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Biglycan Is Required for Adaptive Remodeling After Myocardial Infarction D. Westermann, J. Mersmann, A. Melchior, T. Freudenberger, C. Petrik, L. Schaefer, R. Lüllmann-Rauch, O. Lettau, C. Jacoby, J. Schrader, S.-M. Brand-Herrmann, M.F. Young, H.P. Schultheiss, B. Levkau, H.A. Baba, T. Unger, K. Zacharowski, C. Tschöpe and J.W. Fischer Circulation 2008;117;1269-1276; originally published online Feb 25, 2008; DOI: 10.1161/CIRCULATIONAHA.107.714147 Circulation is published by the American Heart Association. 7272 Greenville Avenue, Dallas, TX 72514 Copyright © 2008 American Heart Association. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circ.ahajournals.org/cgi/content/full/117/10/1269 Subscriptions: Information about subscribing to Circulation is online at http://circ.ahajournals.org/subscriptions/ Permissions: Permissions & Rights Desk, Lippincott Williams & Wilkins, a division of Wolters Kluwer Health, 351 West Camden Street, Baltimore, MD 21202-2436. Phone: 410-528-4050. Fax: 410-528-8550. E-mail: [email protected] Reprints: Information about reprints can be found online at http://www.lww.com/reprints Downloaded from circ.ahajournals.org at SWETS SUBSCR SVC/25338706 on September 15, 2008 Heart Failure Biglycan Is Required for Adaptive Remodeling After Myocardial Infarction D. Westermann, MD*; J. Mersmann, MD*; A. Melchior, MS*; T. Freudenberger, MS*; C. Petrik; L. Schaefer, PhD; R. Lüllmann-Rauch, MD; O. Lettau, MS; C. Jacoby, PhD; J. Schrader, MD; S.-M. Brand-Herrmann, MD, PhD; M.F. Young, PhD; H.P. Schultheiss, MD; B. Levkau, MD; H.A. Baba, MD; T. Unger, MD; K. Zacharowski, MD; C. Tschöpe, MD; J.W. Fischer, PhD Background—After myocardial infarction (MI), extensive remodeling of extracellular matrix contributes to scar formation and preservation of hemodynamic function. On the other hand, adverse and excessive extracellular matrix remodeling leads to fibrosis and impaired function. The present study investigates the role of the small leucine-rich proteoglycan biglycan during cardiac extracellular matrix remodeling and cardiac hemodynamics after MI. Methods and Results—Experimental MI was induced in wild-type (WT) and bgn⫺/0 mice by permanent ligation of the left anterior descending coronary artery. Biglycan expression was strongly increased at 3, 7, and 14 days after MI in WT mice. bgn⫺/0 mice showed increased mortality rates after MI as a result of frequent left ventricular (LV) ruptures. Furthermore, tensile strength of the LV derived from bgn⫺/0 mice 21 days after MI was reduced as measured ex vivo. Collagen matrix organization was severely impaired in bgn⫺/0 mice, as shown by birefringence analysis of Sirius red staining and electron microscopy of collagen fibrils. At 21 days after MI, LV hemodynamic parameters were assessed by pressure-volume measurements in vivo to obtain LV end-diastolic pressure, end-diastolic volume, and end-systolic volume. bgn⫺/0 mice were characterized by aggravated LV dilation evidenced by increased LV end-diastolic volume (bgn⫺/0, 111⫾4.2 L versus WT, 96⫾4.4 L; P⬍0.05) and LV end-diastolic pressure (bgn⫺/0, 24⫾2.7 versus WT, 18⫾1.8 mm Hg; P⬍0.05) and severely impaired LV function (EF, bgn⫺/0, 12⫾2% versus WT, 21⫾4%; P⬍0.05) 21 days after MI. Conclusion—Biglycan is required for stable collagen matrix formation of infarct scars and for preservation of cardiac hemodynamic function. (Circulation. 2008;117:1269-1276.) Key Words: collagen 䡲 myocardial infarction 䡲 extracellular matrix 䡲 fibrosis 䡲 proteoglycans E xtensive remodeling of the extracellular matrix (ECM) occurs during all phases of infarct healing, including initial degradation of ECM by matrix metalloproteinases (MMPs), the formation of provisional ECM by de novo synthesis, and finally the deposition of collagen-forming scar tissue.1 During scar maturation, inflammatory cells and fibroblasts disappear as a result of apoptosis, and collagen becomes increasingly crosslinked to stabilize the scar and to provide tensile strength until a hypocellular, collagen-rich scar is formed.2 The composition of the ECM during the individual phases of infarct healing not only determines the mechanical stability but also modulates the cellular phenotype.3 In the long term, adverse left ventricular (LV) remodeling, including fibrosis and ventricular dilatation, often causes progressive heart failure associated with considerable morbidity and mortality after acute myocardial infarction (MI). Clinical Perspective p 1276 The role of MMPs during postinfarct remodeling has been studied extensively. After MI, an imbalance between MMPs Received May 8, 2007; accepted December 24, 2007. From the Abteilung für Kardiologie und Pneumologie, Charite-Universitätsklinikum Berlin, Campus Benjamin Franklin, Berlin, Germany (D.W., O.L., H.P.S., C.T.); Molecular Cardioprotection and Inflammation Group, Klinik für Anästhesiologie (J.M.) and Molekulare Pharmakologie, Institut für Pharmakologie und Klinische Pharmakologie (A.M., T.F., J.W.F.), Universitätsklinikum Düsseldorf, Düsseldorf, Germany; Center for Cardiovascular Research, Institut für Pharmakologie und Toxikologie, Campus Charite-Mitte, Charite-Universitatsmedizin Berlin, Berlin, Germany (C.P., T.U.); Nephropharmakologie, Allgemeine Pharmakologie und Toxikologie, Klinikum der Johann Wolfgang Goethe Universität Frankfurt, Frankfurt, Germany (L.S.); Institut für Anatomie, Christian-Albrechts-Universität Kiel, Kiel, Germany (R.L.