The American Journal of Pathology, Vol. 179, No. 5, November 2011

Copyright © 2011 American Society for Investigative Pathology.

Published by Elsevier Inc. All rights reserved.

DOI: 10.1016/j.ajpath.2011.07.017

Cardiovascular, Pulmonary, and Renal Pathology

Type I Collagen Cleavage Is Essential for Effective

Fibrotic Repair after Myocardial Infarction

Zengxuan Nong,* Caroline O’Neil,* Ming Lei,†

Robert Gros,*† Alanna Watson,*‡ Amin Rizkalla,§

Kibret Mequanint,§ Shaohua Li,*Matthew J. Frontini,* Qingping Feng,† andJ. Geoffrey Pickering*‡¶�

From the Robarts Research Institute * and the Departments of

Physiology and Pharmacology,† Biochemistry,‡ Chemical and

Biochemical Engineering,§ Medicine (Cardiology),¶ and Medical

Biophysics,� University of Western Ontario, London, Ontario,

Canada

Efficient deposition of type I collagen is fundamental tohealing after myocardial infarction. Whether there isalso a role for cleavage of type I collagen in infarcthealing is unknown. To test this, we undertook coro-nary artery occlusion in mice with a targeted mutation(Col1a1r/r) that yields collagenase-resistant type I colla-gen. Eleven days after infarction, Col1a1r/r mice had alower mean arterial pressure and peak left ventricularsystolic pressure, reduced ventricular systolic function,and worse diastolic function, compared with wild-typelittermates. Infarcted Col1a1r/r mice also had greater30-day mortality, larger left ventricular lumens, andthinner infarct walls. Interestingly, the collagen fibrilcontent within infarcts of mutant mice was not in-creased. However, circular polarization microscopy re-vealed impaired collagen fibril organization and me-chanical testing indicated a predisposition to scarmicrodisruption. Three-dimensional lattices of collage-nase-resistant fibrils underwent cell-mediated contrac-tion, but the fibrils did not organize into birefringentcollagen bundles. In addition, time-lapse microscopy re-vealed that, although cells migrated smoothly on wild-typecollagen fibrils, crawling and repositioning on collage-nase-resistant collagen was impaired. We conclude thattype I collagen cleavage is required for efficient healing ofmyocardial infarcts and is critical for both dynamic posi-tioning of collagen-producing cells and hierarchical as-sembly of collagen fibrils. This seemingly paradoxical re-quirement for collagen cleavage in fibrotic repair shouldbe considered when designing potential strategies to in-hibit matrix degradation in cardiac disease. (Am J Pathol

2011, 179:2189–2198; DOI: 10.1016/j.ajpath.2011.07.017)

Rapid deposition of type I collagen is critical to cardiacrepair after myocardial infarction. When effectively elab-orated, a type I collagen-rich matrix provides a mechan-ically strong network that maintains cardiac integrity, min-imizes infarct expansion, and resists maladaptiveremodeling.1 Elaboration of type I collagen is a multistepprocess that includes expression of the genes encodingpro-�1(I) and pro-�2(I) collagen chains, intracellularwinding of the chains, and secretion of type I procolla-gen. Outside the cell, the propeptide termini are cleavedand the resulting triple helical type I collagen assemblesinto fibrils, which then cross-link and pack.2

Collagen deposition can be opposed by processes thatdegrade type I collagen. Enzymes that destroy the integrityof type I collagen by cleaving the triple helical domain areimportant in this regard. These include the interstitial colla-genases, matrix metalloproteinase (MMP)�1, MMP-8, andMMP-13, that cleave type I collagen three fourths along thelength of its triple helix. Broader-spectrum MMPs, such asMMP-2 and membrane-type (MT)1-MMP/MMP-14, alsohave the capacity to cleave native type I collagen.3,4 Sev-eral studies have highlighted the potential for interstitial col-lagenases to adversely affect the heart. Transgenic ex-pression of MMP-1 promotes ventricular dilatation anddysfunction,5 and serum levels of MMP-1 have been as-sociated with poor patient outcomes after infarction.6 Theexpression of MMP-8 has been associated with post-infarct ventricular rupture as well,7 and MMP-13 is sub-stantially up-regulated in the failing heart.8 There isalso strong evidence linking activation of broader-spectrum MMPs with adverse ventricular remodelingafter infarction.9

Supported by grants from the Canadian Institutes of Health Research(FRN-11715) and the Heart and Stroke Foundation of Canada (T7081).R.G. holds a New Investigator Award from the Heart and Stroke Founda-tion of Canada. J.G.P. holds the Heart and Stroke Foundation of Ontario–Barnett/Ivey Chair.

Accepted for publication July 11, 2011.

Supplemental material for this article can be found at http://ajp.amjpathol.org or at doi: 10.1016/j.ajpath.2011.07.017.

Address reprint requests to J. Geoffrey Pickering, M.D., Ph.D., LondonHealth Sciences Centre, 339 Windermere Rd., University of Western On-

tario, London, Ontario, Canada N6A 5A5. E-mail: gpickering@robarts.ca.

