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Am J Physiol Heart Circ Physiol 292: H1364-H1372, 2007. First published October 27, 2006; doi:10.1152/ajpheart.00355.2006
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Spatial disruption and enhanced degradation of collagen with the transition from compensated ventricular hypertrophy to symptomatic congestive heart failure

H. K. Graham and A. W. Trafford

Unit of Cardiac Physiology, Division of Cardiovascular and Endocrine Sciences, University of Manchester, Manchester, United Kingdom

Submitted 3 April 2006 ; accepted in final form 24 October 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The cardiac extracellular matrix (ECM) maintains the structural and mechanical integrity of the myocardium. We determined the alterations in the composition of the ECM coincident with the transition from compensated left ventricular (LV) hypertrophy (LVH) to symptomatic congestive heart failure (CHF) and the mechanisms underlying such changes. Heart failure was induced in ferrets by aortic banding. Myocardial collagen content was assessed by HPLC and histological analysis. Matrix metalloproteinase (MMP) activity and tissue inhibitor of metalloproteinase (TIMP) expression were evaluated using gelatin zymography and Western blotting, respectively. LV free wall thickness increased by 29% in asymptomatic LVH and was associated with a 20% increase in interstitial fibrosis (P < 0.05). CHF was coincident with increased plasma angiotensin II levels (149 ± 48, 40 ± 19, and 5.6 ± 1 pg/ml for CHF, LVH, and sham, respectively; P < 0.01, CHF vs. sham and LVH), ventricular dilatation (LV internal diameter = 15 ± 0.4 vs. 9 ± 0.1 mm, P < 0.05), increased active MMP-9 (3.0- and 2.2-fold increase over sham and LVH, respectively, n = 5–10 animals per group, P < 0.01), and reduced myocardial total collagen content (3.5 ± 0.4, 2.6 ± 0.3, and 2.2 ± 0.3% in sham, LVH, and CHF, respectively, P < 0.05). In CHF the distribution of collagen was markedly altered, becoming punctate in nature. No difference in MMP-2 activity, TIMP-1, TIMP-2, TIMP-3, or TIMP-4 expression, or collagen cross-linking was found at any time. The present work demonstrates structural reorganization and loss of collagen from cardiac ECM during the transition to decompensated CHF. The enhanced MMP-9 activity coincident with the transition to CHF provides potential therapeutic opportunities for managing the progression from asymptomatic LVH to symptomatic CHF.

connective tissue; extracellular matrix; matrix metalloproteinase

IN THE HEART, an intricate lattice of collagen-containing extracellular matrix (ECM) surrounds individual myocytes and myofibril bundles, ensuring correct tissue geometry and aiding efficient vectoral transmission of force (4). Although contractile dysfunction in heart failure has been attributed to alterations in cellular Ca2+ homeostasis (8, 25), maladaptive remodeling of the cardiac ECM is also noted in various disease states (32, 36) and may contribute to systolic dysfunction through alterations in the tensile properties of the myocardium and in myocyte orientation (1). Histological evidence of reduced interstitial collagen content and/or fragmented collagen distribution exists in diverse models of heart failure with cardiac dilatation (10, 33, 37, 38). Whereas collagen loss and fragmentation in heart failure have been observed in some studies, myocardial fibrosis has been noted in others, particularly where cardiac pathology is of an ischemic or chronic hypertensive origin (2, 3, 13, 16, 24), and as such fibrosis is often considered de rigueur. In these instances, fibrosis is believed to increase ventricular stiffness and reduce compliance and, thus, compound any underlying cellular pathology and further impair myocardial mechanical function. The apparent contradictions regarding the collagen content of the myocardium in disease states possibly result from 1) inappropriate comparisons between different animal models, which result in concentric or eccentric remodeling, or 2) the failure of previous studies to determine the changes in the ECM that lead to the transition from stable compensated left ventricular (LV) hypertrophy (LVH) to symptomatic congestive heart failure (CHF). The primary purpose of the present study was therefore to determine whether the properties of the cardiac ECM change during the transition from compensated LVH to overt CHF.

