A Collagen α2(I) Mutation Impairs Healing after Experimental Myocardial Infarction
2011; Elsevier BV; Volume: 180; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2011.09.033
ISSN1525-2191
AutoresUlrich Hofmann, Andreas Bonz, Stefan Frantz, Kai Hu, Christiane Waller, Katrin Roemer, Jürgen Wolf, Stefan Gattenlöhner, Johann Bauersachs, Georg Ertl,
Tópico(s)Signaling Pathways in Disease
ResumoCollagen breakdown and de novo synthesis are important processes during early wound healing after myocardial infarction (MI). We tested the hypothesis that collagen I, the main constituent of the extracellular matrix, affects wound healing after MI. The osteogenesis imperfecta mouse (OIM), lacking procollagen-α2(I) expression, represents a model of the type III form of the disease in humans. Homozygous (OIM/OIM), heterozygous (OIM/WT), and wild-type (WT/WT) mice were subjected to a permanent myocardial infarction protocol or sham surgery. Baseline functional and geometrical parameters determined by echocardiography did not differ between genotypes. After MI but not after sham surgery, OIM/OIM animals exhibited significantly increased mortality, due to early ventricular rupture between day 3 and 7. Echocardiography at day 1 demonstrated increased left ventricular dilation in OIM/OIM animals. Less collagen I mRNA within the infarct area was found in OIM/OIM animals. At 2 days after MI, MMP-9 expression in the infarct border zone was higher in OIM/OIM than in WT/WT animals. Increased granulocyte infiltration into the infarct border zone occurred in OIM/OIM animals. Neither granulocyte depletion nor MMP inhibition reduced mortality in OIM/OIM animals. In this murine model, deficiency of collagen I leads to a myocardial wound-healing defect. Both structural alterations within pre-existing collagen matrix and impaired collagen de novo expression contribute to a high rate of early myocardial rupture after MI. Collagen breakdown and de novo synthesis are important processes during early wound healing after myocardial infarction (MI). We tested the hypothesis that collagen I, the main constituent of the extracellular matrix, affects wound healing after MI. The osteogenesis imperfecta mouse (OIM), lacking procollagen-α2(I) expression, represents a model of the type III form of the disease in humans. Homozygous (OIM/OIM), heterozygous (OIM/WT), and wild-type (WT/WT) mice were subjected to a permanent myocardial infarction protocol or sham surgery. Baseline functional and geometrical parameters determined by echocardiography did not differ between genotypes. After MI but not after sham surgery, OIM/OIM animals exhibited significantly increased mortality, due to early ventricular rupture between day 3 and 7. Echocardiography at day 1 demonstrated increased left ventricular dilation in OIM/OIM animals. Less collagen I mRNA within the infarct area was found in OIM/OIM animals. At 2 days after MI, MMP-9 expression in the infarct border zone was higher in OIM/OIM than in WT/WT animals. Increased granulocyte infiltration into the infarct border zone occurred in OIM/OIM animals. Neither granulocyte depletion nor MMP inhibition reduced mortality in OIM/OIM animals. In this murine model, deficiency of collagen I leads to a myocardial wound-healing defect. Both structural alterations within pre-existing collagen matrix and impaired collagen de novo expression contribute to a high rate of early myocardial rupture after MI. Osteogenesis imperfecta (OI) is an inherited connective tissue disorder that occurs in 1 out of 20,000 to 1 out of 60,000 live births.1Orioloi I.M. Castilla E.E. Barbosa-Neto J.G. The birth prevalance rates for the skeletal dysplasias.J Med Genet. 1986; 23: 328-332Crossref PubMed Scopus (271) Google Scholar, 2Martin E. Shapiro J.R. Osteogenesis imperfecta: epidemiology and pathophysiology.Curr Osteoporos Rep. 2007; 5: 91-97Crossref PubMed Scopus (112) Google Scholar The clinical phenotype is characterized by blue sclera, bone fractures, high incidence of hearing loss, joint laxity, restrictive pulmonary disease, and growth impairment. Patients with the milder types I and IV of osteogenesis imperfecta may have a normal life span and die of unrelated illnesses, such as myocardial infarction (MI) or malignancy.3McAllion S.J. Paterson C.R. Causes of death in osteogenesis imperfecta.J Clin Pathol. 