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Despite these findings, proof that type I collagen cleav-age adversely affects healing after a myocardial infarction islacking. Moreover, although it seems intuitive that degrad-ing type I collagen is not desired during fibrotic myocardialrepair, it is also possible that proteolytic collagen editing isimportant (eg, to ensure that the developing scar can pro-ductively remodel in the face of changing hemodynamicstresses). Determining whether there is a beneficial role forcollagen degradation is particularly important, given thatinhibiting extracellular matrix degradation is proposed as apotential therapy for myocardial infarction.9

To elucidate the role of collagenase-mediated type Icollagen degradation in the healing response to myocar-dial infarction, we examined the consequences of coro-nary artery occlusion in mice expressing a collagenase-resistant form of type I collagen. These mice express amodified �1(I) collagen chain that prevents the seminalthree-fourths to one-fourth cleavage event within the type Icollagen triple helix.10,11 We specifically investigatedwhether the scarring process was affected, using bire-fringence imaging, microtensile testing, and time-lapseassessment of the cell-collagen fibril interplay in vitro. Wereport that the ability of type I collagen to be cleaved in itstriple helical domain is critical for effective fibrotic repairafter myocardial infarction in mice.

Materials and Methods

Transgenic Mice

Heterozygous mice containing a targeted mutation of thecol1a1 gene (B6,129-Col1a1tm1Jae) were obtained fromJackson Laboratory, Bar Harbor, ME. These mice containtargeted substitutions of three amino acids at the three-fourths to one-fourth collagenase cleavage site of the�1(I) collagen chain, rendering the triple helical domainof type I collagen resistant to proteolytic cleavage.11 Ho-mozygous (Col1a1r/r) offspring and wild-type littermates,on the hybrid B6,129 background, were used for thisstudy. Mice were genotyped by PCR analysis of genomicDNA harvested from the tail.12

Myocardial Infarction

Experiments were performed in accordance with proto-cols approved by the Animal Care and Use Committee ofthe University of Western Ontario, London, Ontario, Can-ada. Myocardial infarctions were generated in 12- to 16-week-old male mice by permanent occlusion of the leftanterior descending (LAD) coronary artery under generalanesthesia [ketamine (80 mg/kg) and xylazine (10 mg/kg)], as described.13 As a control, sham–operated onmice were subjected to surgical anesthesia and theirchests were opened, but they did not receive coronaryartery ligation. Outcome analyses were undertaken byinvestigators (Z.N., M.L.) blinded to treatment groups and

mouse genotype.

Hemodynamic Assessment

The arterial blood pressure and heart rate were recordedfrom anesthetized mice after cannulating the right carotidartery with a Millar-tip transducer catheter (model SPR-261,1.4F; Millar Instruments, Inc., Houston TX). The catheter wassubsequently advanced into the left ventricle (LV) for mea-surement of peak LV systolic pressure, LV end-diastolicpressure, maximal rate of systolic pressure development,and maximal rate of diastolic pressure decline.

Tissue Harvesting and Histological Analyses

Eleven days after surgery, mice were anesthetized andhearts were arrested in diastole by infusion of 1 mL of 0.1mol/L KCl into the inferior vena cava. Hearts were thensubjected to retrograde perfusion through the aorta withPBS at a pressure of 100 mmHg, followed by 3 minutes ofpressurized perfusion with 4% paraformaldehyde. Ex-cised hearts were immersion fixed in 4% paraformalde-hyde for an additional 24 hours and transversely bisectedat the level of the coronary ligation. The distal region wasembedded in paraffin and cross-sectioned from the liga-tion site to the apex, obtaining 15 serial sections (5-�mthick) every 500 �m. Sections were stained with Picro-sirius red by incubating deparaffinized sections with0.1% Sirius red F3BA in saturated picric acid for 30minutes and rinsing twice with 0.01 mol/L HCl. Cardiacmorphometry was assessed in digitally imaged sections(Northern Eclipse, Empix Imaging, Inc., Mississauga, ON,Canada). The infarct size was determined as the endocar-dial circumference of the infarct zone, in absolute terms andrelative to that of the total left ventricular chamber, averagedfrom all sections obtained. The thickness of the infarct zonewas determined by dividing the infarct area measurementby the midline infarct circumference from all sections. Theleft ventricular chamber area was based on the endocardialcircumference, and the largest area served as the index ofchamber size.14 The fraction of collagen in the infarct zonewas quantified as the area of red staining tissue/total infarctwall area, in three low-power (�10 objective) fields persection. Myofibroblast alignment was evaluated in the bor-der regions of the infarct. The relative alignment of myofi-broblasts was assessed from the long axis of approximately100 nuclei within the fibrotic region zone (Northern Eclipse).The diversity of alignment was determined based on the SDof the angle distributions.

Tensile Testing

Microtensile stress tests were conducted using a com-puter-controlled Instron machine (model 3345; InstronCorporation, Canton, MA) at a cross-head speed of 0.5mm/minute and using a 10N load cell. A circumferentialstrip of infarct zone (width, approximately 2 mm) wasfreshly excised from hearts 11 days after LAD coronaryartery occlusion. The specimen was attached to a flatgrip using Vetbond (3M Animal Care Products, St. Paul,MN), and the exact cross-sectional area of each speci-men was determined using a micrometer. Specimens

were positioned parallel to the axis of the grip to reduce

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bending stresses. The modulus of elasticity of each spec-imen was calculated from the slope of the elastic portionof the stress-strain curves.

