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Effect of Ramipril and Furosemide Treatment on Interstitial Remodeling in Post-Infarction Heart Failure Rat Hearts
J Mol Cell Cardiol 34, 151–163 (2002) doi:10.1006/jmcc.2001.1497, available online at http://www.idealibrary.com on
Effect of Ramipril and Furosemide Treatment on Interstitial Remodeling in Post-Infarction Heart Failure Rat Hearts Ute Seeland1, Ichiro Kouchi1, Oliver Zolk2, Gabi Itter1, Wolfgang Linz1 and Michael Bo¨hm1 1
Med. Univ.-Klinik und Poliklinik der Universita¨t des Saarlandes, Innere Medizin III – Kardiologie und Angiologie – 66421 Homburg/Saar, Germany and 2Institut fu¨r Experimentelle und Klinische Pharmakologie und Toxikologie, Universita¨t Erlangen-Nu¨rnberg, Germany
(Received 15 August 2001, accepted for publication 31 October 2001) U. S, I. K, O. Z, G. I, W. L M. B¨ . Effect of Ramipril and Furosemide Treatment on Interstitial Remodeling in Post-Infarction Heart Failure Rat Hearts. Journal of Molecular and Cellular Cardiology (2002) 34, 151–163. Extracellular matrix (ECM) remodeling and increased matrix metalloproteinase (MMP) expression and activity have been observed to be relevant in the development of heart failure (HF). We examined the effects of ramipril alone or with furosemide on ECM in a heart failure model. HF was induced by occlusion of the left coronary artery in spontaneously hypertensive rats (SHR). Rats were assigned to placebo (n=9), ramipril 1 mg/kg/day (n=11), furosemide 2×2 mg/kg/day (n=7) or both (1 mg/kg/day+2×2 mg/kg/day; n= 8). LV-function, collagen content, MMP/TIMP (tissue inhibitor of matrix metalloproteinases) protein- and mRNAexpression were examined in non-infarcted LV tissue. MMP-2/TIMP-4 ratio was increased in HF. Ramipril reduced MMP-2 expression (active form), collagen type I mRNA expression and content and increased TIMP-4 levels associated with decreased left ventricular end diastolic pressure (LVEDP), mortality rate and increased LV pressure (LVP). Combination therapy with furosemide is less efficient with regard to collagen content and MMP-2 (active form) reduction but did not worsen beneficial effects of ramipril on LV function and mortality rate. Furosemide alone had no effect on MMP-2 (active form) expression, collagen content, LV function and mortality rate. Prevention of LV dilatation by ramipril was associated with decreased gelatinolytic activity and increased MMPinhibition in heart failure SHR. Furthermore, ramipril reduced fibrosis by enhanced interstitial collagenase expression. Furosemide did not show the beneficial effects of ramipril on ECM remodeling but did not worsen LV function. Positive effects of furosemide treatment alone on LV remodeling and function were not observed. 2002 Elsevier Science Ltd.
K W: Matrix metalloproteinases; Extracellular matrix; Interstitial remodeling; Heart failure; Diuretic agents; ACE inhibitors.
Introduction Transition from compensatory hypertrophy to heart failure following myocardial infarction (MI) is associated with LV remodeling, characterized by interstitial and perivascular deposition of fibrillar collagen and LV dilatation. Collagen degrading activity leads to myocyte disarray through weakness of the collagen scaffold.1,2 Mediators of myocardial
fibrosis post-MI, including angiotensin II (Ang II) among others, have attracted considerable interest. Chronic administration of an ACE-inhibitor significantly attenuates fibrosis in both infarcted and non-infarcted regions of the myocardium, while improving heart function and survival.3,4 The effects of diuretic therapy, which is often coadministered in this condition, have not been investigated. The mechanisms of the beneficial action of ACE-
∗ Please address all correspondence to: Michael Bo¨hm, Med.Univ.-Klinik und Poliklinik der Universita¨t des Saarlandes, Innere Medizin III, 66421 Homburg/Saar, Germany. Tel: [+49]-6841-16-23372. Fax: [+49]-6841-16-23369. E-mail: [email protected]
0022–2828/02/020151+13 $35.00/0
2002 Elsevier Science Ltd.
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Table 1 Heart weight and hemodynamic parameters Groups
n
LV weight (g)
RV weight (g)
MAP (mmHg)
Heart rate b/min
LVP (mmHg)
+dP/dt (1000 mmHg/s)
Sham C R F R/F
12 9 8 7 8
1.08±0.03 1.17±0.07 1.16±0.03 1.14±0.04 1.05±0.04
0.21±0.02 0.35±0.05† 0.21±0.02∗ 0.22±0.01∗ 0.22±0.01∗
153±8 135±13 124±9 147±12 96±10†
382±11 340±19 315±32† 372±14 310±21†
136±7 98±3† 118±2∗ 108±8 114±4∗
5.32±0.2 3.12±0.4† 4.48±0.2∗ 4.12±0.3 4.21±0.2∗
SHR following myocardial infarction treated with furosemide (F), ramipril (R), ramipril+furosemide (R/F) and no treatment (control=C) compared to Sham operated SHR (Sham). ∗ denotes significant differences from C, † denotes significant differences from Sham; n, number of animals; LV, left ventricular; RV, right ventricular; MAP, mean arterial pressure; LVP, left ventricular pressure; +dP/dt indicates contractility.
inhibitors on extracellular matrix remodeling are not entirely clear. In addition to their inhibitory effect on collagen synthesis, they may affect collagen degradation by influencing matrix metalloproteinase (MMPs) isoforms depending on their substrate specificity.5,6 MMPs are metal-binding proteinases, secreted as a latent proenzyme, which require extracellular activation. MMPs relevant in myocardial remodeling include the interstitial collagenases (MMP-1/MMP-13), cleaving triple helical collagen type I, and the gelatinases (MMP-2, MMP9), degrading favourably denatured collagen type I (gelatine). Endogenous tissue inhibitors of matrix metalloproteinases (TIMPs) function as regulators of MMP expression and activity.7,8 This investigation examines whether diuretic therapy counteracts or further improves the beneficial effects of angiotensin-converting enzyme inhibition on the late extracellular matrix remodeling process. The spontaneously hypertensive rat (SHR) model is characterized by genetic pressure overload and a stimulated renin angiotensin system. The additional effect of experimental myocardial infarction superimposed on genetic hypertension with cardiac hypertrophy and early failure leads to accelerated development of severe heart failure. This study shows that ramipril reduces fibrosis by enhanced interstitial collagenase expression and prevents LV dilatation by decreased expression of gelatinolytic (MMP-2) activity and increased MMPinhibition in heart failure SHR. Additional diuretic therapy with furosemide does not further improve the beneficial effects of ramipril on ECM remodeling but does not worsen LV function.
Materials and Methods Male SHR (250–300 g) were obtained from Harlan Winkelmann (Borchen, Germany), and housed under standardized conditions. The rats had free
access to standard diet (Altromin⊂ Maintenance Diet 1320, sodium content 0.2%) and drinking water ad libitum. The investigation conforms with the Guide for the Care and Use of Laboratory Animals of the National Institute of Health and was performed in accordance with the German animal protection law. All chemicals and reagents were purchased from Sigma (Deisenhofen, Germany), Merck (Darmstadt, Germany) and Roche (Mannheim, Germany) unless otherwise stated.