-R.); Institut für Herzkreislauf-Physiologie, Heinrich-HeineUniversität Düsseldorf, Düsseldorf, Germany (C.J., J.S.); Leibniz-Institute for Arteriosclerosis Research, Department of Molecular Genetics of Cardiovascular Disease, University of Muenster, Muenster, Germany (S.-M.B.-H.); Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Md (M.F.Y.); Institut für Pathophysiologie (B.L.) and Institut für Pathologie (H.A.B.), Universitätsklinikum Essen, Essen, Germany; and Molecular Cardioprotection and Inflammation Group, Department of Anaesthesia, University Hospital Bristol, Bristol Royal Infirmary, Bristol, UK (K.Z.). *The first 4 authors contributed equally to this work. The online Data Supplement, which contains an expanded Methods section and figures, can be found with this article at http://circ.ahajournals.org/ cgi/content/full/CIRCULATIONAHA.107.714147/DC1. Correspondence to Jens W. Fischer, Molecular Pharmacology, Institut für Pharmakologie and Klinische Pharmakologie, Heinrich-Heine-Universität Düsseldorf, Moorenstrasse 5, D-40225 Düsseldorf, Germany. E-mail [email protected] © 2008 American Heart Association, Inc. Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.107.714147 1269 Downloaded from circ.ahajournals.org at SWETS SUBSCR SVC/25338706 on September 15, 2008 1270 Circulation March 11, 2008 and tissue inhibitors of MMPs (TIMPs) is thought to be a key factor in promoting adverse remodeling of the ECM and LV dilatation. However, collagen synthesis and fibrillogenesis also underlie a complex regulation and might in turn be equally important during infarct healing. Self-assembly of procollagen ␣ chains forms triple-helical collagen molecules that associate into collagen fibrils. This process is regulated by accessory ECM molecules such as the family of small leucine-rich proteoglycans, including biglycan and decorin.4 By controlling lateral assembly of triple helixes into fibrils and the arrangement of collagen fibers, biglycan and decorin are key regulators of collagen fibrillogenesis and fibril diameter.4,5 Biglycan and decorin are capable of specific binding to collagen types I, II, III, and VI6,7 via the core proteins near the d band of collagen fibrils.8,9 The role of small leucine-rich proteoglycans in LV remodeling is only partially understood. It has been reported previously that biglycan and decorin are induced after MI in rats and that biglycan associates with cardiac fibroblasts in the infarcted myocardium.10,11 bgn⫺/0 mice have reduced skeletal growth and develop an osteoporosis-like phenotype resulting from a defect in bone formation.12 Furthermore, the diameter of collagen fibrils was reduced in bgn⫺/0 mice.13 Other functions of biglycan include regulation of transforming growth factor -1 activity,14 cell proliferation, apoptosis,15 and activation of inflammatory responses mediated through activation of Tolllike receptor-2 and -4.16 Taken together, biglycan plays important roles in collagen matrix assembly, control of growth factor activity, and cellular phenotype, including inflammatory cells. However, it is still unknown whether biglycan plays a role in cardiac physiology or during infarct healing. The aim of the present study was to investigate the role of biglycan during remodeling and functional adaptation after experimental MI. For this purpose, bgn⫺/0 mice were subjected to permanent occlusion of the left anterior descending coronary artery, followed by analysis of heart morphology, ECM, and hemodynamic function. Hemodynamic Measurements Mice were anesthetized by injection of thiopental (125 mg/kg IP), intubated, and ventilated with a respirator (type 7025, Ugo Basile, Comerio, Italy). A 1.4F microconductance pressure catheter (ARIA SPR-719, Millar Instruments Inc, Houston, Tex) was positioned in the LV via the right carotid artery for continuous registration of LV pressure-volume loops in closed-chest animals.18 Systolic function and myocardial contractility were quantified by LV end-systolic pressure, peak rate of rise in LV pressure (dP/dtmax), ejection fraction, cardiac output, end-systolic volume, and stroke volume. Diastolic performance was measured by LV end-diastolic pressure, peak dP/dtmin, end-diastolic volume, and time constant of isovolumic pressure relaxation. Analysis of Human MIs Tissue sections derived from patients who died of ischemic MI presenting either as an acute, primary event (⬍5 days, n⫽4)) or reoccurring infarction on the basis of old, healed MIs (n⫽4) were analyzed for biglycan by immunostaining (LF 51) and for collagen by Sirius red staining. The samples were collected for diagnostic purposes during autopsy and were examined by a senior pathologist (H.B.). Informed consent was obtained from patients or relatives. The present study was performed according to the Declaration of Helsinki, and the study protocol was approved by the Ethics Committee of the University Hospital of Essen. Statistical Analysis For multiple comparisons, 2-way ANOVA, followed by Bonferroni posttest, was performed. Values of P⬍0.05 were considered significant. Data are expressed as mean⫾SEM. Survival curves were computed by the Kaplan-Meier method and were compared by use of