© 1997 by European Society of Cardiology
Copyright © 1997, European Society of Cardiology
Increased expression of promatrix metalloproteinase-9 and neutrophil elastase in canine dilated cardiomyopathy
aCollagen Research Group, Division of Molecular and Cellular Biology, Department of Clinical Veterinary Science, University of Bristol, Langford, Bristol BS18 7DY, UK
bCollagen Research Group, Division of Companion Animals, Department of Clinical Veterinary Science, University of Bristol, Langford, Bristol BS18 7DY, UK
* Corresponding author. Tel. +44 117 9289235; Fax +44 117 9289505.
Received 19 August 1996; accepted 30 December 1996
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Objective: Canine dilated cardiomyopathy, commonly affecting Doberman pinschers, results in extracellular matrix remodelling within the myocardium. The aim of this study was to examine the proteolytic activity in myocardium from Doberman pinschers with dilated cardiomyopathy. Methods: Samples of myocardium, obtained rapidly post mortem from the left ventricular free wall of Dobermans with dilated cardiomyopathy, clinically normal Dobermans and control dogs (non-Dobermans), were examined for proteolytic activity using substrate gel zymography. Gels were analysed by scanning densitometry. Results: Promatrix metalloproteinase-9 activity was significantly increased in all Doberman myocardium when compared to controls. A significant increase in an enzyme, identified to be neutrophil elastase by inhibition of its activity by Elastatinal and Western blotting, was also detected in all Dobermans when compared to controls. Conclusions: The results indicate that promatrix metalloproteinase-9 and neutrophil elastase, both of which are implicated in inflammatory responses, are present in significantly elevated levels in Doberman dilated cardiomyopathy and are raised in clinically normal Dobermans. Both proteolytic enzymes degrade a wide variety of connective tissue components and thus the increased levels found may play an important role in the structural remodelling seen in the myocardium and subsequent heart failure. Increased proteolytic enzyme levels in clinically normal Dobermans may be indicative of the predisposition of the breed to dilated cardiomyopathy. © 1997 Elsevier Science B.V.
KEYWORDS Cardiomyopathy; Neutrophil elastase; Matrix metalloproteinase; Zymography; Dog, ventricle
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Dilated cardiomyopathy (DCM) is a disease of unknown aetiology, recognised in humans, dogs, cats, and other species. Although idiopathic DCM affects many large breeds of dog, the Doberman pinscher is one of the breeds most commonly afflicted. The disease affects the myocardium and is characterised by dilatation of one or more cardiac chambers, especially the left ventricle. A progressive decrease in systolic (contractile) function results in reduced cardiac output. The outcome is severe and often fatal congestive heart failure, with an expected life span after the onset of the clinical signs of around 6–12 months [1].
The extracellular matrix (ECM) in the heart is largely collagenous in nature and plays a primary role in providing tensile strength and structural integrity to the myocardium [2]. A weave of collagen covers the myocytes and bundles of collagen fibres or struts/tethers (predominantly composed of types I and III collagen) interconnect myocytes and connect myocyte to capillaries. These struts insert into the endomysial weave, thereby preventing cell slippage within a group of myocytes [3, 4]. Type IV collagen is the major component of the basement membranes of myocytes. In view of these crucial roles, it is not unexpected that a disruption or loss of the collagen struts, as reported in DCM [5], will result in a reduction in the tensile strength and subsequent muscle bundle slippage. In such cases the ventricular walls would be less able to resist deformation, resulting in the architectural remodelling seen in ventricular dilatation. These findings, along with a loss of the collagen network in dilated hearts, have been reported by several groups [5–8].
Proteolytic enzymes have been implicated in the degradation and remodelling of collagen in the diseased ventricle [5, 9, 10]. Degradation and remodelling of connective tissue is maintained by a balance of synthesis and degradation by proteolytic enzymes (matrix metalloproteinases, serine and cysteine proteases), their activators and inhibitors, under the control of external stimuli such as cytokines, growth factors and adhesion molecules. Expression of these components is important in both normal physiological and pathological remodelling processes.
The primary aim of the following study is to investigate the potential role that matrix-degrading enzymes play in the remodelling process reported in Doberman DCM. Such enzymes may be important in the aetiology of the disease. Aside from the aetiology, canine DCM is an obvious clinical problem, accounting for a substantial percentage of canine morbidity and mortality. In addition, the natural process of the disease in dogs, particularly Dobermans, closely resembles that seen in human DCM in terms of clinical course and presentation. Identification of factors involved in the aetiopathogenesis of the disease in dogs may have important implications for the disease in humans.