1996; 49: 627-630Crossref PubMed Scopus (127) Google Scholar Human OI results from deficiencies in type I collagen procollagen-α1 or procollagen-α2 chains. The cardiac extracellular collagen matrix consists of more than 80% collagen I.4Robinson T.F. Cohen-Gould L. Factor S.M. Skeletal framework of mammalian heart muscle Arrangement of inter- and pericellular connective tissue structures.Lab Invest. 1983; 49: 482-498PubMed Google Scholar Large coiled perimysial collagen fibers are major determinants of tensile stiffness of myocardial tissue. Ventricular rupture and wound-healing defects after cardiac surgery have been reported in patients with OI.5Lijoi A. Cisico S. Caputo E. Scarano F. Parodi E. Passerone G.C. Left ventricular rupture after mitral valve replacement in a patient with osteogenesis imperfecta tarda.Tex Heart Inst J. 1999; 26: 295-297PubMed Google Scholar, 6Wong R.S. Follis F.M. Shively B.K. Wernly J.A. Osteogenesis imperfecta and cardiovascular diseases.Ann Thorac Surg. 1995; 60: 1439-1443Abstract Full Text PDF PubMed Scopus (64) Google Scholar Incidence of rupture of the left ventricular (LV) free wall as a complication of acute MI is low in humans (1% to 6%), but mortality with this condition is very high (60% to 90%).7Reddy S.G. Roberts W.C. Frequency of rupture of the left ventricular free wall or ventricular septum among necropsy cases of fatal acute myocardial infarction since introduction of coronary care units.Am J Cardiol. 1989; 63: 906-911Abstract Full Text PDF PubMed Scopus (165) Google Scholar, 8McMullan M.H. Maples M.D. Kilgore Jr, T.L. Hindman S.H. Surgical experience with left ventricular free wall rupture.Ann Thorac Surg. 2001; 71: 1894-1898Abstract Full Text Full Text PDF PubMed Scopus (73) A recent retrospective analysis of autopsy records demonstrated that in 13% of cases of sudden death after MI the underlying cause was ventricular rupture.9Pouleur A.C. Barkoudah E. Uno H. Skali H. Finn P.V. Zelenkofske S.L. Belenkov Y.N. Mareev V. Velazquez E.J. Rouleau J.L. Maggioni A.P. Køber L. Califf R.M. McMurray J.J. Pfeffer M.A. Solomon S.D. VALIANT InvestigatorsPathogenesis of sudden unexpected death in a clinical trial of patients with myocardial infarction and left ventricular dysfunction, heart failure, or both.Circulation. 2010; 122: 597-602Crossref PubMed Scopus (175) Google Scholar Collagen degradation starts several hours after MI and last for several days.10Frantz S. Bauersachs J. Ertl G. Post-infarct remodelling: contribution of wound healing and inflammation.Cardiovasc Res. 2009; 81: 474-481Crossref PubMed Scopus (241) Google Scholar, 11Blankesteijn W.M. Creemers E. Lutgens E. Cleutjens J.P. Daemen M.J. Smits J.F. Dynamics of cardiac wound healing following myocardial infarction: observations in genetically altered mice.Acta Physiol Scand. 2001; 173: 75-82Crossref PubMed Scopus (100) Google Scholar, 12Tyagi S.C. Campbell S.E. Reddy H.K. Tjahja E. Voelker D.J. Matrix metalloproteinase activity expression in infarcted, noninfarcted and dilated cardiomyopathic human hearts.Mol Cell Biochem. 1996; 155: 13-21Crossref PubMed Scopus (134) Google Scholar The collagen matrix is degraded by activated serine proteases (eg, plasmin) and matrix metalloproteases (MMPs). Pro-MMP enzymes are stored extracellularly, bound to extracellular matrix (ECM) compounds, and are expressed by all cell types found in the myocardium, especially in response to inflammatory stimuli (for a detailed review, see Vanhoutte et al13Vanhoutte D. Schellings M. Pinto Y. Heymans S. Relevance of matrix metalloproteinases and their inhibitors after myocardial infarction: a temporal and spatial window.Cardiovasc Res. 2006; 69: 604-613Crossref PubMed Scopus (213) Google