The collagen content of the heart is determined by a balance between the synthesis and degradation of collagen. Although collagen synthesis appears to be consistently increased in models of heart disease (10, 26, 41), the role of the specific matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) that control collagen degradation is more equivocal (10, 23). Studies employing transgenic mice clearly show the role of MMPs and TIMPs in maintaining normal cardiac geometry and function (11, 35), as well as determining the remodeling process that occurs with disease (6, 21), and are consistent with less deleterious cardiac remodeling in animal models treated with pharmacological metalloproteinase inhibitors (18, 20, 23). However, little information exists demonstrating how such variables interrelate in cardiac disease states or the temporal alterations in such relations with the progression of cardiac disease states and, in particular, during the transition from compensated LVH to symptomatic CHF. Therefore, we have sought to determine how these parameters change in an animal model of LVH and heart failure by combining measurements of myocardial function, histological evaluation of myocardial collagen distribution, biochemical assessment of tissue collagen content, and zymographic determination of MMP activity and TIMP protein expression. Our data provide an indication of the importance of MMP-9 in the adverse remodeling of the failing heart and, thus, raise the possibility of pharmacological prevention of the degeneration of myocardial structure and function during the transition from compensated cardiac hypertrophy to overt heart failure.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All procedures involving animals were carried out according to the United Kingdom Animals (Scientific Procedures) Act of 1986 and received local ethical approval from the University of Manchester.

Animal model and echocardiographic assessment. LVH and heart failure were induced in 49 weight-matched ferrets (937 ± 15 g), as previously described (8). Perioperative analgesia was provided with meloxicam (4 mg/kg sc). Sham-operated animals served as controls and received the same treatment, except for placement of the ascending aortic occluder. Echocardiography was performed using a 14-MHz linear array probe (Siemens) or a 7.5-MHz sector scanning head (Dynamic Imaging) on animals anesthetized with 1–2% isoflurane in oxygen. In an additional series of experiments, a subset of animals (n = 9) underwent conscious echocardiography. All measurements were obtained from two-dimensional long-axis parasternal images proximal to the papillary muscles and averaged over 15 cardiac cycles.

Analysis of myocardial collagen content and collagen cross-linking. Animals were killed by administration of pentobarbitone sodium (200 mg/kg ip), and full-thickness strips of the intrapapillary LV free wall (~5 x 20 x 5 mm) were fixed for 24 h in PBS containing 4% formaldehyde for histological analysis, or snap-frozen strips were stored in liquid nitrogen for analysis of myocardial hydroxyproline content, substrate zymography, and determination of TIMP protein expression. After the samples were processed and embedded in paraffin, they were cut into 5-µm sections and stained with 0.1% Sirius red in saturated picric acid (17). Some sections (see GoFig. 2D) were counterstained with Miller's reagent. Micrographs were obtained across the entire thickness of the ventricular wall (imaged area ~10.8 mm2 per section), and contiguous regions were chosen to avoid large blood vessels. Polarized images were autothresholded using Scion Image (www.scioncorp.com), and collagen content was expressed as the percentage of nonzero (collagen-containing) pixels per tissue section.


Figure 1
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  		Fig. 1. Chamber dilatation and impaired contractility in heart failure. A–C: representative 2-dimensional long-axis parasternal echocardiograms obtained from sham-operated animals (A) and animals in which left ventricular (LV) hypertrophy (LVH, B) and congestive heart failure (CHF, C) were induced. LV, left ventricular chamber; Ao, aortic outflow tract; LA, left atrial chamber. D–F: M-mode echocardiograms of area proximal to the papillary muscle of sham-operated (D), LVH (E), and CHF (F) animals. Solid arrows, endocardial surface of left ventricular septum; open arrows, endocardial surface of left ventricular posterior wall. Divisions on scale bars, 5 mm.