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2 Methods |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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2.1 Materials
All chemicals were obtained from Sigma Chemical Co. Ltd (Dorset, UK) unless otherwise stated and were of analytical grade.
2.2 Myocardial samples
Samples of myocardium were obtained rapidly, post mortem, from the left ventricular free wall of control dogs (non-Dobermans, n=9), normal (n=5) and DCM (n=12) Doberman pinschers. At the time of death, the age range of the dogs was between 4 and 11 years (Table 1). All dogs were euthanased on humane grounds and control material was obtained from clinical cases euthanased for reasons unrelated to the cardiovascular system. No animal was obtained specifically for the purpose of this study and no laboratory animals were used. The material was frozen, immediately in liquid nitrogen and stored at –80°C until use.
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2.3 Preparation of cardiac tissue extract
Frozen myocardial samples were powdered by dismembrator (B. Braun Biotech International) at 1600 r.p.m. for 2 min and freeze dried overnight. Each sample was normalised according to the original dry mass of the tissue. This was achieved by incubating each of the powdered samples in extraction buffer at the same concentration of 50 mg/ml (0.1% Brij 35 in 20 mM triethanolamine) for 3 h at 4°C with continuous agitation. The homogenates were centrifuged at 50xg, 4°C for 15 min and the supernatants either used immediately or aliquoted and stored at –20°C until required. Before use, extracted supernatants were diluted 1:3 with sample buffer (0.6 M Tris/HCl, pH 6.8, 2% sodium dodecylsulphate, 10% glycerol, 2 mg Bromophenol Blue). Soluble protein was not considered to be an appropriate correlate in this instance when normalising samples because differences in proteolytic activity can fundamentally alter the solubility of the protein.
2.4 Zymography
All zymography was carried out using the Biorad Mini Protean II casting apparatus with 1 mm spacers. Gelatin (type B from bovine skin) or casein (Casein Hammarsten; BDH, Chemicals Ltd, Poole) were incorporated into 10% SDS-polyacrylamide gels [11], adapted for the mini-gel system, at a final substrate concentration of 0.75 and 0.5 mg/ml, respectively. Samples (20 µl), standards (0.135 mg/lane neutrophil elastase, Sigma or 1.25 ng/lane MMP-2, Biogenesis) or rainbow protein molecular weight markers (5 µl; Mr 14–200 kDa, Amersham) were loaded onto each lane of the gels and run in Laemmli electrode buffer at 20 mA per gel and 100 V until the solvent front had reached the bottom of the gel. Following electrophoresis the gels were washed, with agitation, in 2.5% (v/v) Triton X-100 for 1–2 h with at least 3 changes of solution. Gels were incubated in 15 ml of appropriate proteolysis buffer (see below) for 16–20 h at 37°C with agitation. Gels were stained with Coomassie Brilliant Blue R250 (2 g/l) in distilled water, methanol and acetic acid (5:5:2) for at least 1 h, and destained in methanol, acetic acid, water (1:0.75:8.25) until the zones of proteolysis had cleared. The addition of molecular weight markers and standards to the gels facilitated identification of the enzymes. The relative quantities of proteolytic enzymes were analysed by scanning densitometry (Agfa Studioscan II Colour Scanner and Photoshop Imaging software) [12]. Values represent the areas of substrate gel cleared expressed as a percentage of activity of a standard (MMP-2, Biogenesis or a sample containing a constant amount of neutrophil elastase for the gelatin and casein zymography, respectively) to allow comparisons between gels.
The accuracy and sensitivity of the zymographic technique for determining protease levels was shown by analysis of gels loaded with neutrophil elastase or MMP-2 standard. This confirmed the linearity of zymography, detecting levels as low as 1 ng and 0.1 ng of neutrophil elastase and MMP-2 respectively (Fig. 1, a and b, respectively).
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2.5 Proteolysis buffers
MMP proteolysis buffer: 50 mM Tris/HCl, pH 7.8, 50 mM CaCl2 and 0.5 M NaCl. Aminophenylmercuric acetate was added to the MMP buffer (in order to fully activate inactive zymogens) to give a final concentration of approximately 1 mM.
Cysteine protease buffer: 100 mM phosphate, pH 6.8, 8 mM EDTA, 10 mM L-cysteine and 0.2% (v/v) Triton X-100.
Serine protease buffer: 100 mM phosphate, pH 6.8, 8 mM EDTA and 0.2% (v/v) Triton X-100.