Study design One hundred and thirty male spontaneously hypertensive rats (SHR), aged 16 weeks, were randomly assigned to five groups. Severe chronic heart failure was induced in 110 of them by 8 weeks’ occlusion of the left coronary artery 2–3 mm distal to the origin of the aorta, resulting in large infarction of the left ventricular wall. Twenty rats were sham operated. Animals were treated with furosemide (4 mg/kg/bw i.p. for 3 days) to reduce acute lung edema and xylocaine (2 mg/kg i.m. before surgery) to prevent arrhythmias. Mortality within 24 h was less than 10%. Two weeks after surgery, animals with MI were randomized to four groups and treatment was initiated for 6 weeks via drinking water. 1: placebo-treated controls (C; n=9), 2: treatment with 1 mg/kg/day ramipril (R; n=11), 3: treatment with 2×2 mg/kg/day furosemide (F; n=7) and 4: treatment with the combination of ramipril and furosemide 1+2×2 mg/kg/day (R/F; n=8). After 6 weeks of treatment rats were reanesthetized and the carotid artery was cannulated to monitor blood pressure. The hearts were excised and mounted on Langendorff apparatus. Isolated hearts were perfused in the working heart mode as described previously.9 Afterwards, hearts were weighed and left ventricular tissues were stored at −80°C. Scar area was determined by planimetry. Infarct size was
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Figure 1 Functional effects on left ventricular end diastolic pressure (LVEDP) following increases of preload (15 to 30 mmHg) in SHR with post-infarction HF treated with furosemide (F), ramipril (R), ramipril+furosemide (R/F) and no treatment (control=C) compared with Sham-operated SHR (Sham). ∗ denotes significant differences from C.
expressed as percentage of left ventricular mass. Animals with infarct sizes of 20–40% were enrolled.3
Working heart model After anesthesia and transverse thoracotomy, hearts were removed and immersed in physiological buffer (4°C). Aortas were mounted on a cannula attached to the perfusion apparatus. Left atria were cannulated via incision of the left auricle. Pulmonary veins were ligated close to the surface of the atria. Hearts were perfused according to Langendorff’s method with oxygenated (95% O2, 5% CO2) non-recirculating Krebs–Henseleit so-
lution. Working mode with a filling pressure (preload) of 18 mmHg and an afterload pressure of 80 mmHg at baseline conditions was used. For functional LVEDP measurements, the filling pressure was increased stepwise from 15 to 30 mmHg (Hugo Sachs Elektronik, Freiburg, Germany).
Picrosirius red staining LV circumferential sections were stained with picrosirius red for fibrillar collagen measurements using a technique described elsewhere.10 Fractional area of collagen content in percentage of myocardial tissue was measured with Lucia G software (Nikon, Du¨sseldorf, Germany).
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Figure 2 Mortality of HF-SHR treated with furosemide (F), ramipril (R), ramipril+furosemide (R/F) and no treatment (control=C) compared to Sham-operated SHR (Sham). ∗ denotes significant differences from C.
Figure 3 LV cross sections were stained by picrosirius red and viewed under polarized light. Interstitial fibrillar collagen fractional area (%) was measured with Lucia G software; n=3–4. ∗ denotes significant differences from C.
Zymography Zymography detects degradative properties of gelatinases by employing gelatin as substrate into a
gel. Tissue gelatinase activity was measured under non-reducing conditions. Quantification was performed densitometrically by the size-fractionated banding pattern. Frozen adult rat myocardium tissue
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Figure 4 Northern blot of procollagen (1) type I and GAPDH mRNA (upper panel) in myocardium from HF-SHR treated with furosemide (F), ramipril (R), ramipril+furosemide (E/F) and no treatment (control=C) compared with Sham-SHR (Sham). Bar graphs summarize the optical density (OD) in % of Sham from n=5–8 (lower panel). ∗ denotes significant differences from C, † denotes significant differences from Sham.
samples were homogenized in 5× volume extraction buffer containing: 10 mmol/l cacodylic acid, pH 5.0, 0.15 mol/l NaCl, 1 mol/l ZnCl2, 20 mmol/ l CaCl2, 1.5 mmol/l NaN3, and 0.01% (v/v) Triton X100 at 4°C. After centrifugation (10 min, 1200×g) supernatants were collected and pH was raised to 7.5. Protein concentrations were measured using a detergent compatible kit (DC protein assay, Bio-Rad, Munich, Germany) based on the method of Lowry.11 Gelatin type B from bovine skin (1 mg/ml) was added to standard Laemmli acrylamide polymerization mixture.12 Tissue extract (25 g) mixed with sample buffer (10% w/v SDS, 4% w/v sucrose, 0.1% w/v bromphenol blue) was loaded into slots (0.8 mm) of a 10% acrylamide stacking gel. After electrophoretic separation gels were soaked in 2.5% w/v Triton X-100 for 60 min at room temperature (RT). Gels were incubated overnight at 37°C in substrate buffer (50 mmol/l Tris-Cl, pH 8.0, 5 mmol/ l CaCl2). After staining for 2 h in 0.05% Coomassie brilliant blue G-250 (Bio-Rad) the gel was destained in acetic acid/ethanol and dried. Supernatant of HT 1080 (15 l), human fibrosarcoma cells, served as positive control. Each sample lane was scanned and presented as percent of latent MMP-2 HT1080standard.
Immunoblotting Homogenized cardiac tissue samples (25 g/lane), were separated on 8% – (collagen type I), 10% – (interstitial collagenase) and 12% – polyacrylamide gel (TIMP-2 and -4) and transferred to nitrocellulose membranes (Protran⊂, Schleicher & Schuell GmbH, Dassel, Germany) by semi-dry electrophoretic blotting (0.8 mA/cm2). Staining with Poinceau red showed relative loading of total cardiac proteins. Membranes were blocked in PBS-Tween (0.5%) containing 5% (w/v) skim milk. Membranes were probed with primary antibodies for interstitial collagenase (MMP-1, IM35L, Ab-1, monoclonal mouse 1:1000, Calbiochem, Bad Soden, Germany), recognizing the epitope from amino acid 349–357 of the rodent counterpart to human MMP-1,13 TIMP2 (IM56L, Ab-2, monoclonal mouse 1:2000, Calbiochem), TIMP-4 (AB816, polyclonal rabbit 1:1000, Chemicon, Hofheim, Germany) and collagen type I (Dianova, Hamburg, Germany) for 120 min at RT. Peroxidase labeled goat anti-mouse was diluted 1:10 000 and used as secondary antibody for MMP-1 and TIMP-2, goat anti-rabbit was diluted used for TIMP-4 and rabbit anti-goat was used for collagen type I. Incubation was performed
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Figure 5 Collagen turnover. (A) Hydroxyproline/proline ratio determined by HPLC and (B) protein expression of MMP1 (proMMP-1 and active forms) from HF-SHR treated with furosemide (F), ramipril (R), ramipril+furosemide (R/F) and no treatment (control=C) compared with Sham-SHR (Sham). Bar graphs summarize the OD in % of Sham from n= 5–9. ∗ denotes significant differences from C; † denotes significant differences from Sham; a, glycosylated; b, nonglycosylated.
for 90 min at RT. The proteins were visualized by enhanced chemiluminescence (ECL) according to the manufacturer’s guidelines (Amersham Pharmacia Biotech, Freiburg, Germany). Autoradiographs from Western blot analysis were quantified by imaging densitometry and ‘‘ImageQuant-TM’’ software (Image Quant, Molecular Dynamics, Krefeld, Germany).
Northern blot analysis Total RNA from deep frozen left ventricular tissue samples was prepared according to the method of Chomczynski and Sacchi.14 Tissue was homogenized in acid guanidinium thiocyanate solution (peqGOLD
RNAPure⊂, Peqlab Biotechnology, Erlangen, Germany) and extracted with phenol and chloroform. RNA was quantified spectrometically, and ethidium bromide-stained agarose gels were used to check its integrity. Rat MMP-2-, pro-(1) type I collagenand GAPDH (glyceraldehyde-3-phosphate dehydrogenase)- cDNA were generated by RT-PCR of rat heart RNA and confirmed by sequencing. MMLV RT and Taq DNA polymerase were purchased from Gibco BRL (Karlsruhe, Germany) and Roche (Mannheim, Germany). Northern blots were prepared from 10 g total RNA as described previously.15 [32P]deoxy CTP labeled cDNA fragments of rat MMP-2- (404 bp), pro-(1) type I collagen(402 bp) and GAPDH (451 bp) were used. The data were processed using the ‘‘ImageQuant-TM’’ soft-
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Figure 6 Zymographic analysis of gelatinolytic activity from HF-SHR treated with furosemide (F), ramipril (R), ramipril+furosemide (R/F) and no treatment (control-C) compared to Sham-SHR (Sham). Bar graphs summarize OD in % of Sham from n=5–9. ∗ denotes significant differences from C; † denotes significant differences from Sham.
ware. Standardization was performed by hybridization of the same membrane using the cDNA probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Density of bands was expressed relative to the density of the GAPDH band.
High-performance liquid chromatography (HPLC) Ratios of proline and hydroxyproline were analyzed according to Lopez-de Leon and Rojkind.16 In brief, biopsies of the left ventricular tissue were hydrolyzed in 6 N HCl for 24 h at 105°C, freeze-dried and resuspended in 0.5 mol/l NaHCO3. Internal standards (1 mmol/l acetidin-2-carbonacid in 0.4 mol/l K-borate pH 9.0) were added.
Statistical analysis Results are presented as means±... Statistical significance was estimated with Student’s t-test for unpaired observations. Differences among the five groups of rats were tested by one-way analysis of variance (ANOVA) once normality was demonstrated and the Fischer test was used for LVEDPmeasurements. A value of P<0.05 was considered significant. Cumulative survival was analyzed for differences according to the Kaplan–Meier method, after which Cox–Mantel log-rank test was used.
