Alterations in the organisation, ultrastructure and biochemistry of the myocardial collagen matrix in Doberman pinschers with dilated cardiomyopathy

Research in Veterinary Science 2000, 69, 267–274 doi:10.1053/rvsc.2000.0423, available online at http://www.idealibrary.com on

Alterations in the organisation, ultrastructure and biochemistry of the myocardial collagen matrix in Doberman pinschers with dilated cardiomyopathy S. J. GILBERT*, P. R. WOTTON†, A. J. BAILEY*, T. J. SIMS*, V. C. DUANCE‡ *Collagen Research Group, Division of Molecular & Cellular Biology, †Division of Companion Animals, Department of Clinical Veterinary Science, University of Bristol, Bristol BS18 7DY, ‡School of Biosciences, Cardiff University, Cardiff CF10 3US, UK SUMMARY Remodelling of the collagen matrix of the myocardium has been implicated in the pathogenesis of dilated cardiomyopathy, a major cause of heart failure in Doberman pinschers. The aim of this study was to characterise the myocardial collagen matrix of Dobermans. In clinically normal Dobermans there was evidence of focal fibrosis. Collagen cross-links were altered in both diseased and clinically normal Doberman myocardium as compared with myocardium from control dogs. Extensive remodelling, in the form of a loss of collagen tethers, increased collagen synthesis and alterations in the collagen cross-links, occurs in diseased Doberman myocardium. Changes in the collagenous matrix are also present in apparently normal Dobermans. These changes are likely to be involved in the progression of the disease and may explain the predisposition of this breed to dilated cardiomyopathy. © 2000 Harcourt Publishers Ltd

IDIOPATHIC dilated cardiomyopathy (DCM) is a chronic myocardial disease characterised by cardiac dilatation and a reduction in the contractile function. It is a common clinical problem and a major cause of heart failure in several species, but with particular prevalence in humans and certain breeds of dogs. The Doberman pinscher is one of the breeds most commonly afflicted by idiopathic DCM (Hill 1981, Calvert et al 1982, Hazlett et al 1983) and may be a potential large animal model of human DCM (Smucker et al 1990, McCutcheon et al 1992). In the majority of Dobermans, the disease is characterised by a long precongestive ‘occult’ phase, during which there are no overt clinical signs, followed by congestive heart failure (Calvert et al 1996). A large number of Dobermans with occult DCM die suddenly before the onset of heart failure (Calvert 1991). Asymptomatic and apparently healthy dogs of this breed have been shown to have left ventricular dysfunction, despite the fact that radiographically the heart may not be grossly enlarged (Smucker et al 1990). Extracellular matrix components, and collagen in particular, form a network within the myocardium. Despite its relatively low concentration, cardiac collagen is the major structural component determining the architecture and functional integrity of the myocardium (Eghbali and Weber 1990). Since the contractility of the myocardium relies on the supporting collagen framework, alterations in this collagenous matrix will inevitably have profound effects on the

Corresponding author: Dr. S. J. Gilbert, School of Biosciences, Cardiff University, Museum Avenue, PO Box 911, Cardiff CF10 3US, UK. Fax: 02920 874486

0034-5288/00/030267 + 08 $35.00/0

efficient working of the heart. Over the last 10 years, a number of studies have implicated collagen in the pathogenesis and progression of DCM, irrespective of its aetiology. Recent studies in the authors’ laboratory have shown that there are alterations in the levels of extracellular matrix-degrading enzymes in canine DCM (Gilbert et al 1997). Investigations into the myocardial collagen matrix in Dobermans with no clinical signs of DCM may provide insights into why this breed is predisposed to this disease, and may detect the early changes that contribute to the development of DCM. This is of particular importance since it is not possible to study the earliest events in human cardiomyopathy, and thus the Doberman may be a potential large animal model of the disease. The aim of the current study was therefore to characterise the collagen matrix of the myocardium of Dobermans with no clinical signs of DCM and those with naturally occurring DCM, and to compare it with that of control (non-Doberman) myocardium.