the log-rank test. The authors had full access to and take responsibility for the integrity of the data. All authors have read and agree to the manuscript as written. Results Increased Biglycan Expression After MI Real-time reverse-transcription polymerase chain reaction (RT-PCR) revealed strongly increased bgn mRNA expression in the LV after experimental MI, peaking 7 days after MI (Figure 1A). These results were confirmed by immunostaining for biglycan, showing that biglycan accumulation occurs in the infarcted ischemic area and border zone at 7 days (Figure 1C). Furthermore, at 7 days after MI, fibroblasts and inflammatory cells were detected in areas enriched with Methods A A detailed description of the methods and materials is available in the online Data Supplement. Animals bgn⫺/0 mice (bred at the University of Düsseldorf) with a targeted deletion of the bgn gene12 and male wild-type (WT; C57BL/6) littermates were compared in this study. All procedures were carried out in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care guidelines and the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, National Institutes of Health, publication No. 86-23) and were approved by the ethics and research boards of the University of Düsseldorf. Animals (12 to 16 weeks old) were randomized as indicated. Myocardial Infarction At the age of 12 weeks, WT and bgn⫺/0 mice were anesthetized by injection of pentobarbital (100 mg/kg IP). Mice were subjected to permanent left anterior descending coronary artery occlusion, followed by recovery for 7 or 21 days. All procedures were carried out as recently described in detail.17 B C D Figure 1. Time course of biglycan expression after MI. A, Realtime RT-PCR revealed significantly increased bgn mRNA expression, peaking 7 days after MI. B, Immunohistochemistry of biglycan in LV of sham-operated WT mice (inset, negative control) and WT mice after left anterior descending coronary artery occlusion at 7 (C) and 14 (D) days. n⫽6. Magnification ⫻100. *P⬍0.05. Downloaded from circ.ahajournals.org at SWETS SUBSCR SVC/25338706 on September 15, 2008 Westermann et al biglycan (online Data Supplement), in line with previous results in rats.10,11 At 14 days after MI, bgn mRNA levels were still 5-fold higher than in sham animals (Figure 1A), and biglycan immunostaining was still increased in the infarct and peri-infarct zones (Figure 1D). Role of Biglycan During Post-MI Remodeling 1271 A Cardiac Phenotype of Adult bgnⴚ/0 Mice bgn⫺/0 mice were used as a model to study the effect of biglycan on cardiac function. First, the cardiac phenotype was analyzed in healthy, noninfarcted animals. At 6 months of age, morphological and hemodynamic analyses of the heart did not reveal any differences between WT and bgn⫺/0 mice. In 15-month-old mice, the body weight (WT, 48.5⫾4.5 g versus bgn⫺/0, 41.3⫾5.2 g [mean⫾SEM]; P⬍0.05) and ratio of heart to body weight (Data Supplement) were slightly decreased. At 15 months, hemodynamic measurements in these mice revealed no differences in LV end-diastolic pressure, end-diastolic volume, end-systolic pressure, endsystolic volume (not shown), and heart rate (Data Supplement). However, ejection fraction showed a trend toward lower values, and end-diastolic pressure-volume ratios were decreased in bgn⫺/0 mice (end-diastolic pressure-volume ratio: WT, 0.32⫾0.11 mm Hg/L; bgn⫺/0, 0.19⫾0.07 mm Hg/ L [mean⫾SEM]; n⫽6; P⬍0.05; Data Supplement). In addition, 15-month-old bgn⫺/0 mice showed a trend toward decreased collagen type I mRNA expression but increased collagen type III and decorin expression as determined by real-time RT-PCR (data not shown). Morphological analysis and collagen staining showed no obvious difference between WT and bgn⫺/0 mice at 15 months (Data Supplement), suggesting that both the morphology and cardiac ECM showed no major difference in noninfarcted bgn⫺/0 mice. Decreased Survival After MI in bgnⴚ/0 Mice Kaplan-Meier analysis revealed higher mortality in bgn⫺/0 mice after experimental MI. This increase in mortality was obvious after 7 days and continued until 3 weeks at the end of the study. At 3 weeks, the survival rates were 60% and 20% in WT and bgn⫺/0 mice, respectively (Figure 2A). To investigate the cause of death, an additional group of animals (n⫽8) underwent experimental MI and morphological analysis 7 days after MI. During this 7-day period, the animals were closely monitored, and mice that died underwent autopsy immediately. Analysis of this group revealed that the frequency of LV ruptures was ⬇33% in WT and 66% in bgn⫺/0 mice, explaining the lower survival (Figure 2B). Figure 3 shows that the area at risk and infarct size were not different between the 2 genotypes as determined by Evans blue and p-nitro blue tetrazolium 7 days after MI. These data exclude the possibility that increased area at risk or infarct size is responsible for the higher frequency of rupture. B C Figure 2. Survival after experimental MI is decreased in bgn⫺/0 mice. A, Mortality after permanent left anterior descending coronary artery occlusion in WT and bgn⫺/0 mice up to 21 days after MI. Mortality was significantly increased in bgn⫺/0 mice (P⬍0.01, log-rank-test; WT, n⫽20, 8 nonsurvivors; bgn⫺/0, n⫽33, 27 nonsurvivors). B, bgn⫺/0 mice died more often as a result of ventricular rupture than their WT littermates