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1Cardiovascular Research Institute, Xinjiang Medical University, Xinjiang, China; and 2Baker Heart Research Institute, Melbourne, Australia
Submitted 17 July 2007 ; accepted in final form 2 February 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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infarct expansion; collagen; matrix metalloproteinase; inflammatory cytokines
Increased activity of matrix metalloproteinases (MMPs) following acute MI has been demonstrated in various species, including humans (15). Activated MMPs degrade the collagen network and subsequently result in the loss of structural support, distortion of tissue architecture, wall thinning, and infarct expansion (28). Experimental studies using genetically modified mouse models have shown that disruption of MMP-2 or MMP-9 attenuates post-MI rupture (10, 17), indicating a key role of MMPs and collagenolysis in the pathogenesis of cardiac rupture. Recently, Lindsey et al. (16) reported elevated levels of various MMPs by immunoblotting in senescent rat hearts, implying a possible mechanism for age-dependent extracellular matrix remodeling. Furthermore, clinical studies have shown that inflammatory markers such as interleukin (IL)-6 and tumor necrosis factor-
(TNF-) are not only predictors for coronary heart disease in the elderly (2) but also are closely associated with post-MI cardiac remodeling (18), suggesting an important role of inflammation in cardiovascular disease. It remains to be studied whether there is an enhanced inflammatory response and MMP activity in aged vs. young hearts following acute MI.
We hypothesize that an age-related difference in regional inflammation and MMP activity is responsible for a higher incidence of cardiac rupture in elder subjects following acute MI. The mouse MI model has been demonstrated, like in humans, to develop cardiac rupture (9), representing a unique opportunity for testing such a clinically relevant issue. Here we investigated 1) whether there is a difference between old and young mice in the occurrence of LV rupture and early LV remodeling following experimentally induced MI and 2) possible mechanisms responsible for the age-related difference in the risk of cardiac rupture.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Male C57Bl/6 mice, 3- and 12-mo of age with body weight of 23
31 g, were used. Animals were housed in a facility with a 12:12-h dark-light cycle and free access to standard mouse cow and water. All experimental procedures were approved by a local Animal Ethics Committee in accordance with the National Institutes of Health guidelines.
Mice were anesthetized using a mixture of ketamine, xylazine, and atropine (100, 20, and 1.2 mg/kg ip, respectively) and ventilated with a rodent ventilator (model 683; Harvard Apparatus). Under a dissecting microscope, a left thoracotomy was performed, and the left coronary artery was identified and occluded with a 7–0 silk suture, as described previously (9). Sham operation was performed similarly except for occlusion of the artery. Animals were inspected at least four times daily for 7 days.
Echocardiography and Hemodynamic Assessment
Transthoracic echocardiography was performed using a Hewlett-Packard Sonos 5500 ultrasound machine with a 15-MHz linear-array transducer, as described previously (6). Animals were lightly anesthetized with a half dose of the anesthetic mixture for surgery. The following parameters were measured and calculated: LV internal end-systolic and end-diastolic diameters (LVESd and LVEDd, respectively), external LV diastolic diameter (ExLVDd), posterior wall thickness at systole and diastole (PWs and PWd, respectively), and fractional shortening {FS% = [(LVEDd – LVESd)/LVEDd] x 100}.
Hemodynamics were also determined in sham-operated and infarcted mice. After anesthesia (as used for echocardiography), a 1.4-Fr Millar catheter was placed in the LV through the right carotid artery. Arterial blood pressure, LV systolic pressure (LVSP), and the maximal rates of rise and fall in LV pressure (dP/dtmax and dP/dtmin, respectively) were recorded and analyzed with PowerLab Chart 4.1.2 software (ADInstruments). Heart rate was derived from pulse signals.
Pathological and Histological Analyses
Autopsy was performed on each animal either found dead within 1 wk or killed at day 7 after surgery, as described previously (9). The presence of a large amount of blood clot around the heart in the chest cavity and a perforation in the infarcted wall were taken to indicate rupture death. Animals with a large infarct, severe cardiac dilation, lung congestion, and pleural effusion were considered as suffering from heart failure. Body, heart (HW), and lung (LW) weights were obtained, and the tibial length was measured.