Scholar). Importantly, besides transcriptional regulation, post-transcriptional activation and inhibition are subject to tight regulation within the extracellular space. Studies with genetically manipulated mice have demonstrated that MMP activation is a key factor for developing rupture after MI; targeted deletion of MMP-2 and MMP-9 in mice reduced the incidence of myocardial rupture.14Heymans S. Luttun A. Nuyens D. Theilmeier G. Creemers E. Moons L. Dyspersin G.D. Cleutjens J.P. Shipley M. Angellilo A. Levi M. Nübe O. Baker A. Keshet E. Lupu F. Herbert J.M. Smits J.F. Shapiro S.D. Baes M. Borgers M. Collen D. Daemen M.J. Carmeliet P. Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure.Nat Med. 1999; 5: 1135-1142Crossref PubMed Scopus (708) Google Scholar, 15Ducharme A. Frantz S. Aikawa M. Rabkin E. Lindsey M. Rohde L.E. Schoen F.J. Kelly R.A. Werb Z. Libby P. Lee R.T. Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction.J Clin Invest. 2000; 106: 55-62Crossref PubMed Scopus (694) Google Scholar, 16Hayashidani S. Tsutsui H. Ikeuchi M. Shiomi T. Matsusaka H. Kubota T. Imanaka-Yoshida K. Itoh T. Takeshita A. Targeted deletion of MMP-2 attenuates early LV rupture and late remodeling after experimental myocardial infarction.Am J Physiol Heart Circ Physiol. 2003; 285: H1229-H1235Crossref PubMed Scopus (274) Google Scholar In the border zone of the infarct area, myofibroblasts start de novo synthesis of collagen between day 2 and day 3 after MI.11Blankesteijn W.M. Creemers E. Lutgens E. Cleutjens J.P. Daemen M.J. Smits J.F. Dynamics of cardiac wound healing following myocardial infarction: observations in genetically altered mice.Acta Physiol Scand. 2001; 173: 75-82Crossref PubMed Scopus (100) Google Scholar Early collagen type III de novo synthesis is followed by collagen type I deposition, which contributes tensile strength to the infarcted tissue.17Cleutjens J.P. Kandala J.C. Guarda E. Guntaka R.V. Weber K.T. Regulation of collagen degradation in the rat myocardium after infarction.J Mol Cell Cardiol. 1995; 27: 1281-1292Abstract Full Text PDF PubMed Scopus (423) Google Scholar, 18Chipman S.D. Sweet H.O. McBride DJ J.R. Davisson M.T. Marks SC J.R. Shuldiner A.R. Wenstrup R.J. Rowe D.W. Shapiro J.R. Defective pro alpha 2(I) collagen synthesis in a recessive mutation in mice: a model of human osteogenesis imperfecta.Proc Natl Acad Sci USA. 1993; 90: 1701-1705Crossref PubMed Scopus (278) Google Scholar A balance of ECM synthesis and degradation is required for maintaining tissue stability and preventing infarct expansion and rupture. Because animal models have been developed in which MI is induced by coronary ligature, we have gained much insight into inflammatory activation and ECM turnover during wound healing after MI.19Ertl G. Frantz S. Wound model of myocardial infarction.J Am Physiol Heart Circ Physiol. 2005; 288: H981-H983Crossref PubMed Scopus (21) Google Scholar, 20Ertl G. Frantz S. Healing after myocardial infarction.Cardiovasc Res. 2005; 66: 22-32Crossref PubMed Scopus (242) Google Scholar Osteogenesis imperfecta mice (OIM) harbor a recessively inherited guanine deletion on the Col1a2 gene, causing a complete loss of functional α2(I) chains in homozygous mice (OIM/OIM). The OIM/OIM animals form homotrimeric type I collagen fibers, in which the α2(I) chain is replaced by a third α1(I) chain. In OIM/WT animals, both homotrimeric and heterotrimeric type I collagen fibers are found.21McBride DJ J.R. Choe V. Shapiro J.R. Brodsky B. Altered collagen structure in mouse tail tendon lacking the alpha 2(I) chain.J Mol Biol. 1997; 270: 275-284Crossref PubMed Scopus (103) Google Scholar OIM/OIM animals are born with fractures or develop them at an early stage. Joint subluxation, joint hemorrhage, and dorsal kyphosis are phenotypical correlates of a disorganized collagen matrix of the bone found in mice and in humans. Accordingly, both the thermal stability profile of homotrimeric type I collagen and the histological appearance of the bone in OIM/OIM animals is similar to those of