within 7 days after MI. C, Heart of a bgn⫺/0 mouse. Arrows/thread indicate the LV ruptured area. birefringence analysis, Sirius red staining revealed increased density of collagen in WT mice as indicated by the bright red (Figure 4C and 4D; Sirius red birefringence expressed as area fraction: bgn⫺/0, 3.9⫾1% versus WT, 10.8⫾1.7%; n⫽4; P⬍0.05). Furthermore, Movat’s pentachrome staining revealed a higher proportion of proteoglycans in scars of bgn⫺/0 mice at 7 days after MI (Figure 4E and 4F), which was corroborated by Alcian blue staining and subsequent quanti- A B C D Matrix Remodeling 7 Days After MI To investigate the mechanisms that caused LV ruptures, ECM remodeling was characterized in both groups. Collagen content of infarct scars was similar between genotypes as determined by Sirius red staining (bright field, Sirius red expressed as area fraction: WT, 21.9⫾6.7% versus bgn⫺/0, 37.9⫾7%; n⫽4; P⫽1.6). In addition, immunohistochemistry of collagen type 1 revealed no quantitative differences between groups (data not shown). In polarized light using the Figure 3. Infarct size 7 days after MI. Area at risk (A; %LV) and infarct size (B; % LV) were not different between the genotypes and controls (WT, n⫽8; bgn⫺/0, n⫽8). Representative Evans blue/p-nitro blue tetrazolium–stained sections of WT (C) or bgn⫺/0 (D) mice. Downloaded from circ.ahajournals.org at SWETS SUBSCR SVC/25338706 on September 15, 2008 1272 Circulation March 11, 2008 A WT B bgn-/0 C WT D bgn-/0 E WT F bgn-/0 G WT H bgn-/0 in the infarcted area compared with the noninfarcted area in both genotypes (Figure 5). The expression of MMP-2 and -13 was significantly higher in the infarcted area of bgn⫺/0 mice compared with WT. However, MMP-9 was decreased and TIMP-1 and -4 were significantly upregulated in bgn⫺/0 mice after experimental MI. The differences detected by immunostaining were largely paralleled by mRNA expression (Data Supplement). In situ zymography indicating gelatinolytic activity mainly through MMP-2 and -920 revealed no significant difference between genotypes (Figure 5P). Furthermore, the mRNA expression of procollagen-prolyl-4-hydroxylase, procollagenlysyl-hydroxylase 2, procollagen-lysyl-hydroxylase 1, and tissue transglutaminase, key enzymes in the regulation of collagen and ECM crosslinking, was not differentially regulated between the genotypes after MI (Data Supplement). Hemodynamic Parameters Figure 4. ECM remodeling 7 days after MI. A, B, Sirius red staining, bright field. C, D, Sirius red viewed under polarized light revealing less densely packed collagen in the infarct scars of bgn⫺/0 mice. E, F, Movat’s pentachrome staining revealed a proteoglycan-rich ECM in bgn⫺/0 mice vs WT. G, H, Decorin immunostaining (LF-113) was markedly increased in bgn⫺/0 mice. Representative stainings of WT (n⫽8) and bgn⫺/0 (n⫽8). Magnification ⫻100. fication by image analysis (Alcian blue expressed as area fraction: WT, 73.3⫾4.4% versus bgn⫺/0, 87.5⫾3%; n⫽4; P⬍0.05). Decorin was upregulated after MI in WT mice as described previously.19 Notably, post-MI immunostaining of decorin was much stronger in bgn⫺/0 mice (Figure 4G and 4H). Taken together, the infarct scars of bgn⫺/0 mice were characterized by loosely packed collagen fibers and a higher amount of proteoglycans, including decorin. The expression patterns of MMP-2, -8, -9, and -13 and TIMP-1, -2, and -4 were analyzed by immunostaining (Figure 5) and by real-time RT-PCR (Data Supplement). MMP-2, -8, -9, and -13 and TIMP-1, -2, and -4 were strongly upregulated No difference in cardiac hemodynamic parameters was detected between 16-week-old sham-operated WT and bgn⫺/0 animals (Table), confirming the analysis of WT versus bgn⫺/0 mice under normal conditions as mentioned above. In all animals, as predicted, MI caused a decrease in ejection fraction (%) and an increase in LV end-diastolic pressure (mm Hg), end-diastolic volume (L), and end-systolic volume (L) (Figure 6). Additional hemodynamic parameters are presented in the Table, indicating severe impairment of hemodynamic functions of WT and bgn⫺/0 mice 3 weeks after MI such as a decrease in LV end-systolic pressure (mm Hg), dP/dtmax (mm Hg/s), and dP/dtmin (mm Hg/s) and increased (ms). The most important finding is that bgn⫺/0 mice demonstrate significantly increased post-MI LV dilatation with increased LV end-diastolic volume and end-systolic volume and decreased ejection fraction compared with WT controls 21 days after MI (Figure 6A through 6D). Furthermore, bgn⫺/0 mice demonstrated reduced stroke volume and cardiac output despite significantly increased heart rate (bpm) (Table), suggesting compensatory tachycardia. As an additional sign of cardiac dilatation and failure, LV end-diastolic Figure 5. Immunohistochemistry of MMPs and TIMPs. Immunostaining and quantification (area fraction [AF]) in the infarct scar 7 days after MI of MMP-2 (A, B, and I), MMP-8 (C, D, and J), MMP-9 (E, F, and K), and MMP-13 (G, H, and L). For TIMP-1 (M), TIMP-2 (N), and TIMP-4 (O), only the results of the image analysis (AF) are given. In the scar (MI), MMP-2, MMP-13, TIMP-1, and TIMP-4 were increased in bgn⫺/0 mice, whereas MMP-9 was decreased. P, Gelatinolytic activity as determined by in situ zymography (WT, n⫽5; bgn⫺/0, n⫽4). *P⬍0.05, ***P⬍0.001 vs WT-MI; #P⬍0.05 vs respective noninfarcted regions. Downloaded