Results Characterization of the model Following MI, SHR showed symptoms related to
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Figure 7 Northern blot of MMP-2 and GAPDH mRNA (upper panel) in myocardium from HF-SHR treated with furosemide (F), ramipril (R), ramipril+furosemide (R/F) and no treatment (control=C) compared with Sham-SHR (Sham). Bar graphs summarize the OD in % of Sham from n=5–9 (lower panel). ∗ denotes significant differences from C; † denotes significant differences from Sham.
severe congestive heart failure (CHF) signs in humans such as subcutaneus edema, dyspnea and impaired motion. Hydrothoraces and lung edema were demonstrated in NMR imaging (data not shown). Right ventricular heart weight and lung weight increased significantly in SHR with HF (C) compared to Sham. In all treatment groups RV heart weight decreased to Sham levels. Left ventricular heart weights (infarcted area 20–40% by planimetry) showed no difference (Table 1), nor did body weights measured 8 weeks following MI with or without treatment. The data are given in percentage of body weight measured at the beginning (mean±... in %) Sham: 10.9±1.1, C: 17.4±3, R: 11.1±3.4, F: 9.0±1.8, R/F: 14.8±1.8.
LV function At 24 weeks of age, mean arterial pressure (MAP) in SHR-HF was reduced and did not change significantly following treatment with ramipril or furosemide. However, ramipril plus furosemide decreased MAP significantly to 96±10 mmHg (Table 1), which is similar to the MAP of untreated normotensive rats. Working heart data were performed
ex vivo and are summarized in Table 1. LVP and contractility (+dP/dt) were reduced significantly by 42% in untreated SHR-HF (C) compared with Sham (Table 1). In the furosemide treated SHR-HF group, LVP was similar to untreated control group values, whereas ramipril treatment alone and in combination with furosemide normalized contractility (P<0.05, Table 1). For end diastolic pressures, LVEDP were highest in the untreated SHR-HF group depending on linear rise of filling pressure (preload 15 to 30 mmHg) and were reduced in the SHR-HF groups treated with ramipril and with the combination (+furosemide). The LVEDPs were similar to those of Sham-SHR (P<0.05; Fig. 1). In the furosemide treated group LVEDP was reduced but did not reach statistical significance. These data show that coronary ligation led to destruction of myocardial function, which is sensitive to medical therapy.
Survival Acute mortality rate within 24 h of surgery was less than 10%. Arrhythmias and acute lung edema
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Figure 8 Tissue inhibitors of MMP (TIMP). (A) Western blot analysis of TIMP-2 and (B) of TIMP-4 from HF-SHR treated with furosemide (F), ramipril (R), ramipril+furosemide (R/F) and no treatment (control=C) compared with Sham. Bar graphs summarize OD in % of Sham from n=5. ∗ denotes significant differences from C; † denotes significant differences from Sham; ‡ denotes significant differences from R.
were prevented by xylocaine and furosemide, respectively. Mortality rate was 40% in controls. Ramipril treatment alone and in combination with furosemide significantly decreased the mortality rate to 29% and 19%, respectively (P<0.05 v control). Treatment with furosemide alone failed to improve the mortality rate (42%; Fig. 2).
Extracellular matrix proteins Myocardial collagen fractional area (%) increased significantly in untreated HF-SHR (C) compared with Sham-SHR [%, 3.0±0.6 (C) v 0.6±0.08 (Sham); P<0.01]. Ramipril treatment reduced percent fibrillar collagen to 0.8±0.29%, P<0.05. Furosemide treatment alone and in combination with ramipril also reduced collagen content but did not
reach statistical significance (P=0.06/0.13). These results are consistent with western blotting for collagen type I (data not shown; Fig. 3). Procollagen (1) type I mRNA expression was increased in HF-SHR (C) compared with ShamSHR [246±22% (C); P<0.001]. It was reduced by ramipril treatment and in the combination group [127±21% (R), P<0.005; 162±29% (R/F), P<0.046]. Furosemide treatment alone did not alter procollagen (1) type I mRNA expression v untreated HF-SHR [224±32% (F), 246±22% (C); n.s.]; see Figure 4. Hydroxyproline/proline ratio, a parameter of cardiac collagen turnover increased significantly after MI and development of heart failure [P<0.0001; Fig. 5(A)]. Following ramipril and combination treatment, the amount of the ratio was further increased by 44% in both groups in comparison to untreated controls (P<0.05).
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Matrix metalloproteinases (MMPs) Figure 5(B) provides a representative Western blot illustrating the presence of the latent proMMP-1 (57/52 kDa) and the glycosylated (47 kDa) and non-glycosylated active form (42 kDa) of interstitial collagenase. Relative LV myocardial levels of the glycosylated and non-glycosylated active form of interstitial collagenase were increased significantly in the ramipril treated group compared with untreated controls and Sham-SHR (% of Sham: 263±49 (R) v 138±12 (C); P<0.05). The latent (72/69 kDa) and active (66/59 kDa) form of gelatinase MMP-2 was detected in zymography. Latent MMP-2 was induced and activated in failing SHR [% of Sham: latent 267±53, active 209±58 (C); P<0.01]. The increased level of MMP2 active form in untreated HF-SHR (C) was returned to control levels following ramipril treatment and in the combination group [latent: 120±14, active: 124±20 (R); latent 136±31, active 143±38 (R/ F); P<0.05). In contrast, furosemide alone enhanced signals of the latent MMP-2 expression and active form [% of Sham: latent 361±93, active 263±68 (F); Fig. 6]. Autoradiograms of the Northern blots show a representative band at 3.1 kb of MMP-2 mRNA (Fig. 7). Ratios for MMP-2/GAPDH signals were significantly increased in failing myocardium without treatment and following furosemide alone [% of Sham: 254±27 (C), 252±26 (F); P<0.05]. Ramipril alone and in combination with furosemide significantly normalized MMP-2 mRNA expression compared to controls [% of Sham: 254±27 (C), 154±19 (R), P<0.05; 175±14 (R/F), P<0.05].
Tissue inhibitors of matrix metalloproteinases (TIMPs) TIMP-2: except for a 2.5-fold significant increase in the furosemide group (P<0.05), there was a minor increase after myocardial infarction and a reduction following ramipril treatment of TIMP-2 [Fig. 8(A)]. TIMP-4 protein was reduced in the failing hearts to 57±8% (C) of Sham (P<0.01). Treatment with ramipril significantly increased protein levels to 80±4% (R) of Sham; [P<0.05, Fig. 8(B)].
Discussion Fibrosis is regarded as contributing to the development of heart failure in rats after coronary ligation. The model used is characterized by fibrous
tissue accumulation leading to increased myocardial diastolic stiffness and impaired systolic function of the left ventricle with clinical symptoms of severe congestive heart failure. It has been taken into consideration that the degree of fibrosis is very different between the different animals. Spontaneously hypertensive rats preferentially expressed fibrosis, and this was the reason for choosing this model. Other models such as aortic constriction etc. are not so useful for studying the development and remodeling of fibrosis because their fibrosis is only moderate.17 It is useful to study the mechanisms by which ACE- inhibitors and diuretics influence myocardial collagen synthesis and degradation. Differences in MMP/TIMP alterations between normal rats and SHR are described elsewhere.6,18,19 Regulation of the MMP/TIMP system in hypertrophy and heart failure is influenced by the reninangiotensin system. We hypothesized that the activity of the different matrix metalloproteinase isoforms and their tissue inhibitors (TIMP) in heart failure can be influenced by treatment with ramipril and furosemide. While there are abundant data about ACE-inhibitors on LV-remodeling, data on furosemide are lacking. There is only one study on the effect of furosemide on hypertrophy. Kim et al. (1996) reported no difference in collagen type I and III gene expression but a significant reduction of left ventricular hypertrophy following furosemide treatment, presumably caused by blood pressure lowering in SHR without heart failure.20 Data from the present study demonstrate that furosemide influenced neither collagen type I protein concentration and procollagen (1) I mRNA expression nor MMP/TIMP expression. Treatment with furosemide in addition to ramipril did not show any further positive effects on ECM remodeling rather activate MMP-2. However, combination therapy improved hemodynamic parameters like LVEDP, LVP, contractility and mortality rate, and reduced mean arterial pressure compared to hypertensive sham operated rats. Therefore, this is the first evidence that diuretic treatment does not interfere with ACE inhibition with respect to hemodynamic parameters, but it does not improve ECM remodeling as well as ramipril treatment alone in a heart failure model. There are reports that afterload reduction by aortocaval shunt or minoxidil treatment reduces collagen content in rats.21 Our finding that furosemide does not significantly affect interstitial remodeling might evidence that only preload reducing effects (increasing diuretics) are not sufficient to decrease interstitial ventricular remodeling. In this light one also has to view the ACE inhibitor effects, which produce pre- and afterload reduction.