patients with OI.18Chipman S.D. Sweet H.O. McBride DJ J.R. Davisson M.T. Marks SC J.R. Shuldiner A.R. Wenstrup R.J. Rowe D.W. Shapiro J.R. Defective pro alpha 2(I) collagen synthesis in a recessive mutation in mice: a model of human osteogenesis imperfecta.Proc Natl Acad Sci USA. 1993; 90: 1701-1705Crossref PubMed Scopus (278) Google Scholar LV collagen concentration is 45% lower than in wild-type animals, and collagen fiber diameter and number are decreased. LV chamber stiffness is reduced, despite increased collagen cross-linking in OIM/OIM. Heart weight/body weight ratio and basal LV function are similar across the OIM/OIM, WT/WT, and OIM/WT genotypes.22Weis S.M. Emery J.L. Becker K.D. McBride Jr, D.J. Omens J.H. McCulloch A.D. Myocardial mechanics and collagen structure in the osteogenesis imperfecta murine (oim).Circ Res. 2000; 87: 663-669Crossref PubMed Scopus (110) Google Scholar We investigated whether the genetic deficiency in collagen I in the OIM model affects wound healing and facilitates ventricular rupture after MI, assuming that the mechanism required depends on inflammatory cell infiltration and MMP activity. We used male 12- to 18-week-old homozygous OIM/OIM (n = 51) and heterozygous OIM/WT mice (n = 39)18Chipman S.D. Sweet H.O. McBride DJ J.R. Davisson M.T. Marks SC J.R. Shuldiner A.R. Wenstrup R.J. Rowe D.W. Shapiro J.R. Defective pro alpha 2(I) collagen synthesis in a recessive mutation in mice: a model of human osteogenesis imperfecta.Proc Natl Acad Sci USA. 1993; 90: 1701-1705Crossref PubMed Scopus (278) Google Scholar and wild-type (n = 32) littermates, all weighing 22 to 32 g. The cardiac phenotype of OIM has been reported previously.22Weis S.M. Emery J.L. Becker K.D. McBride Jr, D.J. Omens J.H. McCulloch A.D. Myocardial mechanics and collagen structure in the osteogenesis imperfecta murine (oim).Circ Res. 2000; 87: 663-669Crossref PubMed Scopus (110) Google Scholar The OIM model is available from the Jackson Laboratory (no. 001815; Bar Harbor, ME) and was delivered by Charles River Laboratories International (Sulzfeld, Germany). To allow acclimatization, animals were maintained for at least 2 weeks on a 12:12 hours light-dark cycle with free access to standard diet and water. A PCR method was used to determine mouse genotype, as described previously.23Saban J. King D. PCR genotyping of oim mutant mice.Biotechniques. 1996; 21: 190-192PubMed Google Scholar The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the NIH (6th edition). All animal procedures were approved by the applicable local government agency. MI was induced in mice as described previously.24Frantz S. Hu K. Widder J. Bayer B. Witzel C.C. Schmidt I. Galuppo P. Strotmann J. Ertl G. Bauersachs J. Peroxisome proliferator activated-receptor agonism and left ventricular remodeling in mice with chronic myocardial infarction.Br J Pharmacol. 2004; 141: 9-14Crossref PubMed Scopus (65) Google Scholar In brief, anesthesia was induced by isoflurane inhalation. After intubation, mice were ventilated with a volume-cycled rodent respirator with air and isoflurane (2% to 4%, v/v) and were placed on a heating pad to preserve normothermia. After thoracotomy, ligation of the left coronary artery was performed with a 7–0 silk suture, 3 to 4 mm from the tip of the left auricle. Pallor, regional hypokinesia, and enlargement of the left ventricle confirmed the presence of an infarction. The chest wall was closed with a continuous 6–0 Prolene suture. In sham-operated animals, the suture was placed but not ligated. Buprenorphine was given routinely as analgesic during surgery. Antibiotics were not given during the procedure, but no apparent infection had developed by the time of necropsy. For analysis of MMP-9 and collagen I expression, animals were euthanized on day 2. Tissue was collected on day 2 after MI, to avoid loss of infarcted animals to spontaneous death. To investigate the effect of neutrophil depletion or metalloproteinase activity inhibition, animals were monitored for 8 days after permanent MI. Mice