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Ann Thorac Surg 2001;72:1950-1956
© 2001 The Society of Thoracic Surgeons
a Departments of Surgery, Medicine, and Pathology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
b Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, University of Manitoba, Faculty of Medicine, Manitoba, Canada
Accepted for publication August 29, 2001.
* Address reprint requests to Dr Edmunds, 5000 Ravdin Ct, Hospital of the University Pennsylvania, 3400 Spruce St, Philadelphia, PA 19104-4283, USA
e-mail: hank.edmunds{at}uphs.upenn.edu
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Abstract |
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Methods. Fourteen sheep had acute anteroapical infarction that progressively expands into left ventricular aneurysm within 8 weeks. In 7 sheep, infarct expansion was restrained by prior placement of mesh over the area at risk. Fourteen days after infarction, and after hemodynamic and echocardiographic measurements, animals were euthanized for histology, measurements of hydroxyproline, matrix metalloproteinase-1 (MMP-1 or collagenase) and MMP-2 (gelatinase) activity, as well as collagen type I and III in infarcted, borderzone, and remote myocardium.
Results. Restraining infarct expansion does not change collagen content or MMP-1 or MMP-2 activity in the infarct, but significantly increases the ratio of collagen I/III. In borderzone and remote myocardium infarct, restraint significantly increases collagen content and significantly reduces MMP-1 activity. MMP-2 activity is reduced (p = 0.059) in borderzone myocardium only. Between groups, the ratio of type I/III fibrillar collagen does not change in borderzone myocardium.
Conclusions. Fourteen days after acute myocardial infarction, restraining infarct expansion increases collagen accumulation in borderzone and remote myocardium, which may prevent expansion of hypocontractile, fully perfused "remodeling myocardium" adjacent to the infarct. This study demonstrates that changes in regional myocardial wall strain alter the cellular and biochemical processes involved in postinfarction ventricular remodeling.
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Introduction |
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Moderate or large acute myocardial infarctions trigger an immediate neurohormonal response and the Frank-Starling mechanism to maintain the circulation [2], but at the cellular level, initiate a complex series of biochemical events associated with tissue repair and compensation for myocyte dropout [8]. After MI, the infarcted region is replaced by scar tissue characterized by deposition of matrix material, which largely consists of fibrillar collagens [9]; however, cardiac fibrosis also occurs in noninfarcted myocardium and contributes to dysfunction of the failing heart [10]. These biochemical processes, which involve the entire ventricle [8, 10], are primarily controlled and mediated by various local, cell-produced signaling proteins. Angiotensin II (Ang II) and transforming growth factor-ß1 (TGFß-1) are both well-described trophic factors (proteins) involved in the control of cardiac matrix collagen synthesis and degradation [8] and myocyte hypertrophy in viable myocardium [11, 12].
The relationship between postinfarction regional wall strains and stresses and the molecular processes regulating collagen synthesis and degradation is not established. Grossman showed that left ventricle (LV) remodeling in patients with valvular heart disease correlated with changes in calculated myocardial wall stresses [13]. In valvular heart disease, myocardial wall stresses are equal throughout the ventricle at any given time in the cardiac cycle [13]. However, after acute myocardial infarction, regional myocardial wall strains and stresses are not everywhere equal because of differing, postinfarction, regional wall strains and changes in the material properties of the infarct [4, 7, 14, 15]. Our hypothesis states that restraining expansion of an anteroapical infarction, which a priori changes regional wall strains [16], and therefore wall stresses, also changes collagen accumulation and collagenase activity in both infarct and noninfarcted myocardium.
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Material and methods |
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Surgical protocol
Sheep were induced with pentathol (2 mg/kg), intubated, anesthetized with isoflurane (1.5% to 2%), and ventilated with oxygen (Drager anesthesia monitor; North American Drager, Telford, PA). All animals received glycopyrrolate (0.4 mg IV), cefazolin (1 g, IV), and gentamycin (200 mg IV). Sheep were treated in compliance with National Institutes of Health publication No. 85-23, as revised in 1985.