Cell Culture

Cardiac fibroblasts were isolated from the myocardium ofC57BL/6 mice.15 Briefly, minced ventricles were sub-jected to six 10-minute incubations in Hanks’ buffer con-taining type II collagenase (100 U/mL) at 37°C. Releasedcells were pelleted, resuspended in Dulbecco’s modifiedEagle’s medium with 10% fetal bovine serum, and plated.After 3 hours, unattached cells were removed, adherentfibroblasts were grown for 6 to 7 days until confluence,and fibroblasts at passage two or three were studied.Human vascular smooth muscle cells (SMCs) were cul-tured by outgrowth from the internal thoracic artery, aspreviously described.16,17

Collagen Isolation and Analysis of 3D Lattices

Tails of wild-type or Col1a1r/r mice were dissolved in 0.1mol/L acetic acid and collagen purified, as previouslydescribed.18 The collagen concentration was determinedspectrophotometrically (absorbance, 540 nm) from Picro-sirius red–stained aliquots dried onto microtiter wells.Three-dimensional (3D) collagen lattices were generatedby adding a suspension of 6 � 105 cells/mL to an equalvolume of neutralized collagen solution (2 mg/mL). The mix-ture, 1 mL, was then added to wells of a 24-well dish andallowed to polymerize at 37°C for 1 hour, before adding 1mL of Dulbecco’s modified Eagle’s medium with 10% fetalbovine serum. To initiate cell-mediated contraction of thelattice, gels were released from the well edges. Expressionof smooth muscle �-actin was assessed by immunostainingusing a mouse monoclonal antibody (clone 1A4; DAKOCanada Inc., Burlington, ON, Canada), and the presence ofcollagenase-cleaved type I collagen was assessed by us-ing a rabbit polyclonal antibody (C1-2C; IBEX Pharmaceu-ticals, Inc., Mount-Royal, QC, Canada).

Collagen Birefringence Imaging

Collagen organization was assessed using circular po-larization microscopy.19 Picrosirius red–stained sectionsof myocardium and 3D collagen lattices were visualizedusing a Nikon Optiphot-Pol microscope (Nikon, Missis-sauga, ON, Canada) customized for circular polarizationoptics20 or an Olympus BX51 microscope (Olympus Can-ada, Inc., Richmond Hill, ON, Canada) with polarizer-interference filters, a liquid crystal compensator, acharge-coupled device video camera, and commercialsoftware (Abrio LC-PolScope; Cambridge Research andInstrumentation, Inc., Hopkinton, MA). The latter systemenabled measurement of light retardation by collagen, inabsolute terms (nanometers), in defined fields of view. In3D collagen lattices, 6 to 10 randomly selected cells percondition that had their long axis parallel to the sectionplane were studied. The mean collagen retardation wasquantified in two regions of interest defined by the nu-

clear area, on either side of the nucleus, and corrected

for the mean retardation of collagen fibrils in regionsbetween the cells. As an additional approach thatavoided cell selection, total collagen birefringence wasassessed in five full fields of view (objective, �60) to yieldthe sum of retardation signals (in nanometers) for allpixels in the images. This summed value was correctedfor collagen retardation in regions devoid of cells bysubtracting the corresponding summed signal normal-ized for area. The cumulative retardation value was thendivided by the total number of cells in the fields of view.

Migration Analysis

Myofibroblast and SMC motility was evaluated using dig-ital time-lapse video microscopy.21–23 Cells plated ontodishes coated with a polymerized 3D network of mousetail collagen (0.8 mg/mL) were imaged with an invertedmicroscope (Zeiss Axiovert S100; Cornwall, ON, Canadaor Leica DMI6000 B; Concord, ON, Canada) using mod-ulated contrast optics. Images were digitally acquiredevery 5 minutes during a 6- to 8-hour recording period,using a high-resolution charge-coupled device camera(Sony XC-75/75CE; EMPIX Imaging, Inc. or LeicaDFC420 C video camera; Leica Microsystems Canada,Inc.) and time-lapse software (Northern Eclipse, EmpixImaging, Inc., and Leica Application Suite; Leica Micro-systems Canada Inc.). The ambient temperature wasmaintained at 37°C using a temperature control cell [CC-100 (20/20 Technology, Inc., Wilmington, NC) and Live-Cell (Pathology Devices, Westminster, MD)]. To maintainphysiological pH in room air, cells were incubated inbicarbonate-reduced medium M199 with Hanks’ saltsand 25 mmol/L HEPES.

Statistics

Data are expressed as mean � SEM. Comparisons be-tween groups were made with the Student’s t-test. Sur-vival was evaluated with Kaplan-Meier analysis, andcomparisons between groups were made by log-rankand Wilcoxon testing. Frequencies of mechanical micro-disruption were compared using the Fisher’s exact test.