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The development of heart failure may be associated both with alterations in the balance of collagen synthesis and inadequate collagen degradation. The rate limiting step in the extracellular degradation of collagen is the catalytic cleavage by interstitial MMPs.22 Depending on substrate specificity active MMP-2 cleaves non triple helical collagens responsible for collagen cross linking and basement membrane function. Reduction in myocardial collagen cross-linking parallels left ventricular dilatation in rat models23 and in failing human hearts.24 Loss of interstitial integrity impairs myocyte alignment and promotes progressive left ventricular dysfunction. The data presented for zymography suggest that the increase in the active form of MMP-2 expression in untreated heart failure- SHR is relevant in this process of ventricular dilatation. In agreement with that, LV myocardial MMP-2 activity has been demonstrated to be increased in a heart failure-SHR model recently published by Peterson et al.25 MMP-2 activity in failing LV-myocardium is significantly reduced following ramipril treatment. This was not observed for furosemide treatment alone in the present study. ACE is a zinc-metalloenzyme and ACE inhibitors inhibit the active site in this enzyme. A direct binding of ramipril to the MMP-2 active site could be one mechanism responsible for this marked inhibition. However, the transcriptional level is involved in the regulation of MMP-2 expression shown by the MMP-2 mRNA data in this study. Furthermore, the levels of the endogenous inhibitor system (TIMPs) are decreased in untreated control animals with severe heart failure. Following ramipril treatment the reduction of TIMP-4, inhibiting most MMPs in adult cardiac tissue, was reversed. These data support the hypothesis that a lack of inhibitory control of MMPs by TIMP-4 contributes to the development of interstitial remodeling. The data from the present study show that this imbalance was reversed by ramipril but not by furosemide. In contrast to TIMP-4, TIMP-2 expression was enhanced following furosemide treatment. This was not expected because of high levels of MMP-2 activity. In addition to the inhibitory control of most MMPs, TIMP-2 can form a complex with MT1MMP at the cell membrane, which possibly plays a regulatory role in the proteolytic activation of MMP-2.26 Therefore the function of TIMP-2 as an inhibitor or activator of MMP-2 is likely to depend on its concentration. It is known that angiotensin II stimulates collagen synthesis in rat cardiac fibroblasts in a concentration dependent manner27,28 and inhibits MMP-1 activity.29 The present data show a sig-
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nificant reduction of percent fibrillar collagen (fractional area), collagen type I protein content, and procollagen (1) I mRNA expression following ramipril treatment. This was accompanied by a significant increase in the expression of active interstitial myocardial matrix collagenase following therapy with ramipril in contrast to untreated controls. Interstitial collagenase is a key enzyme involved in fibrillar collagen degradation because of its ability to cleave native triple helical collagen i.e. collagen type I. Tyagi and Cleutjens purified and characterized rat interstitial collagenase.30 The antibody used in this study showed specific signals for collagenase zymogen at 57/52 kDA and the proteolytically processed active form at 46/42 kDa. Previous studies have reported regulation of MMP1 or indirectly measured collagenase activity by [14C] collagen assay in the rat heart.18,19 It is conceivable that increased interstitial collagenase activity is responsible for the increased hydroxyproline/proline ratio, a parameter of collagen turnover, measured in non-infarcted myocardium following ramipril treatment and combination treatment with furosemide. In order to correlate extracellular matrix remodeling with diastolic myocardial function, left ventricular end diastolic pressure (LVEDP) was determined in the working heart model. The results show a significantly reduced LVEDP following ramipril and ramipril/furosemide treatment compared to controls, whereas no reduction was achieved in the furosemide group. The normalized LVEDP following ramipril treatment and treatment in combination with furosemide is in agreement with an improved MMP-2/TIMP-4 ratio similar to ShamSHR. Furthermore, activated interstitial collagenase expression and reduced collagen type I concentration support the beneficial diastolic and systolic function measured in this study. Beneficial effects of ramipril are reflected in the Kaplan-Meier survival curves. The lowest mortality rates were achieved following ramipril alone and in combination with furosemide. Conversely, furosemide alone failed to improve survival rate. The beneficial effects on hemodynamic parameters and mortality rate following combined therapy may be caused by decreased mechanical load with reduced myocardial wall stress and improved systolic function. In SHR treated with four mg/bw furosemide the excretion of water was very effective. An increase in dose was not followed by a significantly higher efficacy without a change in osmolality.31 In the present study 4 mg/bw were very effective in reducing peripheral edema of SHR with severe congestive heart failure.
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In summary, ACE-inhibitor treatment positively influenced extracellular matrix remodeling by decreasing the gelatinolytic MMP-2/TIMP-4 ratio to prevent myocyte disarray and by increasing the active form of the interstitial collagenase to reduce collagen deposition in the left ventricle following myocardial infarction. In the present study furosemide alone did not show any direct positive effects on interstitial remodeling. However, the beneficial effects on hemodynamic parameters and mortality rate following ramipril treatment were also shown in the combination group, although further positive effects of furosemide on MMP activation and collagen content were not observed.
10.
11. 12. 13.
14.
Acknowledgments Experimental work was supported by the Deutsche Forschungsgemeinschaft. The expert assistance of Peter Kurschat (Department of Dermatology, University of Cologne) and Carsten Seeland is gratefully acknowledged. We thank C. Tscho¨pe and M. Noutsias for assistance in performing sirius red staining and providing access to the Lucia G software program.
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16.
17.
References 1. C JB, B TK. The collagen network of the heart. Lab Invest 1979; 40: 364–372. 2. S FG. Novel approaches to retard ventricular remodeling in heart failure. Eur J Heart Fail 1999; 1: 17–23. 3. P MA, P JM, S C, F P. Survival after an experimental myocardial infarction: beneficial effects of long-term therapy with captopril. Circulation 1985; 72(2): 406–412. 4. T A I R E (AIRE) S I. Effect of ramipril on mortality and morbidity of survivors of acute myocardial infarction with clinical evidence of heart failure. Lancet 1993; 342: 821–828. 5. B CG, M L, W KT. Advanced hypertensive heart disease in spontaneously hypertensive rats. Lisinopril-mediated regression of myocardial fibrosis. Hypertension 1996; 28: 269–275. 6. L H, S H, B TM, P JT. MMP/TIMP expression in spontaneously hypertensive heart failure rats: the effect of ACE- and MMP-inhibition. Cardiovasc Res 2000; 46: 298–306. 7. G DE, A DF, Y H, T UP. Tissue inhibitors of metalloproteinases: structure, regulation and biological functions. Eur J Cell Biol 1997; 74: 111–122. 8. T SC, K SG, B J, F W. Coexpression of tissue inhibitor and matrix metalloproteinase in myocardium. J Mol Cell Cardiol 1995; 27: 2177–2189. 9. L W, W P, S BA, B RH, M T, W G. Late treatment with ramipril
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increases survival in old spontaneously hypertensive rats. Hypertension 1999; 34: 291–295. W P, K RA, B DR, P JG. Quantitative assessment of myocardial collagen with picrosirius red staining and circularly polarized light. Basic Res Cardiol 1994; 89: 397–410. L OH, R NJ. Protein measurement. J Biol Chem 1951; 193: 265–275. L UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680–685. B M, F A, K¨ V, L JM, A J, S LM, Q V, B J, M G, L-O C. Identification and enzymatic characterization of two diverging murine counterparts of human interstitial collagenase (MMP-1) expressed at sites of embryo implantation. J Biol Chem 2001; 276: 10253–10262. C P, S N. Single-step method of RNA isolation by acid guanidinium thiocyanatephenol-chloroform extraction. Anal Biochem 1987; 162: 156–159. F M, S RHG, S F, F K, S M, K-R F, A G, B¨ M. Evidence for functional relevance of an enhanced expression of the Na+-Ca2+ exchanger in failing human myocardium. Circulation 1996; 94: 992– 1002. L-D L A, R M. A simple micromethod for collagen and total protein determination in formalin-fixed paraffin-embedded sections. J Histochem Cytochem 1985; 33: 737–743. T M, J R. Minoxidil accelerates heart failure development in rats with ascending aortic constriction. Can J Physiol Pharmacol 1998; 76: 613– 620. V N, I MJ, V M, L B, E JC, D J. Chronic AT(1) blockade stimulates extracellular collagen type I degradation and reverses myocardial fibrosis in spontaneously hypertensive rats. Hypertension 2000; 35: 1197–1202. S TM, B E, V V, Q M, T DG, G G, P A. Extracellular matrix gene expression in the left ventricular tissue of spontaneously hypertensive rats. Blood Press 1999; 8: 57–64. K S, H A, S T, K Y, T N, M K, O K, I H. Molecular effects of M17055, furosemide and thiazide on cardiac hypertrophy of spontaneously hypertensive rats. J Pharmacol Exp Ther 1996; 279: 983–990. R M, K FW, L FH. The reninangiotensin system and volume overload-induced changes in cardiac collagen and elastin. Circulation 1994; 90: 1989–1996. W JF J. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 1991; 131: 2145–2154. W AJ, T OJ, S S, L EJ, M T, C ES, M TE, N GR. Reduction in myocardial collagen cross-linking parallels left ventricular dilatation in rat models of systolic chamber dysfunction. Circulation 2001; 103: 155–160. C JP, K JC, G E, G RV, W KT. Regulation of collagen degradation in the rat myocardium after infarction. J Mol Cell Cardiol 1995; 27: 1281–1292.