 

Figure 2
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  		Fig. 2. Altered collagen content and distribution in asymptomatic LVH and symptomatic CHF. Picrosirius red-stained micrographs from sham (A), LVH (D), and CHF (G) animals were viewed by conventional light microscopy at x20 magnification. B, E, and H: circular polarized light images from field of view shown in A, D, and G, respectively. C, F, and I: higher (x40) magnification views for sham, LVH, and CHF sections, respectively. In middle and right images, birefringent collagenous structures are visualized as yellow/red/green pixels. Scale bars, 100 µm.

 
The collagen content of the snap-frozen myocardial samples was determined using precolumn derivatization HPLC and fluorometric detection of 9-fluorenylmethylchloroformate-derivatized 4-hydroxyproline, as previously described (34). 3,4-Dihydroproline (15 µg/ml) was added to each sample/standard as an internal control. Fluorescent 9-fluorenylmethylchloroformate derivatives were separated using reverse-phase HPLC with a 25 cm x 4.6 mm column packed with 5-µm 300-Å C-18 bonded silica and eluted with 50 mmol/l acetic acid and 35% acetonitrile. Fluorometric quantification (260 nm excitation and 330 nm emission) of 4-hydroxyproline was performed against a standard curve, and samples were normalized to the 3,4-dihydroproline peak to control for any loading differences. Collagen content was then calculated with the assumption that collagen contains 13.4% hydroxyproline (22).

Collagen cross-linking was determined from the ratio of pepsin-insoluble to pepsin-soluble collagen by adaptation of previously described methods (27, 28). Approximately 50 mg of frozen myocardium were lyophilized and subjected to pepsin digestion in 0.5 mol/l acetic acid (1 mg/ml) for 24 h at 4°C. Samples were then centrifuged at 15,000 g for 45 min, and the supernatant and pellet were separated and lyophilized. Samples and 4-hydroxyproline standards were then hydrolyzed (2 mol/l NaOH) at 120°C for 20 min before oxidization with chloramine-T (0.028 mol/l) and addition of Ehrlich's reagent (0.5 mol/l) and quantification by absorbance spectrophotmetry at 550 nm.

MMP extraction and gelatin zymography. LV samples (~200 mg) were homogenized in ice-cold extraction buffer [1:10 (wt/vol)] containing 10 mM cacodylic acid, 150 mM NaCl, 20 mM ZnCl2, 1.5 mM NaN3, and 0.1% (vol/vol) Triton X-100 (pH 5.0). Insoluble material was removed by centrifugation at 2,000 g, and protein concentration was determined (Bio-Rad). Samples (15 µg of protein) and standards (2 ng of gelatinase zymography standards; Chemicon) were separated by nonreducing SDS-PAGE (10% acrylamide and 1 mg/ml gelatin). Gels were washed for 1 h in two changes of rinse buffer containing 50 mM Tris and 2.5% (vol/vol) Triton X-100 (pH 7.8) for 15 min in three changes of water before incubation for 16 h at 37°C in incubation buffer containing 50 mM Tris and 5 mM CaCl2 (pH 7.8). Gels were stained in 0.25% Coomassie blue, 50% methanol, and 10% acetic acid for 5–10 min and destained in 30% methanol and 1% acetic acid. Substrate zymography was repeated three times for each sample, and the density of gelatinolytic bands was normalized to that obtained from an internal standard run in parallel on each gel. To confirm the presence of pro- or active MMP, samples and HT-1080-conditioned medium, which served as positive control, were incubated with p-aminophenylmercuric acetate (APMA), as described elsewhere (30).

TIMP protein levels and plasma angiotensin II concentrations. Myocardial samples were homogenized in RIPA buffer containing protease inhibitors, protein concentration was determined, and samples were separated using denaturing SDS-PAGE on a 4–12% Bis-Tris gradient gel and transferred to polyvinylidene difluoride membrane and blocked using 5% nonfat milk, as previously described (8). TIMP-1, TIMP-2, TIMP-3, TIMP-4, and TNF-{alpha} immunodetection was performed using chemiluminescent substrate. Membranes were then stripped and reprobed for GAPDH. Protein loading, sources, and working concentrations of primary and secondary antibodies are summarized in Table 1. TIMP and TNF-{alpha} protein expression was normalized to GAPDH protein expression, and mean data from 3 repeats for each blot and 6–10 animals per experimental group are presented.