Results

Survival after Myocardial Infarction Is Reducedin Mice Expressing a Collagenase-ResistantForm of Type I Collagen

The effect of collagenase-resistant type I collagen on sur-vival after myocardial infarction was assessed in 12- to16-week-old Col1a1r/r mice and wild-type littermates. Forty-six male mice, 23 Col1a1r/r mice, and 23 wild-type litter-mates were subjected to permanent LAD occlusion, andsurvival was tracked over 1 month. One wild-type and onemutant mouse died within 24 hours of surgery. An analysisof the remaining 44 mice revealed that survival of mutantmice was significantly worse than that of control mice (62%versus 20% 30-day mortality; P � 0.05; Figure 1). Two

Col1a1r/r and two control mice displayed left ventricular

2192 Nong et alAJP November 2011, Vol. 179, No. 5

rupture at death. There were no deaths in mice subjected tosham surgery. These findings suggest that the elaborationof collagenase-resistant type I collagen was not protective;instead, it reduced post-infarct survival.

Cardiac Performance after Myocardial InfarctionIs Reduced in Mice Producing Collagenase-Resistant Type I Collagen

To determine whether the increased death after infarctionamong Col1a1r/r mice was associated with worsened car-diac performance, another 34 mice were subjected toLAD occlusion and hemodynamics were evaluated after11 days, using a Millar-tip transducer catheter. Amongsham–operated on mice, there were no differences inheart rate, mean arterial pressure, peak left ventricularsystolic pressure, left ventricular contractile performance,and left ventricular diastolic performance betweenCol1a1r/r mice (n � 8) and wild-type littermates (n � 6)(Table 1). However, among mice subjected to LAD oc-clusion, the mean arterial pressure of Col1a1r/r mice (n �9) was 34.9% lower than that of wild-type mice (n � 11)(P � 0.01). Left ventricular systolic pressure in Col1a1r/r

mice was 23.5% lower (P � 0.05), left ventricular con-

Figure 1. Survival of Col1a1r/r mice and wild-type littermates after myocar-dial infarction. Kaplan-Meier survival curves of male Col1a11r/r mice andmale wild-type littermates over 30 days after induction of myocardial infarc-tion by permanent LAD occlusion. P � 0.03, Wilcoxon log-lank comparison.

Table 1. Hemodynamic Parameters in WT and Col1a1r/r Mice af

Parameter

Sham–operat

WT (n � 6)

Heart rate (beats/minute) 393 � 8MAP (mmHg) 87 � 3LVSP (mmHg) 109 � 4LVEDP (mmHg) 4.2 � 0.6LV dP/dtmax (mmHg/second) 7973 � 484LV �dP/dtmax (mmHg/second) 8171 � 533

Values are given as mean � SEM.*P � 0.05 vs sham and WT LAD occluded.†P � 0.05 vs sham.

dP/dtmax, maximum rate of increase in left ventricular systolic pressure; �dP/d

left ventricular end-diastolic pressure; LVSP, peak left ventricular systolic pressu

tractile performance was 34.4% lower (P � 0.05), and leftventricular diastolic performance was 31.2% lower (P �0.05). Thus, the inability to cleave type I collagen at thethree-fourths to one-fourth site adversely affected bothsystolic and diastolic left ventricular performance aftermyocardial infarction.

Ventricular Distortion after Myocardial InfarctionIs Worsened in Mice Producing Collagenase-Resistant Type I Collagen

Cardiac morphometry was assessed in Picrosirius red–stained sections of hearts arrested in diastole and pressurefixed 11 days after infarction or sham surgery. No differ-ences in left ventricular chamber size or wall thickness werefound between Col1a1r/r and control mice subjected to asham operation (Figure 2). After LAD occlusion, the infarctterritory in wild-type mice occupied 36% � 1.7% of themidventricular myocardium, and this was not different inmutant mice (34% � 2.7%, P � 0.49). However, the infarctzone was significantly thinner, attenuated by 26.8% (P �0.01, Figure 2) and the absolute length of the infarct zonewas greater in mutant than in wild-type mice (6.8 � 0.3versus 5.2 � 0.4 mm; P � 0.01). Also, the left ventricularchamber size was greater (48.7%, P � 0.01).

These morphometric data raised the possibility that thehealing infarct in Col1a1r/r mouse hearts was predisposedto expansion. To further assess this, fresh segments of scarzone 11 days after infarction were excised from the infarctterritory and subjected to tensile testing, using a 10N loadcell. The modulus of elasticity was 2.6 � 0.7 MPa in scarsegments from wild-type hearts (n � 4) and 1.5 � 0.4 MPain scars from Col1a1r/r hearts (n � 5, P � 0.22), suggestinggreater elasticity of mutant scars. The presence of abrupt,but transient, losses of stretch-induced tension in the scarsfrom mutant hearts, well before total mechanical failure wasreached (Figure 3), was more striking, suggesting micro-disruptions in the tissue as it was stretching. This phenom-enon was observed in all five mutant infarcts studied (1.2microfailures per scar) but only in one of four wild-typeinfarcts (0.25 microfailures per scar, P � 0.05). These find-ings indicate that the infarct zone of mice expressing colla-genase-resistant type I collagen has a reduced ability toresist tensile forces.

cardial Infarction

mice LAD-occluded mice

1r/r (n � 8) WT (n � 11) Col1a1r/r (n � 9)