were rigorously observed for morbidity and mortality after MI. Necropsy was performed after each death, to determine the cause of death. When blood in the pericardial sac or mediastinum indicated myocardial rupture, the whole left ventricle was carefully analyzed histologically, after embedding in paraffin and H&E staining. On day 2, the left ventricle was rinsed with saline, opened longitudinally along the septum, and pressed flat between glass plates. For determination of infarct, size 8 mice per genotype were euthanized on day 2. The left ventricle was then photographed and the area of infarction was quantified by planimetry (ImageJ software version 1.44; NIH, Bethesda, MD). Animals with small infarcts ( 30% and a heart rate of >450 beats/minute were included in the final analysis. Hemodynamic measurements were performed according to published protocols under light isoflurane anesthesia (isoflurane 1.5%, v/v).2Martin E. Shapiro J.R. Osteogenesis imperfecta: epidemiology and pathophysiology.Curr Osteoporos Rep. 2007; 5: 91-97Crossref PubMed Scopus (112) Google Scholar LV pressure curves were recorded after catheter placement in the LV cavity; systolic and diastolic blood pressure measurements were obtained on catheter withdrawal in the thoracic aorta. LV performance was assessed by ejection fraction. LV preload was assessed by end-diastolic pressure (LVEDP, mm Hg), contractility by dP/dtmax (mm Hg/second) and (dP/dtmax)/IP, which is the peak rate of rise in ventricular pressure normalized to instantaneous developed pressure and relaxation by −dP/dtmax (mm Hg/second). Formalin-fixed sections of mouse myocardium were prepared according to standard procedures. Slides were labeled by sequential application of the primary polyclonal rat anti-mouse MAC-3 antibody (BD Pharmingen, Heidelberg, Germany), a primary rat anti-mouse neutrophil antibody (clone 7/4; Linaris, Wertheim, Germany), P4HB polyclonal antibody (Abcam, Cambridge, UK), or an irrelevant control antibody. Visualization was performed using a Vectastain avidin-biotin complex ABC kit, as indicated by the manufacturer (Vector Laboratories, Burlingame, CA). The slides were then washed, dehydrated, and mounted for light microscopy. To analyze inflammatory cell infiltration, positively labeled cells were counted in high-power fields (×400) by a blinded observer (technician S.K.). Semiquantitative grading for P4HB+ cell infiltration was performed independently by two researchers (U.H. and technician S.K.) blinded for genotype on high-power fields (×400). Grading was defined as follows: 0 indicates absence of positively stained cells in all fields examined; 1 indicates positive cells in at least one field; 2 and 3 indicate low and high density of positive cells, respectively, on more than one field; and 4 indicates a high density of positive cells in all fields examined. From infarct zones, procollagen-α1(I) and procollagen-α2(I) mRNA was quantified by real-time PCR.24Frantz S. Hu K. Widder J. Bayer B. Witzel C.C. Schmidt I. Galuppo P. Strotmann J. Ertl G. Bauersachs J. Peroxisome proliferator activated-receptor agonism and left ventricular remodeling in mice with chronic myocardial infarction.Br J Pharmacol. 2004; 141: 9-14Crossref PubMed Scopus (65) Google Scholar After synthesis of cDNA with random hexamers (SuperScript; Invitrogen, Carlsbad, CA), real-time PCR was performed (iCycler system; Bio-Rad Laboratories, Hercules, CA) with commercially available TaqMan probes for 18S RNA, collagen type 1 α1, and collagen type 1 α2 (Applied Biosystems, Foster City, CA). PCR parameters were used as recommended for the TaqMan universal PCR master mix kit (Applied Biosystems). RNA samples were normalized to 18S rRNA. Matrix metalloproteinase-9 content in the infarct border zone was quantified with a commercial MMP-9 enzyme-linked immunosorbent assay (R&D Systems; Abingdon, UK) according to the manufacturer's protocol. Picrosirius Red (PSR) polarization microscopy was performed to quantify interstitial collagen. Sections were analyzed