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  		Table 1. Detection of TIMP-1, -2, -3, and -4, TNF-{alpha}, and GAPDH

 
Plasma angiotensin II levels were assessed in a parallel study by ELISA (SPIbio). Whole blood was collected in chilled EDTA tubes, plasma was separated by centrifugation at 3,000 g and passed through phenyl cartridges (Amersham), and angiotensin was eluted with methanol. After evaporation of methanol under nitrogen, angiotensin samples and standards were resuspended in enzyme immunoassay buffer, Ellman's reagent was added, and levels were quantified (absorbance spectrophotometry at 405 nm).

Statistics. Values are means ± SE for n animals. One- or two-way analysis of variance was used to compare experimental groups as appropriate, and P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Myocardial dysfunction and chamber dilatation in heart failure. In 18 of 35 coarcted animals, symptoms of CHF (lethargy, dyspnea, and cachexia) developed after 60 ± 9 days (CHF group), while the remaining 49% remained clinically normal and were killed 209 ± 27 days after coarctation (asymptomatic LVH group). Sham-operated (control) animals were killed 142 ± 15 days after surgery. Table 2 summarizes gross morphological measurements of heart, lung, and liver weights relative to body weight and tibial length. Heart weight-to-body weight ratio increased by 65% and 107% in the LVH and CHF animals, respectively, yet the lung weight-to-body weight ratio increased only in the CHF group (by 280%, P < 0.05), a finding consistent with the lack of clinical symptoms in the LVH animals.


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  		Table 2. Cardiac hypertrophy and pulmonary edema in heart failure

 
Figure 1 illustrates typical echocardiographs obtained immediately before euthanasia in each group and demonstrates the marked chamber dilatation and impaired fractional shortening in the symptomatic CHF group. Table 3 summarizes the changes in cardiac dimensions, fractional shortening, and ejection fraction. Although fractional shortening was maintained in the asymptomatic LVH group, it was reduced by 55% in the symptomatic CHF group and was coincident with a 109% increase in left atrial diastolic diameter compared with the sham group (both P < 0.05). Although fractional shortening and ejection fraction were maintained in the asymptomatic LVH group, there was also an increase, although much less marked (20%), in left atrial diastolic diameter, suggesting that whereas clinical symptoms of heart failure were not observed in this group, considerable underlying pathology/remodeling had occurred. Echocardiographic parameters from a subset of conscious animals (sham and CHF) were very similar to those from anesthetized animals (Table 3).


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  		Table 3. Reduced ejection fraction, fractional shortening, and cardiac dilatation in heart failure

 
Temporal and spatial reorganization of collagen distribution and content in LVH and CHF. Tissue sections stained with picrosirius red and viewed under conventional light microscopy (Fig. 2, A, D, and G) revealed a collagenous network (red) surrounding muscle bundles (yellow) that appeared thicker and more extensive in the LVH group (Fig. 2D) and much less evident in the CHF group (Fig. 2G). Under polarized light, interstitial fibrosis in the LVH group (Fig. 2, E and F) and loss of collagen from the interstitium in the symptomatic CHF group (Fig. 2, H and I) were evident. Furthermore, in heart failure (Fig. 2I), the remaining thinner collagen sheath was disrupted and existed as small punctate islands, rather than as continuous sheets, as observed in sections from sham (Fig. 2C) and LVH (Fig. 2F) animals. The collagen content of the myocardium was quantified histologically in two ways: 1) as the nonvascular myocardial area occupied by collagen, including the endocardial and epicardial surfaces (Fig. 3A), and 2) as the interstitial collagen content, calculated as the fraction of the nonvascular cardiac area occupied by collagen and also excluding the epicardial and endocardial surfaces (Fig. 3B). The total nonvascular collagen content, including the epicardial and endocardial surfaces of the heart, was unaltered in the LVH group (3.0 ± 0.5 and 3.1 ± 0.6% tissue area in sham and LVH, respectively, n = 5–7 animals) but was decreased in the CHF group (1.4 ± 0.4% tissue area, P < 0.05 vs. sham and LVH). When only the interstitial cardiac collagen content was considered (Fig. 3B), an increase in the collagen content was observed in the LVH group, yet a reduction in interstitial collagen was still observed in the CHF group: 1.5 ± 0.2, 1.8 ± 0.1, and 0.7 ± 0.1% tissue area in sham, LVH, and CHF, respectively (P < 0.05, sham vs. LVH and CHF vs. sham and LVH). Because histological methods are potentially susceptible to sampling bias, we also obtained the myocardial collagen content by determining the hydroxyproline content of the tissue. The collagen content, expressed as a percentage of the tissue dry weight of the full-thickness (including vasculature and endocardial and epicardial surfaces) myocardial sections (Fig. 3C), was reduced in the LVH (2.6 ± 0.3 vs. 3.5 ± 0.4%, P < 0.05 vs. sham) and CHF (2.2 ± 0.3%, P < 0.05 vs. sham and LVH) groups.