92 � 9 420 � 13 391 � 987 � 6 86 � 5 56 � 4*12 � 5 106 � 8 81 � 4*.7 � 0.4 11.6 � 2.1† 7.9 � 0.7†

89 � 353 6916 � 453 4607 � 441*04 � 400 6159 � 290† 4239 � 380*

ter Myo

ed on

Col1a

3

13

7684

tmax, maximum rate of decline in left ventricular diastolic pressure; LVEDP,re; MAP, mean artery pressure; WT, wild type.

e plot foating a m

Collagen Cleavage and Myocardial Infarct 2193AJP November 2011, Vol. 179, No. 5

Healing Infarct Zone of Col1a1r/r Mice DisplaysAbnormal Collagen Fibril Organization

We next evaluated the fibrotic response in the hearts.Collagen content in hearts subjected to sham surgerywas not different between wild-type and mutant mice(4.6% � 0.04% versus 4.9% � 0.5%). Interestingly, the

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Figure 2. Impaired left ventricular remodeling after myocardial infarction (Marrested in diastole 11 days after sham surgery or LAD occlusion. B: There wMI compared with wild-type (WT) mice. No differences existed in sham–opafter LAD occlusion in a WT (left) or a Col1a1r/r mutant (right) mouse. Thgenerated stress in the otherwise linear portion of the curve (arrow), indic

r/rCol1a1

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collagen content within the infarct zone was also notdifferent between wild-type and mutant mice (50.8% �5.8% versus 54.2% � 4.4%), indicating that resistance tocollagen cleavage did not lead to an accumulation incollagen fibers. In contrast, distinctions in collagen fibrilmorphological features were noted. This was examinedusing circularly polarized light, an approach that clearly

Sham MI

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l1a1r/r mice. A: Picrosirius red–stained cross sections of pressure-fixed heartseater LV chamber area and a thinner infarct territory in Col1a1r/r mice aftern hearts. *P � 0.05. C: Stress-strain plots of the scar zone harvested 11 daysr the mutant scar reveals greater elasticity and a small abrupt decline in theicrodisruption event.

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Figure 3. Collagen fibers in the infarct zone ofCol1a1r/r mice display less order. A: Circularpolarization microscopy images of Picrosiriusred–stained sections of the LV, 11 days after LADocclusion. Co-aligned bundles of birefringentcollagen fibers are seen in the infarct zone ofwild-type (WT) mice (left), whereas in Col1a1r/r

mice, fibers were more diffusely oriented andthinner (middle). Insets (right): Well-delin-eated wavy appearance of collagen fibers (ar-row) in the infarct zone of WT mice only (toppanel, arrow) versus those in mutant mice(bottom panel). B: Digitally acquired retarda-tion images of the infarct border zone of miceafter LAD occlusion, imaged with polarized lightand liquid crystal compensation. The resultingretardation images (left) are independent of il-lumination intensity, such that images can bedirectly compared. The corresponding heatmaps (right) depict reduced retardation in theinfarct zone of mutant mice, despite copiouscollagen fibers having been deposited. Quanti-tative data from 11 mutant and 9 WT littermatemice are shown. *P � 0.05.

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2194 Nong et alAJP November 2011, Vol. 179, No. 5

delineates fibril morphological features by innate colla-gen birefringence enhanced by the Picrosirius red stain.Collagen fibrils in the infarct zone of mice expressingwild-type type I collagen appeared as well-aligned, cir-cumferentially oriented bands of thick fibers with a wavysinusoidal pattern (Figure 3A). In contrast, in the infarctzone of Col1a1r/r mice, collagen fibers were thinner andless organized. Moreover, these fibers typically did notdisplay sinusoidal periodicity (Figure 3A). These differ-ences were particularly evident in the middle circumfer-ential one third of the scar and the infarct borders.

Collagen birefringence increases as its molecular, su-pramolecular, and fibrillar orders increase, structuralchanges that increase optical anisotropy.19,20,24 There-fore, we quantified collagen retardation as an indicator ofcollagen organization within the fibrotic tissue. Retarda-tion of collagen fibers in noninfarcted hearts was notdifferent between wild-type and mutant mice (4.9 � 0.7versus 4.4 � 0.7 nm; P � 0.55). However, in mice sub-jected to infarction, the mean collagen retardation withinfibrotic regions was 18.0% lower in hearts of Col1a1r/r

compared with wild-type mice (Figure 3B). Together,these morphological and birefringence findings indicatethat the inability to efficiently cleave type I collagen didnot affect collagen accumulation after infarction but sig-nificantly impaired the ability of elaborated collagen tohierarchically organize.