The LV was cut into halves and flattened to expose the endocardial surface. Images were taken using a digital camera and analyzed to quantify the surface areas of infarcted wall and entire LV wall. The infarcted area can be identified by its pale color, intramyocardial hemorrhages, and a reduction of the wall thickness from the noninfarcted myocardium. Infarct size was calculated as a percentage of an infarcted area in the entire LV (9). The extent of infarct expansion was estimated by measuring the largest endocardial circumference and the ratio of the thickness of the thinnest infarcted wall to averaged thickness of the ventricular septum from a single LV section stained with hematoxylin and eosin, as described previously (7).
Quantification of Myocardial Collagen Content, Soluble and Insoluble Collagen, and Collagen Subtypes
Histological measurement. A transverse LV section (5 µm) obtained from the equator of each heart was stained with 0.1% Picrosirius red. Interstitial collagen content in the LV was determined under x20 lens using the Imagetool 3.0 program (Health Science Center, University of Texas). Ten fields were chosen randomly from each specific region, including infarct and noninfarct regions and sham-operated heart. Fields containing blood vessels, epicardium, or endocardium were avoided. Collagen content was calculated and averaged as a percentage of the positive stained area in total selected fields.
Analysis of collagen cross-linking. Myocardial collagen can be fractioned into pepsin-soluble and -insoluble collagens; collagen cross-linking was determined from the ratio of insoluble to soluble collagen (12) by adaptation of previously described methods (20, 24) with minor modifications. Normal LVs were lyophilized, weighed, and pulverized. Samples were digested with 1 mg/ml pepsin in 0.5 M acetic acid for 24 h at 4°C and then centrifuged at 15,000 g for 45 min at 4°C to separate supernatants (soluble collagen) and pellets (insoluble collagen). Supernatants and pellets were hydrolyzed in 6 M HCl at 110°C for 18–20 h. Aliquots of the filtered hydrolysates were lyophilized and resuspended in H2O and determined by spectrophotometry at 560 nm following reaction with chloramine T and Ehrlich's reagents.
Immunohistochemical staining for type I and III collagen. LV sections (2 µm) from paraffin-embedded tissues were incubated with 0.3% hydrogen peroxide, 5% BSA for 15 min, and then anti-mouse monoclonal antibodies against type I and type III collagen (Boster) at a 1:100 dilution and 4°C overnight. The sections were then incubated with biotinylated secondary antibody for 20 min at 37°C followed by the streptavidin-peroxidase complexes tertiary system (Boster) according to the manufacturer's instructions. After being incubated with diaminobenzidine tetrahydrochloride, sections were counterstained using hematoxylin, dehydrated through gradient alcohols, and mounted. Images were obtained using a video camera under a microscope with a x40 lens, and 10–20 fields/section were chosen randomly from infarct and noninfarct regions or sham-operated heart. The content of type I or III collagen was measured and averaged as a percentage of the positive stained area in total selected fields.
Determination of Inflammatory Response and MMPs
Nuclear density. Nuclear density was counted from hematoxylin- and eosin-stained sections using the Imagetool 3.0 program. Ten fields at the infarcted border zone or sham-operated heart at day 3 after surgery were randomly selected under a x20 lens.
Identification of neutrophils and macrophages. LV sections (2 µm) were incubated with 0.3% hydrogen peroxide and normal goat serum and then antimouse monoclonal antibody for neutrophil (Serotec) and CD68 antibody (KP1) for macrophages (Abcam, Jingmei Biotech) at a 1:100 dilution and 4°C overnight. The second antibody was applied for 45 min at 37°C and then followed by an established immunohistochemical protocol as mentioned before.
Real-time PCR for inflammatory cytokine expression.
Total RNA was extracted from the infarcted and noninfarcted LVs using TRIzol. After reverse transcription, real-time quantitative PCR was performed in duplicate using a SYBR green kit (Invitrogen) and an ABI Prism 7700 system (Applied Biosystems). Primers were designed from a known mouse sequence or from the literature. Each reaction was designed to quantitate expression of TNF , IL-1β, and IL-6. Results were normalized to reference gene 18S, as previously described (8).