MMPs in SHR with Heart Failure 25. P JT, H H, J L, L H, O’B PM, S DR, B TMA, C ML, E T, S FG. Matrix metalloproteinase inhibition attenuates left ventricular remodeling and dysfunction in a rat model of progressive heart failure. Circulation 2001; 103: 2303–2309. 26. K P, Z P, N R, B K, S P, K CE, K T, M C. Tissue inhibitor of matrix metalloproteinase-2 regulates matrix metalloproteinase-2 activation by modulation of membrane-type 1 matrix metalloproteinase activity in high and low invasive melanoma cell lines. J Biol Chem 1999; 274: 21056– 21062. 27. B CG, Z G, M L, W KT. Collagen metabolism in cultured adult rat cardiac fibroblasts: response to angiotensin II and aldosterone. J Mol Cell Cardiol 1994; 26: 809–820.
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28. S J, I S. Molecular characterization of angiotensin II-induced hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts. Critical role of the AT1 receptor subtype. Circ Res 1993; 73: 413–423. 29. L P, P V. Antagonism of the renin-angiotensin-aldosterone system and collagen metabolism in cardiac fibroblasts. Methods Find Exp Clin Pharmacol 1999; 21: 215–227. 30. T SC, C JP. Myocardial collagenase purification and structural characterization. Can J Cardiol 1996; 12: 165–171. 31. Z J, K L, B¨ H. Age related effects of furosemide in the rat. Arch Int Pharmacodyn 1975; 218: 167–176.
Effect of Ramipril and Furosemide Treatment on Interstitial Remodeling in Post-Infarction Heart Failure Rat Hearts Ute Seeland1, Ichiro Kouchi1, Oliver Zolk2, Gabi Itter1, Wolfgang Linz1 and Michael Bo¨hm1 1
Med. Univ.-Klinik und Poliklinik der Universita¨t des Saarlandes, Innere Medizin III – Kardiologie und Angiologie – 66421 Homburg/Saar, Germany and 2Institut fu¨r Experimentelle und Klinische Pharmakologie und Toxikologie, Universita¨t Erlangen-Nu¨rnberg, Germany
(Received 15 August 2001, accepted for publication 31 October 2001) U. S, I. K, O. Z, G. I, W. L M. B¨ . Effect of Ramipril and Furosemide Treatment on Interstitial Remodeling in Post-Infarction Heart Failure Rat Hearts. Journal of Molecular and Cellular Cardiology (2002) 34, 151–163. Extracellular matrix (ECM) remodeling and increased matrix metalloproteinase (MMP) expression and activity have been observed to be relevant in the development of heart failure (HF). We examined the effects of ramipril alone or with furosemide on ECM in a heart failure model. HF was induced by occlusion of the left coronary artery in spontaneously hypertensive rats (SHR). Rats were assigned to placebo (n=9), ramipril 1 mg/kg/day (n=11), furosemide 2×2 mg/kg/day (n=7) or both (1 mg/kg/day+2×2 mg/kg/day; n= 8). LV-function, collagen content, MMP/TIMP (tissue inhibitor of matrix metalloproteinases) protein- and mRNAexpression were examined in non-infarcted LV tissue. MMP-2/TIMP-4 ratio was increased in HF. Ramipril reduced MMP-2 expression (active form), collagen type I mRNA expression and content and increased TIMP-4 levels associated with decreased left ventricular end diastolic pressure (LVEDP), mortality rate and increased LV pressure (LVP). Combination therapy with furosemide is less efficient with regard to collagen content and MMP-2 (active form) reduction but did not worsen beneficial effects of ramipril on LV function and mortality rate. Furosemide alone had no effect on MMP-2 (active form) expression, collagen content, LV function and mortality rate. Prevention of LV dilatation by ramipril was associated with decreased gelatinolytic activity and increased MMPinhibition in heart failure SHR. Furthermore, ramipril reduced fibrosis by enhanced interstitial collagenase expression. Furosemide did not show the beneficial effects of ramipril on ECM remodeling but did not worsen LV function. Positive effects of furosemide treatment alone on LV remodeling and function were not observed. 2002 Elsevier Science Ltd.
K W: Matrix metalloproteinases; Extracellular matrix; Interstitial remodeling; Heart failure; Diuretic agents; ACE inhibitors.
Introduction Transition from compensatory hypertrophy to heart failure following myocardial infarction (MI) is associated with LV remodeling, characterized by interstitial and perivascular deposition of fibrillar collagen and LV dilatation. Collagen degrading activity leads to myocyte disarray through weakness of the collagen scaffold.1,2 Mediators of myocardial
fibrosis post-MI, including angiotensin II (Ang II) among others, have attracted considerable interest. Chronic administration of an ACE-inhibitor significantly attenuates fibrosis in both infarcted and non-infarcted regions of the myocardium, while improving heart function and survival.3,4 The effects of diuretic therapy, which is often coadministered in this condition, have not been investigated. The mechanisms of the beneficial action of ACE-
∗ Please address all correspondence to: Michael Bo¨hm, Med.Univ.-Klinik und Poliklinik der Universita¨t des Saarlandes, Innere Medizin III, 66421 Homburg/Saar, Germany. Tel: [+49]-6841-16-23372. Fax: [+49]-6841-16-23369. E-mail: [email protected]
0022–2828/02/020151+13 $35.00/0
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Table 1 Heart weight and hemodynamic parameters Groups
n
LV weight (g)
RV weight (g)
MAP (mmHg)
Heart rate b/min
LVP (mmHg)
+dP/dt (1000 mmHg/s)
Sham C R F R/F
12 9 8 7 8
1.08±0.03 1.17±0.07 1.16±0.03 1.14±0.04 1.05±0.04
0.21±0.02 0.35±0.05† 0.21±0.02∗ 0.22±0.01∗ 0.22±0.01∗
153±8 135±13 124±9 147±12 96±10†
382±11 340±19 315±32† 372±14 310±21†
136±7 98±3† 118±2∗ 108±8 114±4∗
5.32±0.2 3.12±0.4† 4.48±0.2∗ 4.12±0.3 4.21±0.2∗
SHR following myocardial infarction treated with furosemide (F), ramipril (R), ramipril+furosemide (R/F) and no treatment (control=C) compared to Sham operated SHR (Sham). ∗ denotes significant differences from C, † denotes significant differences from Sham; n, number of animals; LV, left ventricular; RV, right ventricular; MAP, mean arterial pressure; LVP, left ventricular pressure; +dP/dt indicates contractility.
inhibitors on extracellular matrix remodeling are not entirely clear. In addition to their inhibitory effect on collagen synthesis, they may affect collagen degradation by influencing matrix metalloproteinase (MMPs) isoforms depending on their substrate specificity.5,6 MMPs are metal-binding proteinases, secreted as a latent proenzyme, which require extracellular activation. MMPs relevant in myocardial remodeling include the interstitial collagenases (MMP-1/MMP-13), cleaving triple helical collagen type I, and the gelatinases (MMP-2, MMP9), degrading favourably denatured collagen type I (gelatine). Endogenous tissue inhibitors of matrix metalloproteinases (TIMPs) function as regulators of MMP expression and activity.7,8 This investigation examines whether diuretic therapy counteracts or further improves the beneficial effects of angiotensin-converting enzyme inhibition on the late extracellular matrix remodeling process. The spontaneously hypertensive rat (SHR) model is characterized by genetic pressure overload and a stimulated renin angiotensin system. The additional effect of experimental myocardial infarction superimposed on genetic hypertension with cardiac hypertrophy and early failure leads to accelerated development of severe heart failure. This study shows that ramipril reduces fibrosis by enhanced interstitial collagenase expression and prevents LV dilatation by decreased expression of gelatinolytic (MMP-2) activity and increased MMPinhibition in heart failure SHR. Additional diuretic therapy with furosemide does not further improve the beneficial effects of ramipril on ECM remodeling but does not worsen LV function.