Collagenase-Resistant Collagen Fails toAssemble into High-Ordered Fibrils in Responseto Cell-Mediated Contractile Forces

To determine whether the reduced collagen organizationwas directly related to collagenase resistance, we eval-uated the interplay between mesenchymal cells and col-lagen fibrils in a 3D network.25 Collagen fibril latticeswere prepared from collagen harvested from tails of wild-type or Col1a1r/r mice and embedded with mouse myo-cardial fibroblasts. Networks embedded with human vas-cular SMCs were similarly prepared, in view of themyofibroblast-like attributes of these cells. When colla-gen fibril lattices are released from the sides of the cul-ture dish, they contract, in association with elaboration ofcollagen-degrading enzymes.26 We observed that bothmouse and human mesenchymal cells contracted thecollagen gels and that contraction did not occur if cellswere not present in the network. Interestingly, networksgenerated from collagenase-resistant collagen con-tracted more than those constructed of wild-type colla-gen (63% � 4% versus 46% � 3% at 48 hours; P � 0.05;Figure 3). Immunostaining using an antibody specific forcollagenase-cleaved type I collagen revealed a signal infibrils immediately adjacent to a few cells (�10%), sug-gesting highly localized collagen editing with a transientcleavage product (Figure 4). Signal was not detected ingels of mutant collagen. The proportion of fibroblasts inmutant collagen that expressed immunodetectablesmooth muscle �-actin was not different from that forfibroblasts in wild-type collagen (33.2% � 2.5% versus

33.8% � 2.2%), suggesting no differences in the intrinsic

contractile properties of the cells. Collectively, these datasuggest a degree of collagen fibril disruption and/or slip-page during the contraction process that did not proceedin gels of mutant collagen.

To evaluate the architecture of the fibrils, lattices wereexamined histologically and fibril retardation was quanti-fied by polarization microscopy. Before contraction, bothwild-type and mutant fibrils appeared as relatively homo-geneous lattices of fibrils (thickness, �0.2 �m). However,after 48 hours of contraction, the collagen fibrils in theimmediate vicinity of the cells had substantially remod-eled into thicker (1 to 8 �m) and highly birefringent bun-dles of fibrils (Figure 4). This fibril reorganization wasobserved for wild-type collagen but markedly less so forcollagenase-resistant collagen (Figure 4B). The mean re-tardation of pericellular collagen fibrils was 29.0% higherin gels of wild-type collagen, when evaluated on fibrilssurrounding randomly selected individual cells, and47.7% higher when assessed as the cumulative retarda-tion index, normalized for background and the number of

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Figure 4. Cell-mediated contraction of collagen fibril lattice and assembly ofpericellular collagen fibrils in vitro. A: The magnitude of contraction ofcollagen lattices embedded with mouse cardiac fibroblasts and constructedof either collagenase-resistant collagen or wild-type (WT) fibrils. B: Sectionsfrom cell-embedded collagen fibril lattices stained after 48 hours with Picro-sirius red and viewed with circular polarized light. Birefringent collagenfibrils assembled on the surface of cell body and projections in the WTcollagen network (arrows). In contrast, only a modest accumulation ofbirefringent fibrils can be seen around the cells in the network of collage-nase-resistant collagen. Immunostaining at 24 hours using the C1-2C anti-body revealed evidence for collagenase-cleaved type I collagen in immediatepericellular regions in WT (arrows, top right panel), but not mutant(bottom right panel), gels. C: Collagen fibril retardation of circularly po-larized light. Retardation was quantified as the average retardation in definedregions of interest associated with selected cells (left) and the averagecell-associated collagen retardation from the entire unselected field of view,and expressed as summed retardation (right). *P � 0.05.

cells in the section (Figure 4C). Taken together, these

Collagen Cleavage and Myocardial Infarct 2195AJP November 2011, Vol. 179, No. 5

findings establish that, in response to cell-mediated con-tractile forces, collagen fibrils organize into high-orderedbundles and that this assembly process requires theproteolytic cleavage of the type I collagen triple helix.

Cell Migration and Positional Alignment AreInhibited by Collagenase-Resistant CollagenFibrils

The ability of collagen-producing cells to crawl into thewound and appropriately orient also determines the effi-cacy of fibrotic healing. To determine whether collage-nase-resistant collagen affected fibroblast/myofibroblastpositioning after infarction, we quantified the alignment ofmesenchymal cells in the infarct border zones, the site offibroblast/myofibroblast streaming. This revealed a61.5% (P � 0.01) greater dispersion in fibroblast orien-tation in the hearts of Col1a1r/r mice compared with that ininfarcted hearts of wild-type mice (Figure 5A). This dis-persed alignment suggests a reduced ability of the infil-trating mesenchymal cells to co-align in an environmentof collagenase-resistant collagen.

To further evaluate motility in a collagen fibril environ-ment, we undertook time-lapse video microscopy of cells

Figure 5. Cell motility and dynamic repositioning are suppressed on collagethe transition zone of infarcted myocardium 11 days after LAD occlusion in(light gray) and human SMCs (dark gray) crawling on a 3D collagen fibril latof mouse fibroblasts crawling on the designated collagen lattices. Tracings decells crawling on collagen fibrils over 150 minutes. Arrows (top panel): A cdegrees from its original path. Arrows (bottom panel): A cell in which the leresulting in stretching of the cell body and tail. The long arrow denotes the

turning or reorientation. These distinctions in the dynamic motility patterns of cells(available at http://aip.ampathol.org). *P � 0.05 versus the corresponding WT value

on crawling along 3D lattices of wild-type or mutant typeI collagen. Mouse myocardial fibroblasts migrated alonga network of wild-type collagen, with a migration speed at32.9 � 4.5 �m/hour, and human SMCs migrated at19.4 � 3.3 �m/hour. However, the migration speeds ofeither mouse or human cells were significantly lowerwhen they crawled on collagenase-resistant collagen fi-brils (P � 0.01, Figure 5B).