Figure 3
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  		Fig. 3. Altered collagen content in LVH and CHF. A: myocardial collagen content determined histologically using picrosirius red staining and circular polarizing light microscopy. Collagen content was determined from nonvascular components of the myocardium. B: interstitial collagen content quantified histologically by exclusion of vascular, epicardial, and endocardial collagen components of the myocardium. C: hydroxyproline determination of myocardial collagen content. Values are means ± SE (n = 5–10 animals in each group). *P < 0.05 vs. sham. $P < 0.05 vs. LVH.

 
Collagen remodeling, cardiac function, geometry, and myocardial gelatinolytic activity. The relation between LV end-diastolic diameter (LVEDD), LV fractional shortening (as an indication of ventricular function), and the collagen content of the LV is summarized in Fig. 4A. The loss of myocardial collagen led to progressive chamber dilatation (slope = –1.88 ± 0.7, P < 0.05) and reduced fractional shortening (slope = 0.13 ± 0.05, P < 0.05). Figure 4B summarizes the relation between fractional shortening and LVEDD and, again, illustrates the progressive deterioration in LV function with ventricular dilatation (slope = –0.05 ± 0.01, P < 0.05). These data demonstrate that the loss of collagen from the myocardium paralleled the progressive deterioration in cardiac function and dilatation. In the next series of experiments, we therefore sought to determine the mechanisms of collagen loss from the myocardium.


Figure 4
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  		Fig. 4. Functional consequences of altered collagen content in LVH and CHF. A: dependence of LV end-diastolic diameter (LVEDD, top) and fractional shortening (bottom) on myocardial collagen content determined using hydroxyproline quantification. B: dependence of fractional shortening on LVEDD. Values are means ± SE (n = 5–10 per group). Circles, sham; squares, LVH; inverted triangles, CHF; solid symbols, paired data points; open symbols, means ± SE for all unpaired data. Regression lines (dashed lines) have been fit to all data points.

 
Gelatin zymography revealed lytic bands at 64, 70, and 86 kDa (Fig. 5A). The 64- and 70-kDa bands correspond to pro- and active MMP-2 human gelatinase standards (data not shown). To establish that gelatinolytic activity is due to MMP activity, samples were incubated with EDTA, which abolished all activity (Fig. 5B). To determine whether the lytic activity at 86 kDa was due to the pro- or the active form of MMP-9, samples and conditioned medium from HT1080 cells were incubated with APMA (Fig. 5C). APMA activation resulted in a shift in the size of the HT-1080-conditioned medium lytic bands from 92 kDa (pro-MMP-9) to 82 kDa (active MMP-9) and the appearance of an additional band at 62 kDa (active MMP-2). In tissue extracts, there was no shift in the 86-kDa band, suggesting that the active form of MMP-9 was present.