Using aseptic technique, polypropylene snares were placed around the homonymous (designated LAD in this article) and second diagonal (D2) coronary arteries approximately 40% from the apex via left anterolateral thoracotomy. Snares were briefly tightened for 30 seconds to identify the area at risk and precisely outline the distinct, but irregular border between ischemic and perfused myocardium with a surgical marker. A piece of Marlex mesh, cut carefully to cover only the area of the subsequent infarct, was tacked to the epicardium with interrupted 5-0 polypropylene sutures in two groups of sheep (Fig 1) [16]. For 14 sheep destined to have infarctions, snares were buried in a subcutaneous pocket and the wound was closed. For the other animals, the snares were cut off, but not removed. The thoracotomy was closed and animals were recovered.
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Baseline data
Ten to 14 days after initial thoracotomy, 14 sheep assigned to groups I and MI were anesthetized with isofluane (1.5% to 2%), intubated, and placed supine. The electrocardiogram and femoral arterial pressure were monitored; a Swan-Ganz catheter was placed to periodically monitor cardiac output by thermodilution in duplicate. A sterile midline laparotomy was made, and subdiaphragmatic two-dimensional echocardiographic images were obtained using a 5-MHz probe (77020A; Hewlett Packard, Palo Alto, CA) [17]. Images were recorded on 0.5-inch videotape at 30 Hz (Panasonic AG-6300 VHS Recorder; Matsushita Electrical Ind Co, Ltd, Tendo City, Japan). Left ventricular short axis images at three levels (at the tips of the papillary muscles, at the bases of the papillary muscles, and at the apex) and two orthogonal long axis views were obtained. These images served to verify the size and location of the infarct and restraint of infarct expansion by the mesh.
Infarction or sham infarction
After baseline echocardiographic studies, during steady-state conditions, the coronary arterial snares were exteriorized, tightened, and tied to completely occlude the two snared arteries in the two infarction groups. Echocardiographic measurements were repeated 50 minutes after onset of infarction. The 4 animals in groups C, N, and MN had anesthetic induction and intubation, but did not have echocardiograms or infarctions.
Periinfarction and postinfarction arrhythmias were treated prophylactically with amiodarone (150-mg IV load, 30 minutes before infarction), lidocaine (2 to 4 mg · kg-1 · min-1), magnesium (1 g), calcium chloride (200 mg), and potassium supplementation to keep the potassium concentration more than 4.5 (mg/dL) before infarction. Mean blood pressure was maintained above 80 mm Hg using small infusions of phenylephrine as needed; cardiac output was maintained greater than 2.0 liters per minute with an epinephrine infusion. Hearts were electrically defibrillated when necessary. Animals were routinely observed in the laboratory suite 2 to 3 days after infarction and daily thereafter.
Terminal measurements and autopsy
Fourteen days after infarction or sham infarction, sheep were anesthetized, intubated, and ventilated. The two infarction groups (n = 11) underwent repeat echocardiographic measurements as described above. Fifteen surviving animals had midline sternotomy and dissection of the anterior heart and great vessels. Five hundred milliliters of cold crystalloid cardioplegic solution was rapidly infused into the ascending aorta below a cross-clamp. Simultaneously, the inferior vena cava was transected. When the heart was arrested, it was rapidly excised and placed into a basin of saline and ice. Extraneous tissue was trimmed; the heart was cut base to apex to expose the entire LV. Full-thickness sections (approximately 1 by 2 cm) were obtained from the center of the infarct, from the muscular borderzone between infarct and muscular myocardium, and from the posterior wall of the LV between the first and second circumflex marginal coronary arteries (remote myocardium). Samples were immediately frozen in liquid nitrogen and stored at –80°C in a monitored freezer. Smaller samples were placed in OCT embedding compound and frozen in liquid nitrogen. The remaining heart was labeled and fixed in 5% buffered formalin.
Preparation of histologic material
Fixed tissue from the infarct, from remote myocardium, and from the muscular borderzone, approximately 5 mm from the edge of the infarct and from the edge of the mesh in group MI, was embedded in paraffin, sectioned at 3 to 4 µm, and stained with either hematoxylin or Masson’s trichrome.