from circ.ahajournals.org at SWETS SUBSCR SVC/25338706 on September 15, 2008 Westermann et al Table. Role of Biglycan During Post-MI Remodeling 1273 Hemodynamic Measurements at 21 Days After MI Heart Rate, bpm Sham WT (n⫽7) bgn⫺/0 (n⫽8) End-Systolic Pressure, mm Hg dP/dtmax, mm Hg/s dP/dtmin, mm Hg/s , ms Stroke Volume, L Cardiac Output, L/min Lung Wet/Dry Weight 442⫾12 105⫾2 7554⫾818 ⫺5231⫾485 13.8⫾0.7 36.6⫾3.4 13160⫾1560 4.48⫾0.1 428⫾8 105⫾5 6890⫾345 ⫺5763⫾429 14.0⫾0.7 35.4⫾3.2 13640⫾1728 4.56⫾0.1 4045⫾414† 3240⫾406† ⫺3446⫾314† ⫺2757⫾346† 17.4⫾0.8† 17.0⫾1.4 19.6⫾1.1† 14.1⫾1.2† 8450⫾441† 6723⫾716† MI WT (n⫽10) bgn⫺/0 (n⫽6) 433⫾11 489⫾25*† 73⫾4† 64⫾2† 5.5⫾0.2† 6.4⫾0.2*† *P⬍0.05, WT vs bgn⫺/0; †P⬍0.05, sham vs MI. pressure was significantly increased compared with WT controls. This increase in LV end-diastolic pressure is further verified by increased ratios of lung wet weight to lung dry weight after MI compared with WT as an additional indicator for LV heart failure (Table). These data clearly indicate that bgn⫺/0 mice develop impaired hemodynamic function in response to ischemic injury. In addition, a subset of animals was investigated with MRI. The longitudinal MRI images (Figure 6E and 6F) showed stronger LV dilation and are consistent with the hemodynamic data. Calculation of LV end-diastolic wall stress revealed increased wall stress in bgn⫺/0 mice after MI compared with WT (14.84⫾1.2⫻103 dynes · mm⫺2 [P⬍0.05 compared with WT MI] versus 9.09⫾1.8⫻103 dynes · mm⫺2). Matrix Remodeling 21 Days After MI Increased frequency of cardiac ruptures and decreased hemodynamic function of bgn⫺/0 mice after MI and less tightly packed collagen at 7 days (Figure 4) point toward perturbed ECM remodeling in the infarct scar of bgn⫺/0 mice. Therefore, ECM remodeling also was characterized at 21 days after MI. MI induced a marked upregulation of collagen type 1 and 3 mRNA as determined by real-time RT-PCR, which was significantly stronger in the bgn⫺/0 mice (Figure 7A). Decorin mRNA showed a trend toward upregulation in WT versus bgn⫺/0 mice 21 days after MI (Figure 7B). The overall collagen content was similar as evidenced by Sirius red staining (Figure 7C and 7D; total Sirius red expressed as area fraction: WT, 41⫾3.9% versus bgn⫺/0, 43.8⫾11.7%; n⫽4; P⬍0.05) and collagen type 1 immunostaining (not shown). A striking difference appeared with respect to collagen organization (Figure 7C and 7D). Under polarized light, Sirius red staining revealed that the collagen present in the scar of bgn⫺/0 mice was characterized by a lower percentage of tightly packed collagen (Figure 7E and 7F; Sirius red birefringence expressed as area fraction: WT, 11.1⫾0.3% versus bgn⫺/0, 5.9⫾1.4%; n⫽4; P⬍0.05). Immunohistochemistry showed still increased decorin accumulation (not shown); however, the difference was not as pronounced as detected at 7 days (Figure 4). Analysis of the collagen matrix by electron microscopy revealed tight and parallel packing of collagen fibrils in the scars of WT mice. In contrast, collagen fibril organization was perturbed in bgn⫺/0 mice (Figure 7H). The tensile force to rupture LV rings ex vivo was determined to investigate whether the differences in matrix composition and organization correlate with changes of tensile strength. Under control conditions without MI, no difference was evident between LV rings from WT and bgn⫺/0 mice that ruptured at 84 and 90 mN, respectively (Figure 7I). However, 21 days after MI, the force to rupture was dramatically decreased in LV rings derived from bgn⫺/0 mice (69 mN) compared with WT mice (146 mN). Biglycan Accumulation in Human MI Figure 6. Hemodynamic parameters 21 days after MI. A through D, Hemodynamic measurements in WT vs bgn⫺/0 under control conditions (sham) and 21 days after MI. After MI, bgn⫺/0 mice were characterized by decreased ejection fraction (EF; A) and increased LV end-diastolic pressure (Ped; B), LV end-diastolic volume (Ved; C), and LV end-systolic volume (Ves; D) (sham WT, n⫽7; sham bgn⫺/0, n⫽8; MI WT, n⫽10; MI bgn⫺/0; n⫽6). Additional hemodynamic parameters are given in the Table. E and F, MRI representative longitudinal sections showing enhanced LV dilatation in bgn⫺/0 mice (F; n⫽7) vs WT (E; n⫽8) at 21 days after MI. Arrows point toward an area of marked ventricular wall thinning. *P⬍0.05, ***P⬍0.001, #P⬍0.05 vs respective noninfarcted controls. Tissue sections derived from patients who died of ischemic MI presenting as either an acute, primary event or reoccurring acute infarction on the basis of old healed MI were examined. Figure 8A through 8D represents an area from an acute MI characterized by acute myocyte necrosis with eosinophilic cytoplasm and without visible cardiomyocyte nuclei. Hardly any immunostaining for biglycan nor Sirius red staining of collagen was detected under these conditions. In contrast, Figure 8E through 8H depicts a healed infarction with fibrotic regions and some remaining vital cardiomyocytes. In these healed infarcts, strong accumulation of both biglycan and densely packed collagen as shown by birefringence analysis Downloaded from circ.ahajournals.org at SWETS SUBSCR SVC/25338706 on September 15, 2008 1274 Circulation March 11, 2008 Figure 7. ECM remodeling 21 days after MI. Quantitative realtime RT-PCR of collagen (coll) type 1 (A) and decorin (dcn) (B) mRNA