Figure 5
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  		Fig. 5. Increased matrix metalloproteinase (MMP)-9 relative abundance in CHF. A: representative zymograms of myocardial extracts from sham, LVH, and CHF hearts. B: zymogram from sham, LVH, and CHF tissue extracts after incubation with EDTA. C: zymogram of HT10180-conditioned media (lanes 1–3) or myocardial tissue extracts (lanes 4–8) incubated in the presence (+) or absence (–) of p-aminophenylmercuric acetate (APMA). D–F: densitometric analysis of collagenolytic activity from sham, LVH, and CHF samples relative to internal control for active MMP-9 (D), pro-MMP-2 (E), and active MMP-2 (F) levels. Values are means ± SE (n = 8–10 hearts per group). *P < 0.01 vs. sham. $P < 0.01 vs. LVH.

 
Figure 5, D–F, summarizes gelatinolytic abundance in sham, LVH, and CHF samples. There was no difference in pro-, active, or total MMP-2 (data not shown) in all the samples. However, the amount of active MMP-9 was dramatically increased in the CHF group only: by 200% compared with the control group and by 175% compared with the LVH group (P < 0.01 vs. sham and LVH).

Figure 6 shows the relation between MMP-9 activity and LVEDD, LV fractional shortening, and the collagen content of the heart. Linear regressions were applied to the mean relations between LVEDD and active MMP-9 abundance (r2 = 0.99, P < 0.01) and fractional shortening and active MMP-9 abundance (r2 = 0.96, P < 0.05). However, the mean relation between myocardial collagen content and active MMP-9 abundance was best described using an exponential function (r2 = 0.9, exponent = 4.2).


Figure 6
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  		Fig. 6. Influence of MMP-9 activity on cardiac function in CHF. Top and middle: dependence of LVEDD and ejection fraction on MMP-9 activity within the myocardium. Dashed lines, linear regressions to all the data points. Bottom: dependence of myocardial collagen content determined by hydroxyproline quantification on MMP-9 activity. Values have been fitted (dashed line) with an exponential function of the following form: y = a + b{exp(–k[MMP-9])}, where a (2.23) and b (41.9) are constants, [MMP-9] is MMP-9 activity relative to internal standard, and k (4.2) is the value of the exponent. Circles, sham; squares, LVH; inverted triangles, CHF; solid symbols, paired data; open symbols, mean ± SE (n = 8–10 per group) for unpaired data points.

 
Collagen cross-linking was determined from the ratio of pepsin-insoluble to pepsin-soluble hydroxyproline. This ratio was unaltered by disease progression (0.62 ± 0.07, 0.66 ± 0.13, and 0.69 ± 0.09 for sham, LVH, and CHF, respectively), thus suggesting that the loss of collagen from the myocardium with disease progression is key to the adverse geometric remodeling and onset of clinical symptoms.

Plasma angiotensin II concentrations were increased in CHF (149 ± 48, 40 ± 19, and 5.6 ± 1 pg/ml for CHF, LVH, and sham, respectively; P < 0.01, CHF vs. sham and LVH, n = 4–7 animals per group). Although there was a trend for the plasma angiotensin II concentration to increase in LVH, this did not reach significance because of the large variance in the results, presumably representing a difference in the extent of disease progression, despite the lack of obvious clinical symptoms.

Unaltered TIMP expression in LVH and CHF. The final series of experiments examined the expression of TIMP-1, TIMP-2, TIMP-3, and TIMP-4 in sham-operated, asymptomatic hypertrophied, and symptomatic CHF LV myocardium. Figure 7A shows representative Western blots. These blots were then stripped and reprobed against GAPDH to control for any loading differences. Figure 7B summarizes the TIMP-to-GAPDH protein ratios expressed relative to sham levels on each blot. Two-way analysis of variance (to analyze between disease states and blots) failed to detect any difference in TIMP levels between sham, LVH, and CHF at the time of sample collection, i.e., death at the end of the experiments. Determination of myocardial TNF-{alpha} expression also failed to detect any differences between the three experimental groups. The ratio of total MMP-2 abundance determined zymographically to TIMP expression was unaltered (Fig. 7C), whereas the ratio of MMP-9 abundance to TIMP-1, TIMP-2, and TIMP-4 was increased only in the CHF group (P < 0.05).