Hydroxyproline quantification
Full-thickness samples from remote, muscular borderzone myocardium and infarcted myocardium were assayed for hydroxyproline to determine collagen content [18]. The modified technique used the standard addition method.
Collagen studies
Collagen types I and III were measured in 5-µm paraffin sections containing both muscular borderzone and infarct taken from three different sheep in groups I and MI (n = 6 sheep) using rabbit anticollagen I and III antibodies (Research Diagnostics Inc, Flanders, NJ) as previously described [19]. For each group, a total of 14 slides were analyzed. Low-power images of stained slides for each collagen type in the infarct and muscular borderzone were obtained using a steromicroscope (Leica DMLB, Wetzlar, Germany) at 10x magnification and digitized with pixel spacing corresponding to 150 pixels/mm, using a polaroid digital camera (DMC LE; Polaroid Corp, Cambridge MA). Sample fields were 1,900,000 pixels. Blue component was selected after red-green-blue image decomposition. The gray-scale mean optical density of remote myocardium was measured in each slide and a histogram was generated (Image J software; National Institutes of Health, Bethesda, MD) [19].
Positive collagen staining was defined as integrated optical density values in excess of the mean background value ± 2 SD [20]. Collagen-stained areas were segmented on each image by interactive thresholding above the 2 SD positive limit. A collagen index for collagen type I and collagen type III [21] was calculated by measuring the number of collagen-staining pixels for each field and dividing by the total number of pixels. An average of four fields of scar tissue or muscular borderzone was sampled for each slide, for a total of 56 fields of each tissue for each group. A collagen type I/III ratio was calculated for each field.
Matrix metalloproteinase (MMP) assays
Cardiac tissue was ground with mortar and pestle under liquid nitrogen. Powdered tissue (50 g) was suspended in 1 mL phosphate-buffered saline (pH 7.4) containing 100 µg/mL phenylmethylsulfonyl fluoride and 2 µg/mL leupeptin, and incubated at 4°C with continuous agitation for 20 hours to extract a protein fraction enriched with MMP activity [22]. The addition of phenylmethylsulfonyl fluoride and leupeptin to the sample buffer was necessary to rule out nonspecific gelatin lysis due to serine proteases. The sample was then centrifuged at 13,000 g at 4°C for 10 minutes. The resulting supernatant was employed for total protein assay and zymographic analysis. Total protein content in each sample was determined using the bicinchoninic acid protein assay kit (Sigma, St. Louis, MO) [23]. MMP activity was determined using zymography [23]. To accomplish this, gelatin (final concentration 1 mg/mL) was added to a standard 7.5% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Gelatin is a substance that is readily cleaved by cardiac MMPs and is easily incorporated in polyacrylamide gels. Thirty micrograms of nonreduced protein was loaded per lane, and samples were run at 15 mA/gel. After electrophoresis, gels were washed 2 x 15 minutes in 25 nmol/L glycine (pH 8.3), 2.5% Triton X-100 with gentle agitation at 4°C to eliminate SDS from the gels. Once rinsed, the gels were incubated at 37°C for 18 hours in substrate buffer (50 nmol/L Tris-HCl, pH 8.0, 5 mmol/L CaCl2). After incubation, gels were stained in 0.05% Coomassie blue (R-250) for 30 minutes and then destained in acetic acid and methanol. Gels were then dried and scanned using a CCD camera densitometer (imaging densitometer GS 670; Bio-Rad, Hercules, CA).
Statistical analysis
Groups C, MN, and N (n = 4) were procedural controls and excluded from statistical analysis. For each myocardial location, groups I and MI were compared using Student’s unpaired t statistic.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Baseline echocardiograms obtained before ischemia demonstrated normal wall motion of the antero-apical region of the heart in every sheep before snares were drawn up (Fig 2). Postinfarction echocardiograms demonstrated immediate bulging of the anteroapical region that was partially restrained in all mesh-treated animals. At 2 weeks, in these animals, anteroapical bulging had not increased and was qualitatively less than group I sheep, in which the bulging had increased (Fig 2). The echocardiograms, which were not quantitative, confirmed those previously observed [16].