MATERIALS AND METHODS Materials All chemicals were obtained from Sigma Chemical Co. Ltd (Dorset, UK) unless otherwise stated and were of analytical grade.

† Present address: Department of Veterinary Clinical Studies, University of Glasgow Veterinary School, Glasgow, G61 1QH, UK

© 2000 Harcourt Publishers Ltd

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Source of tissue samples Canine hearts were obtained immediately following post mortem examination and the right and left ventricles were separated by dissection. Samples of myocardium were obtained from control dogs of mixed breeds and weight with no predisposition to DCM (n = 7, mean age 8.1 ± 1.0), Doberman pinschers with normal myocardium (n = 5, mean age 9.8 ± 1.8) and Dobermans with DCM (n = 9, mean age 8.5 ± 0.4). There were no significant differences in age between these groups. Criteria for clinical diagnosis of DCM included the presence of an abnormal electrocardiogram, a reduction in contractile function, and the absence of other types of cardiac, pulmonary and systemic disease. All cases fulfilled these criteria and all were in congestive heart failure. The diagnosis was confirmed by post mortem. ‘Normal’ Dobermans had no clinical or post mortem evidence of heart disease although no echocardiography was performed. All dogs were euthanased on humane grounds, and control material was obtained from cases euthanased for reasons unrelated to the cardiovascular system. No animal was obtained specifically for the purpose of this study. All analyses were performed on the left ventricle since dilatation of the left ventricle and left heart failure predominates in Doberman DCM (Sisson and Thomas 1995). This enabled a focused study of an anatomically well-defined area of the heart. Preliminary studies (results not shown) indicated 5–10 mm3 samples of myocardium were representative of the left ventricle. Tissue was rapidly frozen in liquid nitrogen cooled isopentane and subsequently stored in liquid nitrogen. For scanning electron microscopy studies, small pieces of fresh heart muscle were fixed in 2.5 per cent glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4. Immunohistochemistry Unfixed, 10 µm cryostat sections were prepared (Bright microtome) on precoated slides (Biobond adhesive; British Biocell International, Cardiff, UK) and non-specific binding sites were blocked with non-immune sera from the appropriate species prior to additon of the primary antibody. The sections were incubated sequentially in antiserum to collagen types I, III, IV, V or VI and fluorescein isothiocyanate (FITC) conjugated secondary antibody, both diluted in phosphatebuffered saline (PBS) (Table 1). The sections were mounted TABLE 1:

in Citifluor (Glycerol/PBS solution; Agar Scientific, Essex, UK) and examined on a fluorescence light microscope (Leitz Dialux). Non-immune sera of the appropriate species were used as negative controls. The specificity of all the antibodies used was confirmed by Western blotting (Towbin et al 1979) using canine heart and placental collagen standards purified using standard techniques (Miller and Rhodes 1982, Avery and Bailey 1995). All antibodies showed reactivity with their appropriate canine collagen and no crossreactivity with other collagen types. Scanning electron microscopy (SEM) Fixed tissue was washed in sodium cacodylate buffer and post-fixed in 1 per cent osmium tetroxide in 0.1 per cent sodium cacodylate buffer. The tissue was dehydrated in a graduated ethanol series and infiltrated with sublimation dehydrant, Peldri II (Ted Pella, Inc., USA) in 100 per cent ethanol (1:1) and left in 100 per cent Peldri overnight at 30˚C. The Peldri was solidified by transferring the vials to a cooled metal plate and sublimed off under vacuum in a tissue drier (Edwards). The dried tissue was mounted on stubs using carbon cement, coated in gold using an SEM sputter coating system (S150A Sputter Coater, Edwards) and examined with a Cambridge Stereoscan 200 scanning electron microscope with an accelerating voltage of 15–20 KV. Estimation of total collagen content Whole heart tissue was lyophilised to a constant weight and digested in 6N HCl (maximum 10 mg ml–1) for 20– 24 hours at 110˚C in sealed tubes. The samples were diluted in distilled water (1:5), freeze dried and resuspended in 1 ml of distilled water. Hydroxyproline was estimated by analysis on a continuous flow hydroxyproline analyser (Chemlab Instruments Ltd, Eindhoven, Netherlands). The method is based on the oxidation of hydroxyproline by chloramine-T followed by coupling with dimethylaminobenzaldehyde (Woessner 1976). The coloured product is measured at 550 nm. Standards of 1–5 µg ml–1 hydroxyproline were used to calculate a standard curve, and the sample solutions were appropriately diluted to within this range. The collagen content, as a percentage of the dry weight, was calculated by multiplying the hydroxyproline values by 7.14 assuming a 14 per cent hydroxyproline content for all collagen types (Etherington and Sims 1981).