Materials and Methods Male SHR (250–300 g) were obtained from Harlan Winkelmann (Borchen, Germany), and housed under standardized conditions. The rats had free
access to standard diet (Altromin⊂ Maintenance Diet 1320, sodium content 0.2%) and drinking water ad libitum. The investigation conforms with the Guide for the Care and Use of Laboratory Animals of the National Institute of Health and was performed in accordance with the German animal protection law. All chemicals and reagents were purchased from Sigma (Deisenhofen, Germany), Merck (Darmstadt, Germany) and Roche (Mannheim, Germany) unless otherwise stated.
Study design One hundred and thirty male spontaneously hypertensive rats (SHR), aged 16 weeks, were randomly assigned to five groups. Severe chronic heart failure was induced in 110 of them by 8 weeks’ occlusion of the left coronary artery 2–3 mm distal to the origin of the aorta, resulting in large infarction of the left ventricular wall. Twenty rats were sham operated. Animals were treated with furosemide (4 mg/kg/bw i.p. for 3 days) to reduce acute lung edema and xylocaine (2 mg/kg i.m. before surgery) to prevent arrhythmias. Mortality within 24 h was less than 10%. Two weeks after surgery, animals with MI were randomized to four groups and treatment was initiated for 6 weeks via drinking water. 1: placebo-treated controls (C; n=9), 2: treatment with 1 mg/kg/day ramipril (R; n=11), 3: treatment with 2×2 mg/kg/day furosemide (F; n=7) and 4: treatment with the combination of ramipril and furosemide 1+2×2 mg/kg/day (R/F; n=8). After 6 weeks of treatment rats were reanesthetized and the carotid artery was cannulated to monitor blood pressure. The hearts were excised and mounted on Langendorff apparatus. Isolated hearts were perfused in the working heart mode as described previously.9 Afterwards, hearts were weighed and left ventricular tissues were stored at −80°C. Scar area was determined by planimetry. Infarct size was
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Figure 1 Functional effects on left ventricular end diastolic pressure (LVEDP) following increases of preload (15 to 30 mmHg) in SHR with post-infarction HF treated with furosemide (F), ramipril (R), ramipril+furosemide (R/F) and no treatment (control=C) compared with Sham-operated SHR (Sham). ∗ denotes significant differences from C.
expressed as percentage of left ventricular mass. Animals with infarct sizes of 20–40% were enrolled.3
Working heart model After anesthesia and transverse thoracotomy, hearts were removed and immersed in physiological buffer (4°C). Aortas were mounted on a cannula attached to the perfusion apparatus. Left atria were cannulated via incision of the left auricle. Pulmonary veins were ligated close to the surface of the atria. Hearts were perfused according to Langendorff’s method with oxygenated (95% O2, 5% CO2) non-recirculating Krebs–Henseleit so-
lution. Working mode with a filling pressure (preload) of 18 mmHg and an afterload pressure of 80 mmHg at baseline conditions was used. For functional LVEDP measurements, the filling pressure was increased stepwise from 15 to 30 mmHg (Hugo Sachs Elektronik, Freiburg, Germany).
Picrosirius red staining LV circumferential sections were stained with picrosirius red for fibrillar collagen measurements using a technique described elsewhere.10 Fractional area of collagen content in percentage of myocardial tissue was measured with Lucia G software (Nikon, Du¨sseldorf, Germany).
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Figure 2 Mortality of HF-SHR treated with furosemide (F), ramipril (R), ramipril+furosemide (R/F) and no treatment (control=C) compared to Sham-operated SHR (Sham). ∗ denotes significant differences from C.
Figure 3 LV cross sections were stained by picrosirius red and viewed under polarized light. Interstitial fibrillar collagen fractional area (%) was measured with Lucia G software; n=3–4. ∗ denotes significant differences from C.
Zymography Zymography detects degradative properties of gelatinases by employing gelatin as substrate into a
gel. Tissue gelatinase activity was measured under non-reducing conditions. Quantification was performed densitometrically by the size-fractionated banding pattern. Frozen adult rat myocardium tissue
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Figure 4 Northern blot of procollagen (1) type I and GAPDH mRNA (upper panel) in myocardium from HF-SHR treated with furosemide (F), ramipril (R), ramipril+furosemide (E/F) and no treatment (control=C) compared with Sham-SHR (Sham). Bar graphs summarize the optical density (OD) in % of Sham from n=5–8 (lower panel). ∗ denotes significant differences from C, † denotes significant differences from Sham.
samples were homogenized in 5× volume extraction buffer containing: 10 mmol/l cacodylic acid, pH 5.0, 0.15 mol/l NaCl, 1 mol/l ZnCl2, 20 mmol/ l CaCl2, 1.5 mmol/l NaN3, and 0.01% (v/v) Triton X100 at 4°C. After centrifugation (10 min, 1200×g) supernatants were collected and pH was raised to 7.5. Protein concentrations were measured using a detergent compatible kit (DC protein assay, Bio-Rad, Munich, Germany) based on the method of Lowry.11 Gelatin type B from bovine skin (1 mg/ml) was added to standard Laemmli acrylamide polymerization mixture.12 Tissue extract (25 g) mixed with sample buffer (10% w/v SDS, 4% w/v sucrose, 0.1% w/v bromphenol blue) was loaded into slots (0.8 mm) of a 10% acrylamide stacking gel. After electrophoretic separation gels were soaked in 2.5% w/v Triton X-100 for 60 min at room temperature (RT). Gels were incubated overnight at 37°C in substrate buffer (50 mmol/l Tris-Cl, pH 8.0, 5 mmol/ l CaCl2). After staining for 2 h in 0.05% Coomassie brilliant blue G-250 (Bio-Rad) the gel was destained in acetic acid/ethanol and dried. Supernatant of HT 1080 (15 l), human fibrosarcoma cells, served as positive control. Each sample lane was scanned and presented as percent of latent MMP-2 HT1080standard.
Immunoblotting Homogenized cardiac tissue samples (25 g/lane), were separated on 8% – (collagen type I), 10% – (interstitial collagenase) and 12% – polyacrylamide gel (TIMP-2 and -4) and transferred to nitrocellulose membranes (Protran⊂, Schleicher & Schuell GmbH, Dassel, Germany) by semi-dry electrophoretic blotting (0.8 mA/cm2). Staining with Poinceau red showed relative loading of total cardiac proteins. Membranes were blocked in PBS-Tween (0.5%) containing 5% (w/v) skim milk. Membranes were probed with primary antibodies for interstitial collagenase (MMP-1, IM35L, Ab-1, monoclonal mouse 1:1000, Calbiochem, Bad Soden, Germany), recognizing the epitope from amino acid 349–357 of the rodent counterpart to human MMP-1,13 TIMP2 (IM56L, Ab-2, monoclonal mouse 1:2000, Calbiochem), TIMP-4 (AB816, polyclonal rabbit 1:1000, Chemicon, Hofheim, Germany) and collagen type I (Dianova, Hamburg, Germany) for 120 min at RT. Peroxidase labeled goat anti-mouse was diluted 1:10 000 and used as secondary antibody for MMP-1 and TIMP-2, goat anti-rabbit was diluted used for TIMP-4 and rabbit anti-goat was used for collagen type I. Incubation was performed
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Figure 5 Collagen turnover. (A) Hydroxyproline/proline ratio determined by HPLC and (B) protein expression of MMP1 (proMMP-1 and active forms) from HF-SHR treated with furosemide (F), ramipril (R), ramipril+furosemide (R/F) and no treatment (control=C) compared with Sham-SHR (Sham). Bar graphs summarize the OD in % of Sham from n= 5–9. ∗ denotes significant differences from C; † denotes significant differences from Sham; a, glycosylated; b, nonglycosylated.
for 90 min at RT. The proteins were visualized by enhanced chemiluminescence (ECL) according to the manufacturer’s guidelines (Amersham Pharmacia Biotech, Freiburg, Germany). Autoradiographs from Western blot analysis were quantified by imaging densitometry and ‘‘ImageQuant-TM’’ software (Image Quant, Molecular Dynamics, Krefeld, Germany).