from hearts of 5 OIM/OIM, 12 OIM/WT, and 4 WT/WT animals with MI that survived the 56-day protocol. From each heart, one PSR-stained section derived from a midpapillary ring was photographed under polarized light with identical exposure times and was digitally analyzed according to a modified Junqueira method, as described previously.25Frantz S. Hu K. Bayer B. Gerondakis S. Strotmann J. Adamek A. Ertl G. Bauersachs J. Absence of NF-kappaB subunit p50 improves heart failure after myocardial infarction.FASEB J. 2006; 20: 1918-1920Crossref PubMed Scopus (116) Google Scholar Gel zymography was performed as described previously.26Fraccarollo D. Galuppo P. Bauersachs J. Ertl G. Collagen accumulation after myocardial infarction: effects of ETA receptor blockade and implications for early remodeling.Cardiovasc Res. 2002; : 559-567Crossref PubMed Scopus (65) Google Scholar For assessment of the inhibitory effect of doxycycline, gels were incubated with 25 μg/mL doxycycline. Neutrophil granulocytes were depleted in 10 OIM/OIM animals by intraperitoneal administration of 250 μg of an anti-Ly6G antibody (clone 1A8; BioLegend, Fell, Germany)27Daley J.M. Thomas A.A. Connolly M.D. Reichner J.S. Albina J.E. Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice.J Leukoc Biol. 2008; 83: 64-70Crossref PubMed Scopus (815) Google Scholar either twice, during infarct surgery (day 0) and on day 2, or once, on day 2. Animals were monitored for 7 days. For control of efficacy, 50 μL of heparin-anticoagulated mouse blood was subjected to fluorescence-activated cell sorting analysis on a FACSCalibur analyzer (BD Biosciences, San Jose, CA; Heidelberg, Germany) on day 2 in separate animals. Neutrophil granulocytes were identified within the leukocyte gate by direct staining of whole blood with biotinylated anti-CD45 and anti-GR1-Cy5 antibodies (BD Biosciences). Before analysis, red blood cells were lysed by addition of 100 μL of FixPerm buffer (eBioscience, San Diego, CA). Ten OIM/OIM animals were treated intraperitoneally with doxycycline 30 mg/kg dissolved in NaCl 0.9% before surgery (day 0) and then orally (100 mg/kg) on days 1 to 7.28Prall A.K. Longo G.M. Mayhan W.G. Waltke E.A. Fleckten B. Thompson R.W. Baxter B.T. Doxycycline in patients with abdominal aortic aneurysms and in mice: comparison of serum levels and effect on aneurysm growth in mice.J Vasc Surg. 2002; 35: 923-929Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar Control animals were treated with NaCl 0.9%. Results are presented as means ± SEM per group. For single comparison of data, a U-test or, as appropriate, an unpaired t-test was performed. Two-way analysis of variance was performed for multiple comparisons. Survival data were analyzed by a log-rank test and visualized as Kaplan-Meier plots. Differences were considered significant at P < 0.05. Data analysis was performed using StatView software verison 3.0 (SAS Institute, Cary, NC) and WinSTAT version 2001.1 add-in to Microsoft Excel 2003 software (R. Fitch Software, Bad Krozingen, Germany). As determined by echocardiographic analysis, baseline LV geometry in OIM/OIM animals did not differ from that of WT/WT or OIM/WT animals (Table 1). Body weight was significantly less for the OIM/OIM animals (Table 1).Table 1Baseline Characteristics in OIM/OIM, WT/WT, and OIM/WT MiceOIM/OIMOIM/WTWT/WTSample sizen = 51n = 39n = 14Body weight (g)24.2 ± 0.6⁎P < 0.05, versus WT/WT.†P < 0.05, versus OIM/WT.28.7 ± 0.531 ± 1.0Heart rate (bpm)469.0⁎P < 0.05, versus WT/WT. ± 7.8462.6 ± 7.7504.9 ± 13.0Echo data, papillary ESA (cm2)4.5 ± 0.34.5 ± 0.33.8 ± 0.5 EDA (cm2)9.0 ± 0.38.9 ± 0.38.7 ± 0.4 LVDD (cm)0.34 ± 0.010.35 ± 0.010.33 ± 0.01 LVSD (cm)0.24 ± 0.010.25 ± 0.010.23 ± 0.01Echo data, apical ESA (cm2)2.9 ± 0.23.1 ± 0.22.9 ± 0.2 EDA (cm2)6.3 ± 0.26.6 ± 0.26.7 ± 0.3 LVDD (cm)0.32 ± 0.010.32 ± 0.010.31 ± 0.01 LVSD (cm)0.22 ± 0.010.25 ± 0.010.20 ± 0.01Data are expressed as means ± SEM.bpm; beats per minute; EDA, end-diastolic area; ESA, end-systolic area; LVDD, left ventricular diastolic diameter; LVSD, left ventricular systolic diameter. P < 0.05, versus WT/WT.