Antibodies used for immunohistochemistry

Antiserum to:

Host animal

Dilution

Primary Type I collagen, human polyclonal (Collagen Research Group, University of Bristol) Type III collagen, human polyclonal (Collagen Research Group) Type IV collagen, human polyclonal (Collagen Research Group) Type V collagen, human polyclonal (Sera Labs International, Salisbury, UK) Type VI collagen, bovine polyclonal (gift from S. Ayad)

Goat

1:50

Goat

1:50

Rabbit

1:500

Goat

1:10

Rabbit

1:100

Rabbit Goat

1:100 1:100

Secondary Anti-goat IgG, FITC conjugate Anti-rabbit IgG, FITC conjugate

Quantification of collagen cross-links Analysis of collagen cross-links was carried out as previously described (Sims and Bailey 1992). Myocardial samples (approximately 1 g wet weight) were finely minced in PBS and left overnight at 4˚C to hydrate. The material was reduced with sodium borohydride (approximately 10 mg g–1 sample) in 1 mM NaOH at room temperature, and the reaction was stopped by the addition of glacial acetic acid. The sample was washed in distilled water, freeze dried and the reduced material was hydrolysed in 6N HCl (at a concentration of 10 mg ml–1) at 110˚C for 24 hours. The hydrolysates were freeze dried, reconstituted in distilled water, and an equal volume of glacial acetic acid was added. An aliquot

The collagen matrix in Doberman pinschers with dilated cardiomyopathy

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FIG 1: (A) Immunohistochemical control section of non-immune goat serum. Non-immune rabbit serum was also negative (results not shown). Immunohistochemical localisation of collagen in control (non-Doberman) myocardium stained with antibodies to types I(B), III(C), IV(D), V(E) and VI(F) collagen. An artery (a) and individual myocyte (m) are indicated. Immunohistochemical localisation of collagen in normal Doberman myocardium (G) showed a similar pattern of staining as control myocardium but there were focal areas of myocyte hypertrophy (→) and fibrosis (*) (H). Immunohistochemical localisation of collagen in dilated cardiomyopathy Doberman myocardium stained with antibodies to types I(I), III(J), IV(K) and VI(L) collagen. Myocyte hypertrophy (→), vacuolation (v), interstitial (*) and replacement fibrosis (>) following myocyte necrosis were common features. Bars = 80 µm (A, D–H), 100 µm (B & C), 50 µm (I–L).

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A

C

B

D

FIG 2: Scanning electron microscopy of control (non-Doberman) myocardium (A,B) showing collagen tethers between muscle bundles (→), individual myocytes (>) and perimysial collagen fibres (p) and dilated cardiomyopathy (DCM) Doberman myocardium (C,D) showing a lack of collagen tethers between muscle bundles (→) and the presence of fibrosis (>). Normal Doberman myocardium was similar to controls (results not shown). Bars = 100 µm (A,D) or 50 µm (B,C).