expressed as fold of sham-operated WT 21 days after MI. C and D Sirius red staining and morphology of the infarct scar in WT mice was similar to that in bgn⫺/0 mice. Magnification ⫻40. E and F, Sirius red staining viewed under polarized light. Compared with WT (E), less densely packed collagen was detected in the infarct scar of bgn⫺/0 mice (F). A through F, Sham WT, n⫽7; sham bgn⫺/0, n⫽8; MI WT, n⫽10; MI bgn⫺/0, n⫽6. Magnification ⫻40. G and H, Ultrastructure of collagen fibrils derived from the infarct scar as visualized by electron microscopy. Infarcts of WT mice showed tightly packed collagen fibrils aligned mostly in parallel (G). In contrast, organization of collagen fibrils in the infarct scar of bgn⫺/0 mice (H) was perturbed. Magnification ⫻12 000. Representative images of 4 for each genotype 21 days after MI. I, Quantitative analysis of collagen fibril arrangement. Collagen confluence indicates the percentage of tight, parallel arranged collagen fibrils viewed by electron microscopy. J, Tension to rupture (mN) measured ex vivo on 1-mm rings of the LV of noninfarcted mice (control: WT, n⫽5; bgn⫺/0, n⫽8) and 21 days after MI (WT, n⫽5; bgn⫺/0, n⫽7). ***P⬍0.001, #P⬍0.05 vs respective noninfarcted controls. of Sirius red staining occurs (Figure 8F and 8H). Notably, biglycan colocalizes with tightly packed collagen. Thus, biglycan accumulates during healing and remodeling and associates with collagen after MI in human hearts. Discussion The key results of this study are increased mortality, high frequency of cardiac ruptures, and aggravated cardiac failure in bgn⫺/0 mice after experimental MI, which were likely the result of perturbed collagen remodeling in the infarct scar. Seven days after experimental MI, thinning of the infarct area and formation of a collagen matrix occur in mice. After 14 days, a A B C D E F G H Figure 8. Immunohistochemistry of biglycan in human MI. A through D, Consecutive sections of acute, human MI characterized by myocyte necrosis. A, Hematoxylin and eosin; B, biglycan (LF51); C, Sirius red; D, Sirius red polarized. Arrows indicate the same coronary artery for orientation. E through H, Consecutive sections of healed MI with collagenous scar tissue showing strong accumulation of biglycan (F) in the fibrotic interstitium in colocalization with densely packed collagen (G and H). Magnification ⫻40. The presented cases are representative of each of 4 cases of acute and healed infarct scars. *Areas of colocalization between biglycan and densely packed collagen as evidenced by polarized light (H). mature collagen-rich scar is established, and at this time point, collagen levels peak.2,21 Collagen type 1 is the major collagen of the myocardial ECM. Much attention has been focused on regulators of collagen expression and the breakdown of collagen matrixes during postinfarct remodeling. However, little is known about the role of small ECM molecules that govern collagen fibril formation. Among these small ECM molecules are the small leucine-rich proteoglycans such as biglycan and decorin. Small leucine-rich proteoglycans can be regarded as accessory ECM molecules at the interface of matrix assembly and signaling, which might have potential for therapeutic interventions. The aim of the present study was to investigate whether biglycan affects cardiac phenotype in healthy animals and, importantly, after experimental MI. No differences were detected with respect to the cardiac phenotype at 6 months in WT versus bgn⫺/0 mice. At 15 months, the relaxation parameter and the end-diastolic pressure-volume ratio were decreased in bgn⫺/0 mice. Although other parameters22 Downloaded from circ.ahajournals.org at SWETS SUBSCR SVC/25338706 on September 15, 2008 Westermann et al may influence the end-diastolic pressure-volume ratio, this finding suggests that the intrinsic myocardial stiffness is reduced in aged bgn⫺/0 mice. Taken together, these findings suggest that during physiological conditions bgn deficiency has only minor effects on LV function, which starts to induce hemodynamic changes only during aging. After experimental MI, biglycan expression was strongly upregulated in WT mice, as shown previously in rats.10,11 Interestingly, the upregulation was transient, peaking at 7 days, at the same time that collagen content also is highest.2 Using immunohistochemistry, we found biglycan in the infarct scar and border zone of the infarct at 7 and 14 days after MI, in line with a role of biglycan in collagen remodeling. During the whole experimental period, bgn⫺/0 mice showed higher mortality after MI, which was due in large part to LV ruptures. In addition, spontaneous death without ruptures occurred more often in bgn⫺/0 mice. This might be a consequence of aggravated cardiac failure and LV dilatation as indicated by increased LV end-diastolic volume, end-diastolic pressure, and ratios of wet lung weight to dry lung weight in bgn⫺/0 mice after MI. bgn⫺/0 mice and WT mice showed no differences in the area at risk and infarct size, which excludes the possibility that decreased survival of bgn⫺/0 mice after MI is a consequence of increased ischemia or necrosis. Birefringence analysis of Sirius red–stained sections revealed that the packing of collagen fibrils was disturbed in bgn⫺/0 mice 7 and 21 days after MI. This was confirmed by analysis of