Gelatin zymography. MMP-2 and MMP-9 activities in the infarcted and noninfarcted LVs were measured separately by gelatin zymography as described previously (25, 27) and with minor modifications. LVs were homogenized with a buffer containing 10 mM cacodylic acid, 0.15 mM NaCl, 20 mM ZnCl, 1.5 mM NaN3, and 0.01% Triton X-100 (pH 5.0). Homogenates were centrifuged at 14,000 rpm at 4°C for 10 min. Equal loading was ensured by a protein concentration reading. Protein (50 µg) was loaded on a 7.5% acrylamide gel containing 0.5 mg/ml gelatin and electrophoresed at 4°C. The gel was washed in 2.5% Triton X-100, incubated in a developing buffer (50 mM Tris, pH 7.6, 5 mM CaCl2, 1 µM ZnCl2, and 0.02% Brij-35) for 16 h at 37°C, stained with 0.5% Coomassie blue, and destained in 10% acetic acid and 30% methanol for 120 min to visualize sharp bands. The gel was scanned, and densitometry levels were quantified using image analysis software (Quantity One; Bio-Rad).
Experiment Protocols
To study the age-related differences in the incidence of LV free-wall rupture and early remodeling during the acute phase of MI, 140 mice (70 each for 3- and 12-mo-old groups) were subjected to the left coronary artery ligation, and 20 mice (10 for each group) underwent sham operation. Echocardiography was performed at day 7 after surgery and then followed by catheterization for hemodynamic measurement. At the end, animals were killed, and hearts were collected for histological and molecular studies.
To study the age-related differences in myocardial collagen content, collagen subtypes and cross-linking, and MMP activities, another cohort of animals was subjected to left coronary artery ligation and sham operation. At day 3 after surgery, mice were killed, and hearts were collected for histological measurement, immunohistochemical study, biochemical assay, real-time PCR, and zymography.
Statistics
Values are expressed as means ± SE or as a percentage. Parametric data were analyzed by one-way or two-way ANOVA for overall significance followed by the Newman-Keul's multiple comparison or unpaired t-test. 
2 or Fisher's exact test was used to compare percentage. P < 0.05 was considered statistically significant.
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RESULTS |
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Operative mortality within 24 h after surgery was similar between the two groups (14% for young vs. 16% for old mice). All 20 sham-operated mice and 119 mice with MI (3 mo old, n = 60, 12 mo old, n = 59) survived 24 h after surgery. During 1 to 7 days after MI, 45.8% of old and 25% of young mice died of either LV rupture or acute heart failure (Fig. 1A). A significantly higher incidence of LV rupture was observed in the old than in the young groups (Fig. 1A) despite a similar incidence of heart failure death and a comparable infarct size between the two groups [43.8 ± 1.3 vs. 41.2 ± 1.6%, P = not significant (NS)]. The time window of rupture was 3–6 days after MI in both age groups (Fig. 1B). Incidence of plural effusion (24 vs. 12%), lung congestion (42 vs. 37%), and atrial thrombosis (29 vs. 14%) was not significantly different between old and young mice.
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The echocardiographic data from surviving mice at day 7 after MI are shown in Table 1. LV dimension and LV contractile function (FS) at baseline were similar between young and old groups. MI for 7 days markedly increased LV dimension as shown from LVEDd, LVESd, and ExLVDd in both age groups, with a more significant chamber enlargement in old vs. young mice (all P < 0.05). FS was reduced in young (60%) and old mice (76%, P < 0.05 vs. young mice) compared with their respective sham values. A significant LV wall thinning relative to the sham values indicated by PWs and PWd at day 7 after MI was observed, which was more pronounced in old than in young groups during both systole and diastole (P < 0.05). Moreover, although there were no difference in HW and LW when normalized by tibial length (TL) between the two groups with sham operation (HW/TL, 4.2 ± 0.1 vs. 4.1 ± 0.1 mg/mm, LW/TL, 5.5 ± 0.2 vs. 5.7 ± 0.1 mg/mm), old mice had greater HW and LW following MI (HW/TL, 8.3 ± 0.2 vs. 6.9 ± 0.3 mg/mm; LW/TL, 11.7 ± 0.7 vs. 9.6 ± 0.9 mg/mm, both P < 0.05), indicating a significant hypertrophic response and lung congestion in old mice.
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Infarct Expansion
For mice that either died of LV rupture or survived to day 7 after MI, infarct size was comparable between the two age groups. However, infarct size measured as a percentage of surface area of the LV was greater in mice dying of rupture than those survived to day 7, especially in old mice (Table 2). Wall thickness ratio and endocardial circumference were not different between the two groups with sham operation. At day 7, wall thickness ratio in infarcted hearts was reduced by 50 and 66%, and endocardial circumference was increased by 30 and 50%, respectively, in young and old groups (Table 2). Compared with mice that survived to day 7, ruptured hearts had a smaller wall thickness ratio and further enlarged endocardial circumference, and these changes were more evident in old mice, indicating a more severe infarct expansion.