2.6 Inhibition profile
In order to determine whether clarified zones were due to MMP, cysteine or serine protease action, duplicate gels were incubated in proteolysis buffer with addition of appropriate inhibitors for the enzyme class. Inhibitors were also added, at the same concentration, to the enzyme extract and incubated for 2 h at room temperature prior to electrophoresis and to the Triton X-100 washing solution. The inhibitors used were: 10 mM EDTA for MMPs; 2 mM NEM and 25 µM E64 for cysteine proteases; 4 mM Pefabloc for general serine proteases (Boehringer Mannheim); and 2 mM Elastatinal for neutrophil elastase. Elastatinal is known to be target-specific and does not inhibit other serine and thiol enzymes [13].
2.7 Western blotting
Immunoblot analysis [14]was used to confirm the findings of casein substrate zymography. Samples (20 µl), an elastase standard (10 µl; Sigma) and rainbow protein molecular weight markers (5 µl) were run on standard 10% SDS-polyacrylamide gels and the proteins transferred electrophoretically (overnight at 30 mA) to polyvinyl difluoride (PVDF) membrane (Millipore). The membrane was incubated in blocking buffer (0.15 M NaCl, 0.05 M Tris/HCl, pH 8.0, 3% (w/v) skimmed milk powder) with 3 changes and probed with rabbit antiserum to human leukocyte elastase (Biogenesis), appropriately diluted in wash buffer (0.15 M NaCl, 0.05 M Tris/HCl, pH 8.0, 0.2% Tween 20), for 2–2.5 h. After extensive washing in buffer, appropriately diluted alkaline phosphatase conjugated anti-rabbit IgG was added and the membrane incubated for 2 h. Further extensive washing was carried out before a final rinse in substrate buffer (0.2 M Tris/HCl, pH 9.69, 0.2 M MgCl2). The antibody localisation was visualised with the alkaline phosphatase substrate, 3-bromo-4-chloro-5-indolyl phosphate, p-toluidine salt plus nitro blue tetrazolium.
2.8 Statistical analysis
Data are presented as means±standard error mean (SEM). Student's t-test was used to analyse the data. Differences were considered significant at any P-value <0.05.
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3 Results |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Relative levels of proteolytic enzymes were determined using casein and gelatin substrate zymography.
3.1 Gelatin substrate zymography
Bands of proteolysis, seen at approximately Mr 66 and 90 kDa in all samples, were identified as MMPs 2 and 9 by eliminating activity with 10 mM EDTA (E64, Pefabloc and NEM had no effect). By comparison with MMP standards, the Mr 66 and 90 kDa bands were identified to be coincident with latent or proMMP-2 and -9, respectively (Fig. 2a). Fig. 2b shows the relative activity of proMMP-2 and -9. No significant differences in the levels of proMMP-2 were detected in the left ventricles of control dogs (43.2±3.8), normal (30.1±5.1) or DCM (52.1±8.5) Doberman myocardium.
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P=0.012 vs. control.By contrast, there was a significant increase in the levels of proMMP-9 in the myocardium of Dobermans with DCM in comparison to controls (DCM 61.9±8.0 vs. control 23.3±1.8; P=0.0005) and normal Dobermans (DCM 61.9±8.0 vs. normal Dobermans 36.6±3.3; P=0.012). Normal Doberman myocardium also contained significantly higher levels of proMMP-9 than controls (P=0.012).
3.2 Casein substrate zymography
One enzyme, with an approximate molecular weight of Mr 30 kDa, was detected by casein zymography (Fig. 3a). Levels of this protease were significantly increased in myocardium from Dobermans with DCM when compared to control dogs (DCM 18.9±1.8 vs. control 6.7±1.1; P<0.0001) and higher, although not significant, in normal Doberman myocardium when compared to controls (10.7±3.6) (Fig. 3b). Due to its apparent molecular weight of 30 kDa and its ability to digest casein in the presence of EDTA, the enzyme was provisionally identified as neutrophil elastase.
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An inhibition profile and Western blotting were carried out to confirm the identity of the 30 kDa enzyme. No inhibition of the enzyme activity occurred following treatment with cysteine (E64 or NEM) protease inhibitors. Some inhibition resulted from incubation with Pefabloc, a serine protease inhibitor, and approximately 86% inhibition of its activity occurred with the addition of Elastatinal, a selective inhibitor of elastase-like serine proteases (Fig. 4).
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Western blot analysis, probed with appropriate antibodies, was used to confirm the findings of casein substrate zymography. Evidenced by its immunodetection and by comparison with an elastase standard, the band seen at Mr 30 kDa was verified as neutrophil elastase (Fig. 5).