Figure 7
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  		Fig. 7. Unaltered expression of tissue inhibitor of metalloproteinase (TIMP) in CHF. A: representative Western blots for TIMP-1, TIMP-2, TIMP-3, TIMP-4, TNF-{alpha}, and GAPDH. B: mean protein expression data, normalized to sham levels on each blot, for TIMP-1, TIMP-2, TIMP-3, TIMP-4, and TNF-{alpha}. C: mean total MMP-2-to-TIMP ratio. D: mean MMP-9-to-TIMP ratio. For C and D, MMP levels were obtained from Fig. 5 and TIMP levels from Fig. 7B. *P < 0.05 vs. LVH.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Remodeling of the ECM is believed to influence myocardial structure and function in health and disease. To our knowledge, this is the first report directly examining the alterations in the relations between myocardial collagen content, collagen distribution, cardiac function, and MMP activity during the transition from compensated asymptomatic LVH to decompensated symptomatic CHF. The principal observation is that of considerable disruption to collagen distribution within the cardiac ECM and a loss of collagen coincident with chamber dilatation and onset of clinical symptoms. We propose that the loss of collagen from the myocardial interstitium occurs as a consequence of an increase in MMP-9 activity.

Reduced cardiac collagen content and altered distribution with progression to symptomatic CHF. Hydroxyproline quantification and histological analysis were used to quantify myocardial collagen content in this study. Both methods yielded quantitatively similar collagen contents. In agreement with other models of heart failure and clinical observations (10, 29, 33, 39), collagen content was reduced at CHF coincident with disrupted distribution evident histologically. In the asymptomatic LVH group, a moderate interstitial fibrosis was observed histologically when the endocardial, epicardial, and vascular regions were excluded from analysis. This is consistent with a number of other studies and the accepted dogma of myocardial fibrosis in cardiac disease states (15, 16, 24, 26, 41). Nevertheless, the hydroxyproline quantification demonstrated a reduction in total myocardial collagen content in the asymptomatic LVH group that likely reflects differential remodeling of collagen within the interstitial, vascular, and surface layers of the heart at this time point.

We propose that the loss of collagen from the interstitium is a critical step in the development of cardiac dilatation and onset of clinical symptoms of heart failure. In agreement with this assertion, data from a recent study by Heymans et al. (15) demonstrated an increased myocardial collagen content 2 wk after thoracic aortic banding in mice that decreased 7 wk after banding, when cardiac dilatation and reduced ejection fraction were observed. Additionally, Fedak et al. (10) using the cardiomyopathic hamster, and Spinale et al. (33), in tachypacing heart failure in the pig, reported that the collagen weave was markedly reduced and disrupted. Weber et al. (37) noted similar loss of collagen and altered distribution of collagen in explanted failing human hearts, suggesting a commonality of mechanism between diverse species and disease models in end-stage heart failure. The apparent discrepancy between the present study and those studies of human hypertension (16, 26) or animal models of pressure-overload cardiac dysfunction (24), where interstitial fibrosis is considered the norm, may simply reflect the end-stage nature of the disease process in the present study.