was removed for hydroxyproline analysis before the addition of butan-1-ol and fractionation on CF1-cellulose (Whatman International Ltd, Kent, UK). The adsorbed cross-linking amino acids were removed by washing with distilled water and the effluent was collected and freeze dried. The samples

were reconstituted in 0.01N HCl, filtered through a 0.2 µm filter, and applied to a Pharmacia Alpha Plus II amino acid analyser configured for the separation of cross-links. Amino acids were post-column derivatised with Ninhydrin and detected at 540 nm. A standard cross-link mixture was run in

The collagen matrix in Doberman pinschers with dilated cardiomyopathy

A 0·5 (mol mol–1 collagen)

5

4

3

HH-PYR

Collagen content (%)

271

2

1

0·4 0·3 0·2 0·1 0 Controls (non-Dobermans)

Normal Dobermans

DCM

Dobermans

0 DCM

Dobermans

FIG. 3: Collagen content (%) in the left ventricle of control (non-Doberman), normal and dilated cardiomyopathy (DCM) Doberman myocardium. *P = 0.011 vs control.

parallel. The method enabled the detection and quantification of the enzyme-mediated reducible cross-links dihydroxylysinonorleucine (DHLNL) and hydroxylysinonorleucine (HLNL), normally prevalent in immature tissues, and the nonreducible cross-links hydroxylysyl-pyridinoline (HL-Pyr) and histidinohydroxymerodesmosine (HHMD), more prevalent in mature tissues.

B 2 (mol mol–1 collagen)

Normal Dobermans

DHLNL

Controls (non-dobermans)

1·5

1

0·5

0

Statistical analysis Data are presented as mean ± standard error mean. Student’s t-test and Spearman’s Rank correlation were used to analyse the data (Minitab). Differences were considered significant at any P value less than 0.05.

Controls (non-Dobermans)

Normal Dobermans

Dobermans

Controls (non-Dobermans)

Normal Dobermans

Dobermans

DCM

C

Immunohistochemistry Non-immune controls were negative (Fig 1A). Granular staining was observed in the majority of sections, possibly representative of lipofuchsin (Schaper and Speiser 1992). The pattern of staining in control (non-Doberman) myocardium can be seen in Fig 1 B–F. Type I collagen was prominent in the perimysium, separating small groups of myocytes, and present in the endomysium, surrounding individual myocytes. The adventitia of medium-sized arteries also contained type I collagen. Type III collagen localisation was similar to that of type I collagen and was abundant around small capillaries and in the adventitia of medium-sized arteries. Type IV collagen was absent from the perimysium and located in the basement membrane of myocytes and arteries. Type VI collagen was seen in the endomysium and adventitia of small arteries but did not surround the cells entirely. Staining for type V collagen was weak but followed a similar distribution to type VI collagen. It was not seen in large septa. Normal Dobermans were found to have a pattern of staining similar to that of control (non-Doberman) myocardium (Fig 1G). However, in some areas of tissue, myocyte

(mol mol–1 collagen)

RESULTS

1·5

HHMD

2·5

0·5

2

1

0 DCM

FIG 4: (A) Hydroxylysyl-pyridinoline (HL-Pyr) content in the left ventricle of control (non-Doberman), normal and dilated cardiomyopathy (DCM) Doberman myocardium. +P = 0.013 vs normal Doberman. (B) Dihydroxy-lysinorleucine (DHLNL) content in the left ventricle of control (non-Doberman), normal and DCM Doberman myocardium. (C) Histidinohydroxymerodesmosine (HHMD) content in the left ventricle of control (non-Doberman), normal and DCM Doberman myocardium. *P=0.011 vs control; +0.034 vs control.

hypertrophy was observed along with a degree of fibrosis (Fig 1H). In DCM Doberman myocardium, cellular hypertrophy and fibrosis were common features (Fig 1 I-L). Fibrosis was predominantly interstitial, surrounding myocytes, but replacement fibrosis, following myocyte necrosis, was also

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evident. Fibrosis resembled an exaggerated normal collagen matrix with increased type I and III collagen in both the endomysium and perimysium, as well as an increase in the staining for type IV and VI collagen. Staining for type V collagen was similar to that of control myocardium (results not shown). Disruption of the collagen matrix and cellular vacuolation were observed in sections taken from DCM hearts.

young Dobermans with DCM (less than 5 years old) exhibiting similar changes to older Dobermans with DCM (greater than 5 years old). No correlation (Spearman’s Rank correlation) was found between age and any of the factors examined in this study, thereby suggesting that the alterations in matrix were indicative of disease remodelling.