the ultrastructure of collagen in the infarct scar, showing that the array of collagen was heavily perturbed in bgn⫺/0 mice. The quantity of collagen accumulating in the scar was not decreased as analyzed by immunohistochemistry of collagen type 1 and Sirius red staining. In contrast, mRNA expression of collagen type 1 and collagen type 3 was increased even in bgn⫺/0 mice, which might represent a compensatory mechanism. Increased MMP activity is known to contribute to ventricular dilatation after MI, and expression of MMPs is highly increased in the infarcted area.23,24 The upregulation of MMP-2 and MMP-13 was opposed by upregulation of TIMP-1 and TIMP-4 and downregulation of MMP-9. MMP-9 is a key player in adverse ventricular remodeling after MI because MMP-9 – dependent collagen degradation is associated with adverse LV myocardial remodeling in animal models and patients.25,26 An additional mechanism of how the absence of biglycan could increase collagen cleavage is conceivable. It was recently demonstrated in vitro that the noncovalent binding of decorin and biglycan to fibrillar collagen type 1 inhibits the cleavage of collagen type 1 by collagenase-1 (MMP-1) and collagenase-3 (MMP-13).27 The interaction of biglycan core protein with collagen fibrils renders collagen type 1 fibrils less accessible to MMPmediated cleavage. Of note, biglycan inhibited collagen breakdown without changing the activation or expression of MMPs, which would obscure this mechanism even after analysis of the MMP/TIMP system as we performed in the present study. Taken together, it is conceivable that changes in the MMP/TIMP levels and/or increased susceptibility to breakdown in the absence of biglycan contribute to the cardiac phenotype of bgn⫺/0 mice described in the present study. Role of Biglycan During Post-MI Remodeling 1275 Taken together, in bgn⫺/0 mice, disturbed collagen matrix assembly might result in decreased mechanical strength of myocardial scar tissue after MI. Indeed, ex vivo measurements revealed severely reduced tensile strength of LV rings derived from bgn⫺/0 mice compared with WT mice. The reduced tensile strength of LV and the increase in LV end-diastolic wall stress might explain the frequent ruptures in bgn⫺/0 mice. Interestingly, the dcn⫺/⫺ mice also have a strong cardiac phenotype after experimental MI.19 dcn⫺/⫺ mice developed impaired LV function after experimental MI that was attributed to abnormal collagen fibril formation. However, ventricular ruptures did not occur in dcn⫺/⫺ mice.19 Therefore, from the present study, it appears that the cardiac phenotype is even stronger in bgn⫺/0 mice compared with dcn⫺/⫺ mice. Furthermore, upregulation of decorin as observed in bgn⫺/0 mice 7 days after MI was not sufficient to compensate for the loss of biglycan as it does with respect to the osteoporosis phenotype.28 Analysis of scars of MI from humans revealed a strong accumulation of biglycan in close association with collagen in fibrotic areas of healed scars, whereas biglycan was not detected after acute human MI. It is therefore possible that the present findings are of clinical relevance and that biglycan might participate in the evolution of a collagenous ECM during healing of human MI. However, the present data do not allow a conclusion to be drawn as to whether biglycan deposition is characteristic for postinfarct remodeling or is associated with fibrotic remodeling and hypertrophy in general. LV wall rupture as observed in bgn⫺/0 mice also is a complication of acute MI in patients that is responsible for ⬇20% of infarct-related deaths.29,30 Therefore, it might be of interest to investigate the patterns of biglycan expression in human MI and the relation to cardiac rupture in future studies. From the present study, it is obvious that the complete absence of biglycan is deleterious and prevents the establishment of a stable infarct scar and hemodynamic adaptation, suggesting that biglycan is required for proper infarct healing. However, future studies should address how much biglycan is needed for proper scar formation and whether too much biglycan might also have adverse effects such as enhanced fibrosis. Sources of Funding This study was in part supported by Deutsche Forschungsgemeinschaft SFB612, SFB TR19, GRK1089, and GRK 754; the Interdisciplinary Center for Clinical Research, Münster; and the Excellence Cluster Cardio-Pulmonary System. Disclosures None. References 1. 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Corsi A, Xu T, Chen XD, Boyde A, Liang J, Mankani M, Sommer B, Iozzo RV, Eichstetter I, Robey PG, Bianco P, Young MF. Phenotypic effects of biglycan deficiency are linked to collagen fibril abnormalities, are synergized by decorin deficiency, and mimic Ehlers-Danlos-like changes in bone and other connective tissues. J Bone Miner Res. 2002;17:1180–1189. 6. Douglas T, Heinemann S, Bierbaum S, Scharnweber D, Worch H. Fibrillogenesis of collagen types I, II, and III with small leucine-rich proteoglycans decorin and biglycan. Biomacromolecules. 2006;7:2388 –2393. 7. Wiberg C, Hedbom E, Khairullina A, Lamande SR, Oldberg A, Timpl R, Morgelin M, Heinegard D. Biglycan and decorin bind close to the n-terminal region of the collagen VI triple helix. J Biol Chem. 2001;276:18947–18952. 