† P < 0.05, versus OIM/WT. Open table in a new tab Data are expressed as means ± SEM. bpm; beats per minute; EDA, end-diastolic area; ESA, end-systolic area; LVDD, left ventricular diastolic diameter; LVSD, left ventricular systolic diameter. After MI, there was a significant difference in mortality between OIM/OIM (65%) and WT/WT (13%) animals (Figure 1A). The most frequent cause of death during the first week after MI was LV rupture (Figure 1B). Ventricular rupture was identified in 13/22 OIM/OIM animals, compared with only 1/9 WT/WT animals. Histology typically revealed transmural rupture slits with white blood clots and intramural hemorrhage within the infarcted area (Figure 1D). The site of rupture was always confined to the infarct zone/border zone within the LV free wall. Incidence of rupture was independent of infarct size and was observed even in the absence of transmural infarction. In OIM/OIM animals subjected to sham surgery, mortality was significantly lower than in infarcted OIM/OIM animals, and no rupture was detected (Figure 1C). Echocardiography on day 1 demonstrated increased end-diastolic LV area (EDA) in OIM/OIM animals (OIM/OIM 16.3 ± 1.2 mm2; OIM/WT 10.3 ± 0.5 mm2; WT/WT 13.3 ± 1.5 mm2; P < 0.05) (Figure 2, A and C) and diameter (Figure 2, B and D) on apical short-axis sections reflecting the area of infarction. Infarct size did not differ among genotypes on day 2 (OIM/OIM 37.4 ± 1.9%; OIM/WT 38.4 ± 1.7%; WT/WT 38.5 ± 4.9%). Invasive hemodynamic characterization on day 2 also did not reveal any significant difference (see Supplemental Table S1 at http://ajp.amjpathol.org). Two days after MI, there was significantly lower procollagen α1(I) and α2(I) mRNA expression in the infarct border zone in OIM/OIM animals. Expression of α1(I), in arbitrary units, was 1.16 ± 0.11 for WT/WT and 0.45 ± 0.04 for OIM/OIM; expression of α2(I) was 1.22 ± 0.16 for WT/WT and 0.50 ± 0.05 for OIM/OIM (P < 0.05) (Figure 3A). To determine whether this reduced collagen expression was due to lesser numbers of collagen-expressing cells, we analyzed the frequency of collagen-producing cells on day 2 by immunohistological staining for prolyl 4-hydroxylase (P4HB), an enzyme involved in hydroxylation of prolyl residues in pre-procollagen. The frequency of P4HB+ cells did not differ significantly between WT/WT and OIM/OIM animals on day 2 (Figure 3B). On day 7, histology within the infarct zone of OIM/OIM animals indicated only sparse collagen (Figure 4, D and E); furthermore, histology barely detected collagen in intact septal OIM/OIM myocardium (Figure 4F). These findings were confirmed by quantitative collagen analysis based on PSR staining (Figure 4, G–I).Figure 4Extracellular collagen in WT/WT (left) and OIM/OIM (right) myocardium. On day 7 after myocardial infarction, Ladewig staining reveals any collagen fibers in blue within the infarcted myocardium (A–D) and within the intact septal myocardium (E and F). Collagen fibers can be readily identified within the infarct border zone (A and B) and infarct zone (C and D) in WT/WT animals (A, C, and E), but are barely detected in OIM/OIM animals (B, D, and F). In septal myocardium, collagen can be found mainly in the perivascular space (E and F). G and H: PSR-stained sections. I: Quantitative analysis of collagen fiber area based on PSR-stained sections indicated only sparse collagen fibers within the infarct zone of OIM/OIM (H), compared with WT/WT (G). *P < 0.05. a.u., arbitrary units. Scale bars: 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Analysis of the mature scar 56 days after MI demonstrated a modest but nonsignificant reduction in collagen fiber density (Figure 5E). The altered birefringent appearance of PSR-stained sections illustrates a qualitative difference in scar collagen composition (Figure 5, A–D), most likely because predominantly homotrimeric α1(I) collagen was deposited in the myocardi
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