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Cross sections of the muscular borderzone from the sheep with and without mesh are shown in Figure 3. Figure 3A is a section from the muscular borderzone region in a sheep that did not have mesh. Two weeks after infarction, the muscular borderzone contains thickened strands of fibrous tissue between bundles of viable myocytes. Figure 3B is a section from a sheep that had mesh placed before infarction over the area at risk. Infarction occurred 14 days later. Thickened strands of fibrous tissue also interlace bundles of viable myocytes. No inflammatory cells are present in either photomicrograph.
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The results of the hydroxyproline assays are shown in Table 1. The amount of collagen is significantly higher in muscular borderzone and remote myocardium of mesh-treated sheep as compared with sheep with unrestrained infarcts (group I). The collagen content in the infarct does not significantly differ between groups. In both groups, the amount of collagen is significantly less in remote myocardium than in either borderzone myocardium or the infarct, but there is no significant difference between infarct and borderzone within either group. The procedural controls indicate that these observed differences in collagen content are not due to application of the mesh or to the surgery.
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MMP-1 activity is significantly less in both the borderzone and remote myocardium of hearts with restrained infarctions, but there is no difference in infarcted regions (Table 2).
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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, a protein necessary for myocardial hypertrophy, and phospholipase C activity disproportionately increased in rat myocardium adjacent to an infarction as compared with more remote myocardium [11]. In unpublished studies, Jackson and colleagues found that perfused myocardium adjacent to an expanding infarction becomes progressively hypocontractile and that the amount of this myocardium extends by recruiting adjacent, normally contractile myocardium into a zone of hypocontracting myocardium contiguous with the infarct. Therefore, in this article, "borderzone myocardium," which is perfused and biochemically and functionally distinct from more remote myocardium, is defined by both location and function and not by location alone. The present study shows that restraining infarct expansion reduces metalloproteinase activity and increases collagen content in both borderzone and remote myocardium. Although changes in regional wall strain were not quantitated in this study, the echocardiograms in both groups did not differ qualitatively from those reported previously [16]. In that study, mesh-restrained infarcts attenuated ventricular dilatation and preserved LV geometry and resting function as compared with sheep without mesh [16].
This study establishes a relationship between infarct expansion (strain) and biochemical processes that effect postinfarction LV remodeling of the cardiac extracellular matrix. The mesh does not change collagen content and metalloproteinase activity in the infarct; the most important changes occur in viable borderzone and remote myocardium. The net result is to increase the amount of fibrous tissue in remaining contracting tissue and to attenuate ventricular dilatation and wall stresses. Whether net fibrosis in borderzone and remote myocardium increases, decreases, or remains the same beyond 2 weeks is not known; these preliminary studies only provide one snapshot of an ongoing, complex cellular and biochemical process. The importance of the study is the connection between mechanical restraint (strain) of infarcted myocardium and perturbation of the cellular and biochemical responses manifest as altered matrix deposition.
By LaPlace’s law, acute myocardial infarction immediately increases regional end systolic wall stress in the infarct by decreasing systolic wall thickening [24]. As the infarct bulges and thins, the increase in radius of curvature and decrease in wall thickness further increase wall stress in the infarct and stretch neighboring, perfused, borderzone myocardium [4, 7, 25]. The bulging infarct is akinetic or dyskinetic. The stretched, thinned, borderzone myocardium is hypokinetic and wall stress is increased [4, 25]. Prior application of mesh to just the myocardium at risk tightly binds the mesh to the epicardium and greatly stiffens and slightly thickens the subsequent infarct [16]. As demonstrated, infarct expansion and ventricular deformation and dilatation are attenuated, and by LaPlace’s law, regional wall stresses in infarct and borderzone myocardium must be less than those in corresponding locations in infarcted sheep without mesh.