Differential changes in MMP and TIMP expression in CHF. In the present study, gelatin zymography was used to identify the MMPs likely responsible for the disruption of the interstitial collagen weave in CHF and chamber dilatation. An increased level of the active MMP-9 was noted in the CHF samples, but this was not associated with any change in total, pro-, or active MMP-2 levels at the time of death. Analogous results have been reported in canine dilated cardiomyopathy and in spontaneously hypertensive and Dahl salt-sensitive rats (12, 23). MMP expression and conversion from pro- to active MMP can be upregulated by several different mechanisms, including angiotensin II and cytokines such as TNF-{alpha} (14, 39). In the present study, no changes in myocardial TNF-{alpha} were observed at the time of sample collection. However, involvement of other cytokines in mediating increased MMP-9 abundance cannot be excluded. A potential mechanism by which MMP-9, rather than MMP-2, is selectively upregulated in this study and elsewhere (12, 23) may reside in the unique NF-{kappa}B transcription binding site in the promoter region of MMP-9 (for review see Ref. 7). The NF-{kappa}B pathway is known to be activated by angiotensin II and increases MMP-9 transcription (31), which, together with the increase in collagen type I synthesis in cardiac fibroblasts by the same pathway (5), may underlie much of the beneficial effect of angiotensin-converting enzyme inhibitors in the management of cardiac disease states (19). Further evidence in support of the role of MMP-9 in the deleterious remodeling processes of heart disease is provided by the targeted deletion of MMP-9 whereby cardiac remodeling was substantially attenuated after infarction (9) and after aortic banding (15) in mice. Furthermore, the use of less specific pharmacological interventions that target MMP-9 activity also results in improved cardiac function and limits structural remodeling (18, 40).

In the present study, neither myocardial TIMP protein levels nor collagen cross-linking was found to be altered at the time of death. Since this study is not designed longitudinally because of the impracticality of obtaining serial samples from the same heart, we cannot completely rule out the possibility that changes in TIMP expression, activation of other MMPs, or changes in collagen cross-linking occur at intermediate stages of disease progression. However, the increase in MMP-9 observed in this study at the time of chamber dilatation, reduced fractional shortening, and onset of clinical symptoms of heart failure are indicative of the role of MMP-9 in the deleterious progression from compensated LVH to symptomatic CHF.

Study limitations. Since we are unable to obtain serial samples from the same heart, an important consideration is whether the symptomatic CHF animals undergo a disease process different from that of the LVH group, which remained asymptomatic for considerably longer. To address this issue, we performed echocardiography at an intermediate time point in animals that later developed CHF and found similar changes in free wall thickness (5.0 ± 0.3 and 3.1 ± 0.1 mm at LVH and CHF, respectively) and LVEDD (20.5 ± 0.1 and 23.1 ± 0.3 mm at LVH and CHF, respectively), suggesting that, indeed, those animals that later become symptomatic do experience an asymptomatic hypertrophic phase of cardiac disease in the first instance. We believe that, in the present study, some animals remain in a compensated LVH phase, because the degree of aortic compression and, therefore, overload on the LV is subtly different between animals due to the extent of swelling of the ameroid material in the occluder placed around the ascending aorta.

MMPs are tightly regulated by a family of tissue inhibitors (TIMPs) (32). Although our data suggest that the expression level of TIMPs is unaltered, we cannot address the stoichiometric relation between TIMPs and MMPs. Additionally, zymograms do not evaluate potential contributions to the overall collagenolytic activity of the heart by other serine and cysteine proteases. Nevertheless, two important points support our suggestion that MMP-9 is critical to the remodeling process observed in the present study: 1) there was no shift in the total MMP-2 levels or the ratio of pro- to active MMP-2, and 2) only active MMP-9 was observed.

In conclusion, it is becoming clear that the myocardial matrix is a dynamic structure and that its continuous turnover is under fine control. Furthermore, a maladaptive response to injury or increased workload, resulting in adverse myocardial extracellular matrix abundance and distribution, can contribute to LV dilation and heart failure. We conclude that an increased MMP-9 activity, resulting in a loss of the collagenous interstitium, may play a role in the deterioration of contractile function, LV dilatation, and the progression to heart failure and may provide a novel pharmacological target to prevent the progression from compensated asymptomatic LVH to symptomatic CHF.


   GRANTS
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 ABSTRACT
 METHODS
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 DISCUSSION
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REFERENCES
 
This work was supported by the British Heart Foundation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. W. Trafford, Unit of Cardiac Physiology, Division of Cardiovascular & Endocrine Sciences, Univ. of Manchester, 3.08 Core Technology Facility, 46 Grafton St., Manchester M13 9NT, United Kingdom (e-mail: Andrew.W.Trafford{at}manchester.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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