DISCUSSION Scanning electron microscopy The ultrastructure of the myocardium was examined by SEM (Fig 2). In control (non-Doberman) myocardium, collagen tethers of the endomysium were seen between myocytes and between muscle bundles. A complex weave of collagen surrounded groups of myocytes and thick, wavy perimysial collagen fibres were also evident. Normal Doberman myocardium resembled that of control (non-Doberman) myocardium with evidence of collagen tethers between bundles of myocytes (results not shown). A loss of collagen tethers was observed in some areas of DCM myocardium and areas of fibrosis in others. Total collagen content The amount of total collagen was calculated by hydroxyproline analysis, and results were expressed as a percentage of the dry weight of the tissue (Fig 3). The total collagen content was significantly increased in myocardium from Dobermans with DCM when compared with control dogs (DCM 3.94 ± 0.73 vs control 1.48 ± 0.16; P = 0.011), and higher, although not significantly, than in normal Doberman myocardium (DCM 3.94 ± 0.73 vs normal Doberman 2.39 ± 0.47; P = 0.10). Although the collagen content of normal Doberman myocardium was higher than controls, the difference was not statistically significant (P = 0.14). Quantification of collagen cross-links Cross-links were identified by comparison of peaks to a standard mixture. Results were expressed as moles of cross-link/mole of collagen. The mature cross-link, HL-Pyr was significantly increased in DCM Doberman myocardium compared with normal Doberman myocardium (DCM 0.39 ± 0.03 vs normal Doberman 0.26 ± 0.03; P = 0.013; Fig 4A), and higher, although not significantly, than controls (0.32 ± 0.02; P = 0.053). The level in normal Dobermans was lower than that in controls (P = 0.15). The level of DHLNL in DCM Doberman myocardium was higher, although not significantly, than that of normal Dobermans (DCM 1.65 ± 0.34 vs normal Dobermans 1.33 ± 0.63; P = 0.65) (Fig 4B). Doberman myocardium contained lower levels of DHLNL than controls (1.87 ± 0.35; P = 0.65). Both DCM and normal Dobermans had significantly decreased amounts of a crosslink identified as HHMD (1.21 ± 0.24; P = 0.011 and 1.37 ± 0.27; P = 0.034, respectively) when compared with control dogs (2.33 ± 0.28) (Fig 4C). No HLNL was detected. Age-related changes The changes that were found in Doberman myocardium were consistent throughout the range of ages examined, with