Northern blot analysis Total RNA from deep frozen left ventricular tissue samples was prepared according to the method of Chomczynski and Sacchi.14 Tissue was homogenized in acid guanidinium thiocyanate solution (peqGOLD
RNAPure⊂, Peqlab Biotechnology, Erlangen, Germany) and extracted with phenol and chloroform. RNA was quantified spectrometically, and ethidium bromide-stained agarose gels were used to check its integrity. Rat MMP-2-, pro-(1) type I collagenand GAPDH (glyceraldehyde-3-phosphate dehydrogenase)- cDNA were generated by RT-PCR of rat heart RNA and confirmed by sequencing. MMLV RT and Taq DNA polymerase were purchased from Gibco BRL (Karlsruhe, Germany) and Roche (Mannheim, Germany). Northern blots were prepared from 10 g total RNA as described previously.15 [32P]deoxy CTP labeled cDNA fragments of rat MMP-2- (404 bp), pro-(1) type I collagen(402 bp) and GAPDH (451 bp) were used. The data were processed using the ‘‘ImageQuant-TM’’ soft-
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Figure 6 Zymographic analysis of gelatinolytic activity from HF-SHR treated with furosemide (F), ramipril (R), ramipril+furosemide (R/F) and no treatment (control-C) compared to Sham-SHR (Sham). Bar graphs summarize OD in % of Sham from n=5–9. ∗ denotes significant differences from C; † denotes significant differences from Sham.
ware. Standardization was performed by hybridization of the same membrane using the cDNA probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Density of bands was expressed relative to the density of the GAPDH band.
High-performance liquid chromatography (HPLC) Ratios of proline and hydroxyproline were analyzed according to Lopez-de Leon and Rojkind.16 In brief, biopsies of the left ventricular tissue were hydrolyzed in 6 N HCl for 24 h at 105°C, freeze-dried and resuspended in 0.5 mol/l NaHCO3. Internal standards (1 mmol/l acetidin-2-carbonacid in 0.4 mol/l K-borate pH 9.0) were added.
Statistical analysis Results are presented as means±... Statistical significance was estimated with Student’s t-test for unpaired observations. Differences among the five groups of rats were tested by one-way analysis of variance (ANOVA) once normality was demonstrated and the Fischer test was used for LVEDPmeasurements. A value of P<0.05 was considered significant. Cumulative survival was analyzed for differences according to the Kaplan–Meier method, after which Cox–Mantel log-rank test was used.
Results Characterization of the model Following MI, SHR showed symptoms related to
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Figure 7 Northern blot of MMP-2 and GAPDH mRNA (upper panel) in myocardium from HF-SHR treated with furosemide (F), ramipril (R), ramipril+furosemide (R/F) and no treatment (control=C) compared with Sham-SHR (Sham). Bar graphs summarize the OD in % of Sham from n=5–9 (lower panel). ∗ denotes significant differences from C; † denotes significant differences from Sham.
severe congestive heart failure (CHF) signs in humans such as subcutaneus edema, dyspnea and impaired motion. Hydrothoraces and lung edema were demonstrated in NMR imaging (data not shown). Right ventricular heart weight and lung weight increased significantly in SHR with HF (C) compared to Sham. In all treatment groups RV heart weight decreased to Sham levels. Left ventricular heart weights (infarcted area 20–40% by planimetry) showed no difference (Table 1), nor did body weights measured 8 weeks following MI with or without treatment. The data are given in percentage of body weight measured at the beginning (mean±... in %) Sham: 10.9±1.1, C: 17.4±3, R: 11.1±3.4, F: 9.0±1.8, R/F: 14.8±1.8.
LV function At 24 weeks of age, mean arterial pressure (MAP) in SHR-HF was reduced and did not change significantly following treatment with ramipril or furosemide. However, ramipril plus furosemide decreased MAP significantly to 96±10 mmHg (Table 1), which is similar to the MAP of untreated normotensive rats. Working heart data were performed
ex vivo and are summarized in Table 1. LVP and contractility (+dP/dt) were reduced significantly by 42% in untreated SHR-HF (C) compared with Sham (Table 1). In the furosemide treated SHR-HF group, LVP was similar to untreated control group values, whereas ramipril treatment alone and in combination with furosemide normalized contractility (P<0.05, Table 1). For end diastolic pressures, LVEDP were highest in the untreated SHR-HF group depending on linear rise of filling pressure (preload 15 to 30 mmHg) and were reduced in the SHR-HF groups treated with ramipril and with the combination (+furosemide). The LVEDPs were similar to those of Sham-SHR (P<0.05; Fig. 1). In the furosemide treated group LVEDP was reduced but did not reach statistical significance. These data show that coronary ligation led to destruction of myocardial function, which is sensitive to medical therapy.
Survival Acute mortality rate within 24 h of surgery was less than 10%. Arrhythmias and acute lung edema
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Figure 8 Tissue inhibitors of MMP (TIMP). (A) Western blot analysis of TIMP-2 and (B) of TIMP-4 from HF-SHR treated with furosemide (F), ramipril (R), ramipril+furosemide (R/F) and no treatment (control=C) compared with Sham. Bar graphs summarize OD in % of Sham from n=5. ∗ denotes significant differences from C; † denotes significant differences from Sham; ‡ denotes significant differences from R.
were prevented by xylocaine and furosemide, respectively. Mortality rate was 40% in controls. Ramipril treatment alone and in combination with furosemide significantly decreased the mortality rate to 29% and 19%, respectively (P<0.05 v control). Treatment with furosemide alone failed to improve the mortality rate (42%; Fig. 2).
Extracellular matrix proteins Myocardial collagen fractional area (%) increased significantly in untreated HF-SHR (C) compared with Sham-SHR [%, 3.0±0.6 (C) v 0.6±0.08 (Sham); P<0.01]. Ramipril treatment reduced percent fibrillar collagen to 0.8±0.29%, P<0.05. Furosemide treatment alone and in combination with ramipril also reduced collagen content but did not
reach statistical significance (P=0.06/0.13). These results are consistent with western blotting for collagen type I (data not shown; Fig. 3). Procollagen (1) type I mRNA expression was increased in HF-SHR (C) compared with ShamSHR [246±22% (C); P<0.001]. It was reduced by ramipril treatment and in the combination group [127±21% (R), P<0.005; 162±29% (R/F), P<0.046]. Furosemide treatment alone did not alter procollagen (1) type I mRNA expression v untreated HF-SHR [224±32% (F), 246±22% (C); n.s.]; see Figure 4. Hydroxyproline/proline ratio, a parameter of cardiac collagen turnover increased significantly after MI and development of heart failure [P<0.0001; Fig. 5(A)]. Following ramipril and combination treatment, the amount of the ratio was further increased by 44% in both groups in comparison to untreated controls (P<0.05).
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Matrix metalloproteinases (MMPs) Figure 5(B) provides a representative Western blot illustrating the presence of the latent proMMP-1 (57/52 kDa) and the glycosylated (47 kDa) and non-glycosylated active form (42 kDa) of interstitial collagenase. Relative LV myocardial levels of the glycosylated and non-glycosylated active form of interstitial collagenase were increased significantly in the ramipril treated group compared with untreated controls and Sham-SHR (% of Sham: 263±49 (R) v 138±12 (C); P<0.05). The latent (72/69 kDa) and active (66/59 kDa) form of gelatinase MMP-2 was detected in zymography. Latent MMP-2 was induced and activated in failing SHR [% of Sham: latent 267±53, active 209±58 (C); P<0.01]. The increased level of MMP2 active form in untreated HF-SHR (C) was returned to control levels following ramipril treatment and in the combination group [latent: 120±14, active: 124±20 (R); latent 136±31, active 143±38 (R/ F); P<0.05). In contrast, furosemide alone enhanced signals of the latent MMP-2 expression and active form [% of Sham: latent 361±93, active 263±68 (F); Fig. 6]. Autoradiograms of the Northern blots show a representative band at 3.1 kb of MMP-2 mRNA (Fig. 7). Ratios for MMP-2/GAPDH signals were significantly increased in failing myocardium without treatment and following furosemide alone [% of Sham: 254±27 (C), 252±26 (F); P<0.05]. Ramipril alone and in combination with furosemide significantly normalized MMP-2 mRNA expression compared to controls [% of Sham: 254±27 (C), 154±19 (R), P<0.05; 175±14 (R/F), P<0.05].