8. Vogel KG, Paulsson M, Heinegard D. Specific inhibition of type I and type II collagen fibrillogenesis by the small proteoglycan of tendon. Biochem J. 1984;223:587–597. 9. 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Targeted disruption of the biglycan gene leads to an osteoporosis-like phenotype in mice. Nat Genet. 1998;20:78 – 82. 13. Ameye L, Aria D, Jepsen K, Oldberg A, Xu T, Young MF. Abnormal collagen fibrils in tendons of biglycan/fibromodulin-deficient mice lead to gait impairment, ectopic ossification, and osteoarthritis. FASEB J. 2002;16:673– 680. 14. Hildebrand A, Romaris M, Rasmussen LM, Heinegard D, Twardzik DR, Border WA, Ruoslahti E. Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor beta. Biochem J. 1994;302:527–534. 15. Schaefer L, Beck KF, Raslik I, Walpen S, Mihalik D, Micegova M, Macakova K, Schonherr E, Seidler DG, Varga G, Schaefer RM, Kresse H, Pfeilschifter J. Biglycan, a nitric oxide-regulated gene, affects adhesion, growth, and survival of mesangial cells. J Biol Chem. 2003; 278:26227–26237. 16. Schaefer L, Babelova A, Kiss E, Hausser HJ, Baliova M, Krzyzankova M, Marsche G, Young MF, Mihalik D, Gotte M, Malle E, Schaefer RM, Grone HJ. The matrix component biglycan is proinflammatory and signals through Toll-like receptors 4 and 2 in macrophages. J Clin Invest. 2005;115:2223–2233. 17. Petzelbauer P, Zacharowski PA, Miyazaki Y, Friedl P, Wickenhauser G, Castellino FJ, Groger M, Wolff K, Zacharowski K. The fibrin-derived peptide Bbeta15-42 protects the myocardium against ischemiareperfusion injury. Nat Med. 2005;11:298 –304. 18. Westermann D, Knollmann BC, Steendijk P, Rutschow S, Riad A, Pauschinger M, Potter JD, Schultheiss HP, Tschope C. Diltiazem treatment prevents diastolic heart failure in mice with familial hypertrophic cardiomyopathy. Eur J Heart Fail. 2006;8:115–121. 19. Weis SM, Zimmerman SD, Shah M, Covell JW, Omens JH, Ross J Jr, Dalton N, Jones Y, Reed CC, Iozzo RV, McCulloch AD. A role for decorin in the remodeling of myocardial infarction. 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Evolution of matrix metalloprotease and tissue inhibitor expression during heart failure progression in the infarcted rat. Cardiovasc Res. 2000;46:307–315. 25. Ducharme A, Frantz S, Aikawa M, Rabkin E, Lindsey M, Rohde LE, Schoen FJ, Kelly RA, Werb Z, Libby P, Lee RT. Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. J Clin Invest. 2000;106:55– 62. 26. Webb CS, Bonnema DD, Ahmed SH, Leonardi AH, McClure CD, Clark LL, Stroud RE, Corn WC, Finklea L, Zile MR, Spinale FG. Specific temporal profile of matrix metalloproteinase release occurs in patients after myocardial infarction: relation to left ventricular remodeling. Circulation. 2006;114:1020 –1027. 27. Geng Y, McQuillan D, Roughley PJ. SLRP interaction can protect collagen fibrils from cleavage by collagenases. Matrix Biol. 2006;25:484 – 491. 28. Young MF, Bi Y, Ameye L, Chen XD. Biglycan knockout mice: new models for musculoskeletal diseases. Glycoconj J. 2002;19:257–262. 29. Moreno R, Lopez de Sa E, Lopez-Sendon JL, Garcia E, Soriano J, Abeytua M, Elizaga J, Botas J, Rubio R, Moreno M, Garcia-Fernandez MA, Delcan JL. Frequency of left ventricular free-wall rupture in patients with acute myocardial infarction treated with primary angioplasty. Am J Cardiol. 2000;85:757–760. 30. Hutchins KD, Skurnick J, Lavenhar M, Natarajan GA. Cardiac rupture in acute myocardial infarction: a reassessment. Am J Forensic Med Pathol. 2002;23:78 – 82. CLINICAL PERSPECTIVE Known mechanisms leading to heart failure after myocardial infarction include subsequent cardiac dilatation and infarct thinning, also called infarct expansion, which may lead to the development of aneurysm and left ventricle rupture. Therefore, adaptive changes in cardiac remodeling are essential so that the ventricle forms a stable scar rather than rupturing after myocardial infarction. Collagenous networks elaborated by cardiac fibroblasts provide the mechanical support for the scar tissue. It seems important to consider that the stability and 3-dimensional arrangement of cardiac collagen matrixes are controlled by accessory molecules such as biglycan. Biglycan is a proteoglycan that binds to the d bands of collagen type 1 fibrils and controls fibrillogenesis and proteolysis of collagen by matrix metalloproteinases. The present study reveals that after myocardial infarction, the scar tissue of biglycan-deficient mice was characterized by perturbed collagen fibril arrangement and that ventricular ruptures occur more frequently compared with wild-type mice. Furthermore, the surviving biglycan-deficient mice showed impaired hemodynamic function with subsequent cardiac dilatation, suggesting that in the absence of biglycan, adverse ventricular remodeling is aggravated. Therefore, biglycan appears to play a critical role in the formation of stable infarct scars and the preservation of hemodynamic function after myocardial infarction. Because biglycan accumulates in the scar tissue of human ischemic infarctions, it is possible that the present findings extend to human postinfarct remodeling. In the future, it will be of interest to determine whether differences in cardiac biglycan expression may be modulated by pharmacological treatment and whether this may change clinical outcome. Downloaded from circ.ahajournals.org at SWETS SUBSCR SVC/25338706 on September 15, 2008