Immunohistochemistry and SEM studies on myocardium from Dobermans with DCM were typical of findings previously reported to be present in ‘end-stage’ DCM hearts (Schaper and Speiser 1992, Wynne and Braunwald 1992, Yoshikane et al 1992). Cell hypertrophy and fibrosis in the form of an increase in collagen were evident. Fibrosis is usually associated with an increase in types I and III collagen but a general increase in matrix proteins including fibronectin, collagen types I, III, IV and VI, and laminin, has been observed in human end-stage DCM (Schaper and Speiser 1992). From this study it was concluded that an accumulation of these proteins within the extracellular space could separate the cells, preventing electrical coupling, diffusion of oxygen and thereby impair heart function. The increased fibrosis would also decrease compliance and predispose the myocardium to the generation of ventricular arrhythmias, which are a notable clinical feature of the disease in this breed. Collagen localisation within normal Doberman myocardium was similar to that found in controls, although there was evidence of focal fibrosis in some cases. Scanning electron microscopy of control (nonDoberman) and normal Doberman myocardium showed the presence of collagen tethers between myocytes. This collagen is responsible for tethering muscle cells to capillaries, and myocyte to myocyte (Borg and Caulfield 1981). Dilated cardiomyopathy myocardium was found to have an increased collagen weave surrounding the myocytes as well as an increase in the thickness of perimysial collagen fibres, both indicative of fibrosis. As well as this increase in collagen, a loss of collagen tethers was observed in several areas of the diseased ventricle. This is the first report of such findings in naturally occurring DCM, and supports findings previously seen in myocardium from rapid pacing models of congestive heart failure which have been postulated to reproduce the remodelling that is seen in naturally occurring DCM (Weber et al 1990). A loss of collagen tethers was observed in the early stages of the disease, whilst in the later adaptive stages, an increase in collagen synthesis was reported. It is likely that a disruption or loss of the collagen tethers will result in a reduction in the tensile strength and subsequent muscle bundle slippage. Due to this, the ventricle would be less able to resist deformation, contributing to the architectural remodelling that is observed (Weber et al 1988). Collagen tethers were present in some areas of diseased myocardium but this is not unexpected since the degradation of collagen is likely to be a focal event coinciding with areas of proteolytic enzyme activation. Matrix metalloproteinase-9 and neutrophil elastase, enzymes known to be involved in the dissolution of collagen in diseased states, are known to be significantly increased in Dobermans with DCM and raised in normal Dobermans (Gilbert et al 1997).

The collagen matrix in Doberman pinschers with dilated cardiomyopathy

The elevated levels of these enzymes are likely to contribute to the structural remodelling seen in the myocardium, such as the loss of tethers, and may be fundamental in the pathogenesis and progression of canine DCM. Biochemical analyses provided further evidence of the structural remodelling that was observed by immunohistochemistry and SEM. The presence of fibrosis was supported by a 2.7- and 1.6-fold increase in the total collagen content in DCM and normal Doberman myocardium, respectively. A fibrotic response in supposedly normal myocardium could be indicative of early remodelling that contributes to the development of DCM. The increased deposition of collagen impairs electrical conduction between myocytes, which predisposes the heart to arrhythmias and thus increases the risk of sudden death, a feature of Doberman DCM. As well as changes in the actual amount of collagen in DCM, it has also been hypothesised that an alteration in the collagen cross-links may also contribute to cardiac dilatation (Gunja-Smith et al 1996, Okada et al 1996). Crosslinks are crucial for the collagen fibril to function optimally as a framework structure since fibrils with an absence of cross-links have no mechanical strength (Bailey et al 1974). There are limited reports characterising the types and amounts of cross-links in myocardium, and none specifically on canine myocardium. The cross-links in control myocardium were therefore characterised and compared with those in both normal and DCM Doberman myocardium. The enzyme-mediated cross-links, HL-Pyr, DHLNL and HHMD were clearly detected in cardiac tissue. Levels of HL-Pyr in control canine myocardium (0.32 mol mol–1 collagen) were considerably lower than those reported previously for control human myocardium (2.07 mol mol–1 collagen) (GunjaSmith et al 1996). Such high levels of HL-Pyr are unusual in soft tissues. The amount of HL-Pyr in bovine whole muscle collagen varies between 0.02 and 0.47 mol mol–1 collagen depending on the muscle type and the method of analysis (Avery 1996). A similar value to that found in this study, of 0.254 ± 0.047 mol mol–1 collagen was reported to be present in the left ventricle of rats (Werman and David 1996). The majority of the differences observed in the actual levels of each cross-link were not statistically significant, perhaps reflecting the low numbers of samples. Hydroxylysylpyridinoline was, however, significantly increased in DCM Doberman myocardium when compared with normal Dobermans, and was higher than that in controls, reflecting the increased synthesis and extension of cross-linking of collagen. The higher levels of DHLNL in DCM Dobermans compared with those in normal Dobermans are consistent with fibrosis. A similar cross-link profile has been found in scar granulation tissue (Bailey et al 1973). Lower levels of HL-Pyr, DHLNL and HHMD were found in normal Dobermans compared with controls. It must be noted that there is some debate on the authenticity of HHMD as a cross-link with the suggestion that it is in fact an artifact of reduction (Robins and Bailey 1977). It is possible that Dobermans, in general, have lower levels of this cross-link compared to nonDobermans and that it is not affected by DCM but, in view of the conflicting evidence regarding its status, no definite proposals can be made at this time. The lower levels of crosslinks in normal Dobermans compared with controls, despite an increase in the total collagen content, suggests that the collagen fibrils in Dobermans are mechanically weaker than