Tissue inhibitors of matrix metalloproteinases (TIMPs) TIMP-2: except for a 2.5-fold significant increase in the furosemide group (P<0.05), there was a minor increase after myocardial infarction and a reduction following ramipril treatment of TIMP-2 [Fig. 8(A)]. TIMP-4 protein was reduced in the failing hearts to 57±8% (C) of Sham (P<0.01). Treatment with ramipril significantly increased protein levels to 80±4% (R) of Sham; [P<0.05, Fig. 8(B)].
Discussion Fibrosis is regarded as contributing to the development of heart failure in rats after coronary ligation. The model used is characterized by fibrous
tissue accumulation leading to increased myocardial diastolic stiffness and impaired systolic function of the left ventricle with clinical symptoms of severe congestive heart failure. It has been taken into consideration that the degree of fibrosis is very different between the different animals. Spontaneously hypertensive rats preferentially expressed fibrosis, and this was the reason for choosing this model. Other models such as aortic constriction etc. are not so useful for studying the development and remodeling of fibrosis because their fibrosis is only moderate.17 It is useful to study the mechanisms by which ACE- inhibitors and diuretics influence myocardial collagen synthesis and degradation. Differences in MMP/TIMP alterations between normal rats and SHR are described elsewhere.6,18,19 Regulation of the MMP/TIMP system in hypertrophy and heart failure is influenced by the reninangiotensin system. We hypothesized that the activity of the different matrix metalloproteinase isoforms and their tissue inhibitors (TIMP) in heart failure can be influenced by treatment with ramipril and furosemide. While there are abundant data about ACE-inhibitors on LV-remodeling, data on furosemide are lacking. There is only one study on the effect of furosemide on hypertrophy. Kim et al. (1996) reported no difference in collagen type I and III gene expression but a significant reduction of left ventricular hypertrophy following furosemide treatment, presumably caused by blood pressure lowering in SHR without heart failure.20 Data from the present study demonstrate that furosemide influenced neither collagen type I protein concentration and procollagen (1) I mRNA expression nor MMP/TIMP expression. Treatment with furosemide in addition to ramipril did not show any further positive effects on ECM remodeling rather activate MMP-2. However, combination therapy improved hemodynamic parameters like LVEDP, LVP, contractility and mortality rate, and reduced mean arterial pressure compared to hypertensive sham operated rats. Therefore, this is the first evidence that diuretic treatment does not interfere with ACE inhibition with respect to hemodynamic parameters, but it does not improve ECM remodeling as well as ramipril treatment alone in a heart failure model. There are reports that afterload reduction by aortocaval shunt or minoxidil treatment reduces collagen content in rats.21 Our finding that furosemide does not significantly affect interstitial remodeling might evidence that only preload reducing effects (increasing diuretics) are not sufficient to decrease interstitial ventricular remodeling. In this light one also has to view the ACE inhibitor effects, which produce pre- and afterload reduction.
MMPs in SHR with Heart Failure
The development of heart failure may be associated both with alterations in the balance of collagen synthesis and inadequate collagen degradation. The rate limiting step in the extracellular degradation of collagen is the catalytic cleavage by interstitial MMPs.22 Depending on substrate specificity active MMP-2 cleaves non triple helical collagens responsible for collagen cross linking and basement membrane function. Reduction in myocardial collagen cross-linking parallels left ventricular dilatation in rat models23 and in failing human hearts.24 Loss of interstitial integrity impairs myocyte alignment and promotes progressive left ventricular dysfunction. The data presented for zymography suggest that the increase in the active form of MMP-2 expression in untreated heart failure- SHR is relevant in this process of ventricular dilatation. In agreement with that, LV myocardial MMP-2 activity has been demonstrated to be increased in a heart failure-SHR model recently published by Peterson et al.25 MMP-2 activity in failing LV-myocardium is significantly reduced following ramipril treatment. This was not observed for furosemide treatment alone in the present study. ACE is a zinc-metalloenzyme and ACE inhibitors inhibit the active site in this enzyme. A direct binding of ramipril to the MMP-2 active site could be one mechanism responsible for this marked inhibition. However, the transcriptional level is involved in the regulation of MMP-2 expression shown by the MMP-2 mRNA data in this study. Furthermore, the levels of the endogenous inhibitor system (TIMPs) are decreased in untreated control animals with severe heart failure. Following ramipril treatment the reduction of TIMP-4, inhibiting most MMPs in adult cardiac tissue, was reversed. These data support the hypothesis that a lack of inhibitory control of MMPs by TIMP-4 contributes to the development of interstitial remodeling. The data from the present study show that this imbalance was reversed by ramipril but not by furosemide. In contrast to TIMP-4, TIMP-2 expression was enhanced following furosemide treatment. This was not expected because of high levels of MMP-2 activity. In addition to the inhibitory control of most MMPs, TIMP-2 can form a complex with MT1MMP at the cell membrane, which possibly plays a regulatory role in the proteolytic activation of MMP-2.26 Therefore the function of TIMP-2 as an inhibitor or activator of MMP-2 is likely to depend on its concentration. It is known that angiotensin II stimulates collagen synthesis in rat cardiac fibroblasts in a concentration dependent manner27,28 and inhibits MMP-1 activity.29 The present data show a sig-
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nificant reduction of percent fibrillar collagen (fractional area), collagen type I protein content, and procollagen (1) I mRNA expression following ramipril treatment. This was accompanied by a significant increase in the expression of active interstitial myocardial matrix collagenase following therapy with ramipril in contrast to untreated controls. Interstitial collagenase is a key enzyme involved in fibrillar collagen degradation because of its ability to cleave native triple helical collagen i.e. collagen type I. Tyagi and Cleutjens purified and characterized rat interstitial collagenase.30 The antibody used in this study showed specific signals for collagenase zymogen at 57/52 kDA and the proteolytically processed active form at 46/42 kDa. Previous studies have reported regulation of MMP1 or indirectly measured collagenase activity by [14C] collagen assay in the rat heart.18,19 It is conceivable that increased interstitial collagenase activity is responsible for the increased hydroxyproline/proline ratio, a parameter of collagen turnover, measured in non-infarcted myocardium following ramipril treatment and combination treatment with furosemide. In order to correlate extracellular matrix remodeling with diastolic myocardial function, left ventricular end diastolic pressure (LVEDP) was determined in the working heart model. The results show a significantly reduced LVEDP following ramipril and ramipril/furosemide treatment compared to controls, whereas no reduction was achieved in the furosemide group. The normalized LVEDP following ramipril treatment and treatment in combination with furosemide is in agreement with an improved MMP-2/TIMP-4 ratio similar to ShamSHR. Furthermore, activated interstitial collagenase expression and reduced collagen type I concentration support the beneficial diastolic and systolic function measured in this study. Beneficial effects of ramipril are reflected in the Kaplan-Meier survival curves. The lowest mortality rates were achieved following ramipril alone and in combination with furosemide. Conversely, furosemide alone failed to improve survival rate. The beneficial effects on hemodynamic parameters and mortality rate following combined therapy may be caused by decreased mechanical load with reduced myocardial wall stress and improved systolic function. In SHR treated with four mg/bw furosemide the excretion of water was very effective. An increase in dose was not followed by a significantly higher efficacy without a change in osmolality.31 In the present study 4 mg/bw were very effective in reducing peripheral edema of SHR with severe congestive heart failure.
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In summary, ACE-inhibitor treatment positively influenced extracellular matrix remodeling by decreasing the gelatinolytic MMP-2/TIMP-4 ratio to prevent myocyte disarray and by increasing the active form of the interstitial collagenase to reduce collagen deposition in the left ventricle following myocardial infarction. In the present study furosemide alone did not show any direct positive effects on interstitial remodeling. However, the beneficial effects on hemodynamic parameters and mortality rate following ramipril treatment were also shown in the combination group, although further positive effects of furosemide on MMP activation and collagen content were not observed.
10.
11. 12. 13.
14.
Acknowledgments Experimental work was supported by the Deutsche Forschungsgemeinschaft. The expert assistance of Peter Kurschat (Department of Dermatology, University of Cologne) and Carsten Seeland is gratefully acknowledged. We thank C. Tscho¨pe and M. Noutsias for assistance in performing sirius red staining and providing access to the Lucia G software program.
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