chain, detailed structural studies will be required to determine the exact nature of the carbohydrate units.

PAS and immunostaining

of heart sections

In normal heart sections, the PAS reagent stained fine lines of material outlining some of the capillaries and separating some muscle fibers (Fig. 8). In the diabetic heart, in contrast, marked reactivity was noted Type

VI

Collagen

component

205 kDa

component

nmol

KS

nmol

KS

22.9 11.9 17.9 2.8 20.6 2.2

3.7 1.9 2.9 0.41 4.2 0.64 13.8 12.1

18.1 7.3 14.5 1.6 25.7 0.7

2.9 1.2 2.4 0.23 5.2 0.20 12.1 10.7

aValues are expressed both as nmol and as gg of monosaccharide an aliquot of the same sample. The percentage represented monosaccharide residue weights divided by the peptide plus

per 100 gg of peptide based on amino by the sugars was determined from monosaccharide residue weights.

acid analysis of the sum of the

406

M. J. Spiro

et al.

FIGURE 7. N-glycanase treatment of rat myocardial type VI collagen. After treatment of the purified component with or without N-glycanase as described in the methods section, polyacrylamide gel electrophoresis electroblotting were performed prior to reaction with anti rat type VI collagen. Molecular weight standards same as described in Figure 1. Arrows indicate the migrations of the native and N-deglycosylated type VI subunits. A faster migrating component probably representing a carbohydrate-free degradation product is also by the antibody in this preparation.

throughout the tissue in the form of nodular deposits as well as fibrous strands (Fig. 8) Treatment of normal and diabetic heart sections with anti-heart type VI collagen indicated a distribution similar to that observed by PAS staining. Reaction of the normal heart was minimal, while the diabetic tissue showed both nodules as well as strands of immunoreactive material (Fig. 8). No reaction was obtained with preimmune serum. The localization of type IV collagen in these sections was quite distinct from type VI pattern, with the basement membranes surrounding the muscle fibers being delineated (data not shown).

142 kDa and are the collagen detected

Type VI collagen in normal and diabetic heart extracts In order to further examine the apparent increase in type VI collagen detected by immunostaining of tissue sections of diabetic rat hearts, the matrix extracts prepared from normal and diabetic hearts were electrophoresed and immunoblotted with anti-type VI collagen. In the diabetic extracts, not only is the amount of type VI perg of tissue increased, but the pattern observed compared to normal hearts is altered, with the amount of the 205 kDa component being preferentially elevated (Fig. 9). A detailed study is currently underway in our laboratory to quantitate the

Myocardial

Type

VI

Collagen

in Diabetes

FIGURE 8. PAS and anti-type VI reactivity of rat heart sections. Normal and diabetic using a cryostat and stained either with the PAS reagent or with anti-rat heart type VI of the autofluorescence with Eriochrome Methods section (magnification, 500x). Q uenching in this experiment was less effective than the diabetic, but it is nevertheless clear that there the anti-type VI antibody in the normal section.

FIGURE 9. Type VI collagen in normal and diabetic diabetic rat hearts, proteins solubilized with SDS-mercaptoethanol human fibroblast type VI collagen; each sample represents weight standards are as in Figure 1.

heart

407

rat ventricles were sectioned collagen as described in the black of the normal heart is little specific staining with

extracts. After sequential extraction of normal and were electrophoresed and immunoblotted with antithe extract from 0.075 g of ventricular tissue. Molecular

408

M. J. Spiro

effect of diabetes with and without hypertension of the level of this collagen in the myocardium. Discussion Since the characteristic feature observed in the heart in diabetic cardiomyopathy is the PAS reactivity of the expanded extracellular matrix [l-5], we have investigated the carbohydratecontaining components of the myocardium in order to evaluate which might be affected by this disease. In the extracts prepared from normal and diabetic rat hearts by a sequential procedure, only a limited number of glycoprotein components were observed after electrophoretic separation and these occurred either associated with the cell membranes or as constituents of the extracellular matrix. Radiolabeling with tritiated mannose indicated a relative decrease in formation of membrane associated glycoproteins in the perfused diabetic heart and enhanced synthesis of extracellular matrix material. Moreover, from this latter fraction, three proteins with Mr values of 205, 142 and 90 kDa were identified which were radiolabeled with [3H]mannose and stained by the PAS reagent. Since preparative Bio-Gel A-5m filtration of nonperfused rat ventricle extracts yielded primarily the 205 and 142 kDa glycoproteins, the 90 kDa component is believed to be derived by degradation of the strongly PASreactive 142 kDa glycoprotein under the conditions of perfusion. Our demonstration that the isolated myocardial matrix glycoproteins react with antiserum to type VI collagen agrees with the previously reported presence of carbohydrate in this molecule. Its original identification was as a 140 kDa transformation-sensitive fibroblast glycoprotein [la] and the collagenous nature of the protein was detected only subsequently [28]. Type VI collagen has now been shown to represent an important fraction of the connective tissues in a variety of organs [24, 28-301 and to be made up of three subunits, with the (it and CQ chains having similar molecular weights of approximately 140 kDa and the cyg chain varying in size from 260 to 200 kDa [31-331. The chain sizes detected for rat myocardial type VI collagen (Mr values = 205 and 142 kDa) are consistent with these reported values.

et

al.

In addition to the reactivity of PAS-positive bands with antibodies to type VI collagen, the 205 and 142 kDa chains separated by gel filtration were shown to be sensitive to collagenase treatment. Both fractions had similar total carbohydrate contents despite the finding that the PAS staining of the electrophoretically separated 205 kDa band was much less than that of the 142 kDa band, probably due to its smaller content of sialic acid, a constituent which is particularly sensitive to the periodic acid reagent. The presence of asparaginelinked carbohydrate units was indicated both by the presence of mannose in these glycoproteins as well as by the N-glycanase sensitivity demonstrated for the 142 kDa protein. Type VI is distinguished from the major collagens of myocardial connective tissue, namely types I and III, not only by its substantially higher carbohydrate content but also by the importance of disulfide bonds to its cross-linking. This permits its extraction by urea, guanidine or SDS in the presence of reducing agents, while types I and III can only be solubilized by proteolytic digestion because of their high degree of lysine-derived crosslinking. In both of these regards type VI is similar to basement membrane collagen (type IV) which contains substantial carbohydrate and can be solubilized by reduction of disulfide bonds [34]. The location of type VI collagen suggested by our immunofluorescent studies is different from that of type IV collagen, occurring not in the myocyte basement membranes but rather in the matrix surrounding the capillaries and separating the muscle fibers. Its increase in the diabetic heart appears to reflect another manifestation of the diabetic microvascular pathology which has been most thoroughly studied in the renal glomerulus with the finding that a number of macromolecular derangements occur, including the increased levels of both types IV and VI collagen together with reductions in the amounts of other extracellular matrix components such as laminin and the heparan sulfate proteoglycan [II, 351. Further studies are currently underway in our laboratory to determine the extent to which both types IV and VI collagen in rat heart are affected by this disease. The causative factors present in the diabetic heart which could lead to an increase in type

Myocardial

Type

VI

VI collagen are presently unclear; it is likely that the changes may represent a response to the cellular injury which is believed to occur in diabetes [36] and which is apparently an important factor in cardiomyopathies in general [371. Studies are in progress using cells in culture to determine which myocardial cell types are affected by the diabetic state as

Collagen

in Diabetes

well as what factors in the diabetic be responsible for such injury.

409

milieu

may

Acknowledgements This work was supported by grant HL31315 from the National Institutes of Health.

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