After infarction, the balance between collagen degradation and collagen synthesis determines collagen content of the extracellular matrix as the heart remodels. For the heart to dilate, the collagen network must expand, which it does by simultaneously degrading and synthesizing collagen. In hearts with expanding infarcts, MMP-1 and MMP-2 activity and, paradoxically, hydroxyproline content increase in borderzone myocardium as compared with remote myocardium, but the amount of collagen in viable and infarcted myocardium is not sufficient to arrest either infarct expansion or LV dilatation. If the infarct expansion is arrested, hydroxyproline content in borderzone myocardium is significantly greater, and MMP-1 and MMP-2 are significantly or nearly significantly less than similarly located myocardium in group I sheep. From our previous study, we know that the mesh arrests infarct expansion, stabilizes LV dilatation, and preserves LV geometry [16]. If confirmed by more extensive studies, the increased collagen in borderzone and remote myocardium may reduce diastolic compliance, but also may be the mechanism for stabilizing LV size and shape and producing a compensated ventricle.
Increased angiotensin II and TGFß-1 activity, which were not measured, may explain the moderate increase in collagen content in borderzone myocardium of animals with expanding infarctions despite the increase in MMP-1 and MMP-2 activity. The role of regional wall stresses in this process is strongly inferred by this study. Increased regional wall stresses probably initiate this process, but once initiated, may not control the final amount of collagen produced until regional stresses are everywhere equalized by stiffening and thickening viable myocardium until dilatation (strain) has stopped and the ventricle becomes compensated. If infarct expansion is restrained, wall strains and stresses in borderzone myocardium are reduced, and this in turn may permit more collagen to accumulate, stiffen the borderzone, and further reduce borderzone wall strain and stress. This process may arrest the transformation, as shown by Jackson and colleagues (in unpublished studies), of remote myocardium into "remodeling myocardium." A study that carefully correlates regional wall strains and stresses with regional collagen accumulation over the remodeling period is needed to prove this hypothesis. Meanwhile, this preliminary study supports the notion that there is a strong correlation between regional wall strain and stress and MMP-1 and MMP-2 activity and collagen accumulation.
Adverse postinfarction LV remodeling of moderate or large transmural infarctions produces progressive LV dilatation, decreasing ejection fractions, eventual heart failure, and shortened survival [26–28]. Cardiac size and ejection fraction, initially augmented by neurohumoral responses and the Frank-Starling mechanism, are better in the first few weeks after infarction than after weeks and months of remodeling [2, 26–28]. Increasing evidence indicates that "remodeling myocardium" defined as hypocontractile, fully perfused myocardium appears after infarction in the borderzone of regional infarctions (7, 29, Jackson et al, submitted for publication). This myocardium is differentiated from stunned and hibernating myocardium and may represent a myocyte response to increased regional wall strain and stress. An attractive hypothesis is that increased regional wall strain and stress may initiate apoptosis; if so, this mechanism is sufficient to explain the gradual dilatation and loss of myocardial contractility that over time leads to heart failure and death in some patients despite no further reduction in coronary blood supply.
Large animal models of an expanding and restrained anteroapical infarction offer an opportunity to investigate and eventually understand the relationships between regional wall strains and stresses and the biochemistry of postinfarction left ventricular remodeling. This understanding introduces the possibility of interventions [16, 19, 30] to control the postinfarction remodeling process so that progressive conversion of normal or even hypercontracting myocardium into remodeling myocardium is arrested. By arresting postinfarction remodeling (ie, cardiac matrix remodeling), a compensated ventricle may be produced that can confer more longevity. The best way or ways to intervene remain to be determined, but any successful intervention offers a more attractive alternative to current therapy for heart failure or rescue operations after remodeling occurs.
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Acknowledgments |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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The editor thanks Dr Thomas B. Ferguson for managing the blinded peer review.
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References |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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and PLC-ß in scar and border tissue in heart failure due to myocardial infarction. Circulation 1998;97:892-899.1-adrenergic stimulation. J Biol Chem 1994;269:13490-13496.This article has been cited by other articles:
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 F. G. Spinale Myocardial Matrix Remodeling and the Matrix Metalloproteinases: Influence on Cardiac Form and Function Physiol Rev, October 1, 2007; 87(4): 1285 - 1342. [Abstract] [Full Text] [PDF]
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