Tracking the migration paths of individual cells re-vealed striking differences in the patterns of migration. Asillustrated in Figure 5, C and D, fibroblasts on wild-typecollagen typically translocated in a given direction for oneor more cell lengths and then turned and migrated inanother direction. This pattern reflects the lack of anexternal chemotactic gradient but also indicates an abil-ity of the cells to quickly reorient as they explore theirenvironment. In contrast, cells on collagenase-resistantcollagen displayed only minor shifts in the position of thecell centroid and little change in the overall direction ofcrawling. Moreover, although cells on wild-type collagenglided smoothly over the fibrils, on mutant collagen theyexhibited a halting translocation pattern. This was asso-ciated with stretching and, at times, bending of the trail-ing aspect of the cell, which appeared to effectively with-

sistant collagen. A: The SD of alignment of fibroblasts/myofibroblasts withinnd Col1a1r/r mice. B: The migration speed of mouse myocardial fibroblasts

ved from the tails of either wild-type (WT) or Col1a1r/r mice. C: Spider plotshourly translocation of the cell centroid over 6 hours. D: Images of individualtranslocates in a given direction and then turns to reorient approximately 90ge protrudes forward but the rear aspect does not release from the substrate,ment of a bend in the stretched tail, suggesting additional restriction to cell

nase-recontrol atice deripict theell thatading eddevelop

on WT and mutant collagen can be seen in Supplemental Videos S1 and S2.

2196 Nong et alAJP November 2011, Vol. 179, No. 5

hold the cell from changing position and orientation(Figure 5D; see also Supplemental Video S1 at http://aip.ampathol.org). The mutant collagen network itself wasseen to fold and compress underneath the cells, indicat-ing that the cells were generating contractile forces nec-essary for migration but were incapable of releasing fromthe collagen substrate to effectively translocate (see Sup-plemental Video S2 at http://aip.ampathol.org). Thesefindings reveal a collagen cleavage–based cellular re-lease phenomenon that is necessary for collagen-pro-ducing cells to efficiently translocate and reorient in theirenvironment.

Discussion

We have studied the role of type I collagen cleavage inthe healing process after myocardial infarction in mice.The findings reveal that mice that produce a collagenase-resistant form of type I collagen undergo maladaptiveventricular remodeling after myocardial infarction, withsuppressed cardiac performance and increased mortal-ity compared with littermate control mice. These adverseevents were associated with impaired hierarchical order-ing of the collagen fibrils deposited in the infarct zone.We also established that mesenchymal cells that wereotherwise fully capable of assembling collagen fibrils intocoherent bundles could not execute this function effec-tively with collagenase-resistant collagen fibrils. More-over, the ability of mesenchymal cells to crawl, orient,and co-align during tissue remodeling was impaired inan environment of collagenase-resistant collagen.These findings suggest that, despite the potential toweaken a developing scar, proteolysis of type I colla-gen is required for key steps in fibrotic healing aftermyocardial infarction.

Col1a1r/r mice contain a targeted mutation in the Col1a1gene that eliminates the Gly775-Ile776 cleavage site lo-cated approximately three fourths along the length of thetriple helical domain of type I collagen. This is an importantproteolysis site because, once cleaved, the collagen triplehelix unfolds, rendering the collagen chains susceptibleto further proteolytic degradation. Several enzymes arecapable of mediating this cleavage, and several, includ-ing MMP-8, MMP-13, and MMP-14, are expressed aftermyocardial infarction.8,27,28 Thus, Col1a1r/r mice consti-tute a powerful tool for elucidating the role of collagendegradation during infarct healing, and their use circum-vents compensation or redundancy among collagen-de-grading enzymes. In addition, collagenases can havetargets other than type I collagen.9 Therefore, the find-ings observed with Col1a1r/r mice reflect only the alteredtype I collagen and not enzymatic cleavage states ofother protein targets. Furthermore, because type I colla-gen in Col1a1r/r mice can still be cleaved at a secondaryN-telopeptide site,11 the current findings specifically es-tablish the importance of three-fourths to one-fourth triplehelix cleavage in infarct repair.

Previous studies have established that Col1a1r/r micehave a propensity to accumulate excess collagen fibers.

Aged Col1a1r/r mice accumulate collagen fibers in the

skin,11 and increased fibrosis has been found in athero-sclerotic lesions29 and aneurysmal arteries.30 Giventhese findings, it is noteworthy that collagen accumula-tion in the LV of Col1a1r/r mice after infarction was notgreater than that in control mice. This finding is consistentwith a previous report31 and probably reflects the factthat the balance of production and degradation of type Icollagen after acute myocardial injury is heavily weightedto the production side. However, the current findings alsoestablish that, despite this production bias, collagen deg-radation plays a key role in the infarct healing process.This role was evidently not to control the amount of col-lagen deposited but to regulate the architecture of thedeposited fibrils. Qualitatively, wavy collagen fibers typ-ical of well-organized fibrous tissue emerged in the in-farct zone of control mice but generally not in the fibroticterritory of Col1a1r/r mice. Quantitatively, fibrils in thehearts of Col1a1r/r mice were less birefringent and had areduced ability to retard circularly polarized light. Thus,the hierarchical collagen ordering process fundamentalto fibrotic healing requires three-fourths to one-fourth col-lagen cleavage.