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controls. It is likely that some of the ‘normal’ Dobermans in this study are in the early stages of DCM or predisposed, and the lower levels of cross-links observed supports this. The lower levels of DHLNL and HHMD, and the corresponding increase in HL-Pyr in end-stage DCM compared with controls, suggests accelerated cross-link maturation of the fibrotic tissue, probably reflecting adaptive responses to initial cardiac damage such as a loss of collagen tethers, in an attempt to strengthen the ventricular wall. This hypothesis is supported by a recent study of hamster DCM (Okada et al 1996) where a decrease in reducible cross-links was found, and it was suggested that during cardiac enlargement and subsequent congestive heart failure, these cross-links have matured to tightly connect collagen fibrils. This study has demonstrated changes in the myocardial collagenous matrix of ‘normal’ Dobermans when compared with that of control (non-Doberman) dogs. The total collagen content was increased but alterations in the cross-links suggest that the strength of this newly synthesised collagen is compromised. In the authors’ previous study (Gilbert et al 1997), both matrix metalloproteinase-9 and neutrophil elastase were found to be elevated in normal Doberman myocardium, albeit at lower levels than those in Dobermans with diagnosed DCM. The presence of elevated degradative enzymes in fibrotic diseases is not unusual and, although there is a net accumulation of extracellular matrix, both anabolic and catabolic mechanisms are increased. These changes suggest that either Dobermans possess abnormalities in their collagenous matrix that may predispose them to DCM or, alternatively, the animals used in this study may already have been in the early stages or ‘occult’ phase of the disease. The authors’ current and previous (Gilbert et al 1997) studies have shown remodelling of the collagen matrix in DCM similar to that reported to occur in human DCM. These matrix changes were found to be independent of age and are therefore indicative of disease remodelling. These results provide evidence to support the idea that the Doberman is a naturally occurring large animal model of the human disease (Smucker et al 1990, McCutcheon et al 1992), and may provide an insight into the biochemical mechanisms involved in the early and progressive stages of DCM.

ACKNOWLEDGEMENTS We gratefully acknowledge Dr Malcolm Cobb for the provision of control material, Dr Gordon Paul for his guidance and help with the cross-link analysis, and Dr Shirley Ayad for the kind gift of antisera to type VI collagen. REFERENCES AVERY, N. C. (1996) The use of solid phase extraction cartridges as a pre-fractionation step in the quantitation of intermolecular collagen crosslinks and advanced glycation end-products. Journal of Liquid Chromatography & Related Technology 19, 1831–1848 AVERY, N. C. & BAILEY, A. J. (1995) An efficient method for the isolation of intramuscular collagen. Meat Science 41, 97–100 BAILEY, A. J., BAZIN, S. & DELAUNAY, A. (1973) Changes in the nature of the collagen during the development and resorption of granulation tissue. Biochimica Biophysica Acta 328, 383–390 BAILEY, A. J., ROBINS, S. P. & BALIAN, G. (1974) Biological significance of the intermolecular crosslinks of collagen. Nature 251, 105–109 BORG T. K. & CAULFIELD J. B. (1981) The collagen matrix in the heart. Federation Proceedings 40, 2037–2041

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Accepted August 30, 2000