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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The present study examined the levels of proteolytic enzymes in the left ventricles of Dobermans with and without DCM and compared them to levels in control, non-Doberman left ventricles. The major findings of this study were:
- a significant, 2.7-fold elevation in the levels of proMMP-9 (P=0.0005) in diseased Doberman myocardium
- a significant, 2.8-fold elevation in the levels of neutrophil elastase (P<0.0001) in diseased Doberman myocardium
- a significant, 1.6-fold elevation in the level of proMMP-9 (P=0.012) in clinically normal Doberman myocardium
- a 1.6-fold elevation in the level of neutrophil elastase in clinically normal Doberman myocardium.
Substrate specific inhibitors and/or Western blotting probed with appropriate antibodies confirmed the identity of these enzymes. The changes that were found were consistent throughout the range of ages examined, thereby suggesting that the increased levels of enzymes found were indicative of disease changes and not a change seen with age.
Only the latent forms of the MMPs were detected. The presence of the latent form alone is indicative of an inflammatory response. Such controlled proteolysis is seen with cellular infiltrations (MMP-9) and normal turnover and remodelling (MMP-2). Activation of MMP-9 and MMP-2 occurs in a focused pericellular environment by the plasminogen activator/plasmin cascade and MT-MMP, respectively, leaving much of the secreted MMP in its pro form. In light of this, the presence of the activated form of the enzyme on a zymogram would be characteristic of a chronic disease state such as is seen in chronic wounds.
The finding of increased levels of proMMP-9 [15]and neutrophil elastase [16], both of which have been implicated in inflammatory disease and are consistent with a neutrophil infiltrate, suggests that DCM is correlated with an inflammatory cell response. It is not clear, however, whether this is a reaction to damaged tissue or is causal to the disease. Despite this finding, histologically (results not shown) there is no marked presence of inflammatory cells and indeed, to date, reports of such an inflammatory response in canine DCM are rare. In the light of this conflicting evidence it will, therefore, be important in future studies to confirm which cells are responsible for producing this anomaly.
Recent studies have reported increased MMP-1 activity and decreased levels of tissue inhibitors of metalloproteinases (TIMPs) in DCM hamsters [17]. Similar results have been reported in endomyocardial biopsy samples obtained from human patients with DCM [18]. Armstrong et al. [19]found an increased level of MMP-9 (92 kDa gelatinase) in the left ventricle of dogs after rapid ventricular pacing. Since MMP-9 degrades collagen types IV and V, it was concluded that the raised levels found in this model of congestive heart failure could be involved in altering the composition of the basement membrane and thus result in dilatation, wall thinning and a globular heart shape. More recently, Tyagi et al. [20]have reported on increased MMP activity in DCM human hearts. They detected a band at 
92 kDa which they mis-identified to be MMP-2.
The loss and disruption of the collagen struts previously reported in DCM (see Section 1) could be due to raised levels of proteolytic enzymes such as those seen in this study. Since both MMP-9 and neutrophil elastase have been shown to degrade a wide variety of connective tissue components [21–23]the raised levels found are likely to contribute to the structural remodelling seen in the myocardium and subsequent heart failure.
The results reported in this study, of an increase in proMMP-9 in canine DCM, reflect those found in pacing models of congestive heart failure characterised by ventricular dilatation. This model is thought to reproduce the remodelling that is seen in DCM. Increased enzyme levels in clinically normal Dobermans may be indicative of a predisposition of the breed to DCM. Indeed, 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 [24]. Zymography and Western blotting provides direct evidence of an increased expression of neutrophil elastase in canine DCM, the first to our knowledge to do so. The raised levels of these proteolytic enzymes, known to be involved in the dissolution of collagen in diseased states, may be fundamental in the pathogenesis and progression of canine DCM or they may represent a secondary response to tissue damage, cell and matrix debris consequential to the disease onset. Further studies are required to determine the levels of other important proteolytic enzymes such as MMP-1 and MMP-8 (collagenases) in DCM. Since it is often an imbalance between levels of proteolytic enzymes and their inhibitors that results in the accelerated and uncontrolled ECM remodelling and degradation that is observed in many diseased states, it will be important to measure the levels of MMP and neutrophil elastase inhibitors in DCM. Such studies are currently being undertaken.
Time for primary review 17 days.
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Acknowledgements |
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The provision of control material by Malcolm Cobb is gratefully acknowledged.
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Notes |
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1 Present address: Connective Tissue Biology Laboratories, School of Molecular and Medical Biosciences, University of Wales College of Cardiff, Cardiff, UK.
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