The poor response to myocardial infarction in Col1a1r/r

mice could reflect a primary defect in collagen organiza-tion in the infarct zone or a secondary effect of a gener-alized perturbation in cardiovascular function in Col1a1r/r

mice. Although the latter possibility cannot be excluded,we did not identify baseline cardiovascular abnormalitiesin the Col1a1r/r mice studied. Heart rate, mean arterialpressure, peak left ventricular systolic pressure, left ven-tricular chamber size, and left ventricular wall thicknesswere similar in sham–operated on wild-type and mutantmice. Thus, for example, baseline differences in afterloadare an unlikely explanation for the worsened response ofCol1a1r/r mice to infarction. Pre-infarction collagen fibrilcontent and organization were also not different betweenmutant and wild-type mice.

Therefore, we propose that the maladaptive post-in-farct remodeling in Col1a1r/r mice was because of theelaboration of mutant collagen in the infarct zone, result-ing in impaired fibrotic healing. This was supported bymechanical testing, which identified a predisposition ofthe scar zone in mutant mice to tension-induced micro-failure. Moreover, in vitro analyses identified a direct re-lationship between the ability of collagen to undergocleavage and collagen fibril assembly. There may also bemodifier genes in the B6,129 strain used that heightenedthe impact of the collagen mutation in vivo. Mice with a129 background have been found to have more vulner-able infarct healing than inbred C57BL/6 mice32 andgreater activation of the renin-angiotensin system.33

Our 3D culture analyses identified two components oftissue remodeling that depend on collagenase-compe-tent collagen fibrils. The first component is the phenom-enon of cell contraction-mediated assembly of collagenfibrils. We identified that collagen fibril assembly pro-ceeds at the cell surface and found that this fiber-buildingprocess required collagen fibrils that can be cleaved atthe three-fourths to one-fourth proteolysis site. The factthat collagen fibril birefringence decreased when colla-

gen cleavage was abrogated seems paradoxical but

Collagen Cleavage and Myocardial Infarct 2197AJP November 2011, Vol. 179, No. 5

suggests a process by which site-specific collagencleavage enables collagen fibrils to reassemble intothicker fibrils. The fact that contraction of mutant collagengels was greater than that of wild-type gels further sug-gests that this augmented fibril assembly process isbased on a highly functional collagen slippage phenom-enon. Therefore, we propose that type I collagen fibrilsproductively organize through a cleave-slide-and-reas-semble cascade and that this cascade is critical to opti-mizing fibrosis after myocardial infarction.

The second process for which collagen fibril cleavagewas determined to be critical was optimizing the motilityand positioning of the collagen-producing cell. We foundthat a 3D network of type I collagen fibrils was a suitablesubstrate for motility and enabled a smooth, gliding mi-gration pattern with efficient shifting of cell orientation.However, on collagenase-resistant collagen, cell migra-tion was slow and halting, with marked stretching of thetrailing aspect of the cell. Moreover, there was accentu-ated deformation and compression of the surroundingcollagen network. These findings suggest a role for col-lagen cleavage in loosening cell-substrate connectionsto allow the cell body and tail to efficiently and appropri-ately follow the leading edge. Previous studies22,34 havesuggested that products of type I collagen cleavage maybe promigratory. Collagen cleavage might also be impor-tant for ensuring that other promigratory matrix mole-cules, such as minor collagens or fibronectin, can appro-priately interact with the cell. Collagen synthesis issuppressed in cells on or in a 3D lattice18,35; thus, in thissystem, the dominant target for editing is likely pre-exist-ing collagen fibrils. In vivo, cleavage of recently depositedcollagen may also be relevant, particularly because col-lagen deposition and maturation during wound healingcan proceed over days to weeks.20,36

The ability of fibroblasts to quickly reorient and fine-tune their direction of traveling within remodeling tissuecould serve two purposes. First, it could ensure that thecell can explore and test its new environment efficiently.Second, there may be a need for cells to rapidly shiftposition in response to ongoing structural changes andmechanical forces, a circumstance highly relevant to theremodeling infarct zone.36 We found that in hearts ex-pressing collagenase-resistant collagen there was re-duced alignment of myofibroblasts entering the infarctzone. Because fibroblast/myofibroblast alignment de-fines collagen fibril alignment, this impaired ability to re-orient could be an additional means by which the fidelityof scar formation was reduced.

In summary, despite the potential deleterious conse-quences of degrading collagen fibrils, cleavage of type Icollagen is required for effective fibrotic healing aftermyocardial infarction in mice. Both the hierarchical as-sembly of collagen fibrils and the positioning of the col-lagen-elaborating cells depend on the capacity withwhich type I collagen can be cleaved. The seeminglyparadoxical requirement of type I collagen cleavage forcardiac repair should be considered when designingtherapeutic strategies to block matrix degradation in the

heart.

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