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Figure 2A shows the changes of LV collagen content in young and old mice measured histologically from Picrosirius red-stained sections at 3 and 7 days after surgery. In sham-operated mice, total collagen content was 58% higher in old than in young mice (P < 0.05). Following MI, collagen content in the noninfarct region was increased by 23-fold in both age groups. In the infarct region, a 715-fold and 510-fold increase of collagen content were observed in young and old groups from 3 to 7 days after MI vs. respective sham values. There was no significant difference in collagen content between the two groups at all time points after MI.
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3-fold increase in both type I and III collagen was observed in the two groups over the respective sham values (Fig. 2, B and C). In the infarct region, both type I and III collagen was significantly increased in old mice at day 3, whereas only a minor change was seen in young mice compared with their sham values. At day 7 after MI, a 56-fold increase in both type I and III collagen content was detected in the two groups (Fig. 2, B and C). The content of type I collagen in both infarct and noninfarct regions at day 7 was significantly higher in old than in young mice. The ratio of collagen type I/III was comparable between the two groups at all time points (data not shown). Biochemical assay from normal LVs showed that insoluble collagen was 60% higher while soluble collagen was 22% lower in old vs. young mice (both P < 0.05), resulting in the ratio of insoluble/soluble collagen being significantly higher in old than in young mice (Fig. 2D).
Inflammatory Response
Cell density was measured by nuclear counting in sham-operated hearts or in the infarcted border zone at 3 days after MI. In sham control hearts, there was no difference in cell density between young and old groups (1,431 ± 70 vs. 1,516 ± 80 nuclei/mm2). A markedly increased cell density was detected after MI, which was significantly greater in old than in young mice (5,804 ± 173 vs. 4,045 ± 298 nuclei/mm2, P < 0.001).
Immunohistochemical study showed that densities of both neutrophils and macrophages in the border zone of infarcted hearts were 20 and 30% higher in old mice vs. young mice at day 3 after MI (both P < 0.05; Fig. 3A). Compared with neutrophils, the number of macrophages was significantly higher in both age groups.
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, IL-1β, and IL-6 was measured in both infarct and noninfarct myocardium from young and old mice at 3 days after MI. The levels of TNF-, IL-1β, and IL-6 in the noninfarct myocardium tended to be higher in old than in young mice (P = NS; Fig. 3B). However, cytokines in the infarcted tissues increased markedly in both age groups with more profound increment seen in old mice (Fig. 3), in keeping with a denser inflammatory cell infiltration (Fig. 3B). Activation of MMP-2 and -9
MMP-2 and MMP-9 activities were determined by zymography from infarct and noninfarct myocardium collected at 3 and 7 days after surgery. In sham-operated hearts, levels of MMP-2 and -9 were not significantly different between the two groups (Fig. 4). Following MI, MMP-2 activity in both infarct and noninfarct myocardium increased in a time-dependent manner and was comparable between young and old groups (Fig. 4). Relative to the sham values, MMP-9 activity in the noninfarct myocardium was increased similarly at day 3 and returned to baseline levels at day 7 in both age groups. In contrast, MMP-9 activity in the infarct myocardium increased by 14-fold in old and 11-fold in young mice at day 3 (P < 0.05) and was comparable between the two groups at day 7 (Fig. 4).
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DISCUSSION |
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Compared with young mice, old mice had a higher incidence of post-MI rupture and a more severe infarct expansion, evidenced by a significant reduction in LV wall thickness ratio and a greater enlargement of endocardial circumference. Severe infarct expansion in aged mouse hearts is the most likely reason for the larger infarct size measured in old compared with young mice that died of rupture. Histological studies have revealed that infarct expansion is due to a slippage between muscle bundles and is associated with rupture of the infarcted wall (30). Post-MI LV remodeling is a progressive process involving chamber dilation, infarcted wall thinning, and compensatory hypertrophy in the noninfarcted myocardium (1). Echocardiography at day 7 after MI showed that LV dimension was enlarged, whereas wall thickness and contractile function were reduced in both young and old groups. The absolute values of LVEDd, LVESd, and ExLVDd were significantly increased, and LV wall thickness and FS were markedly decreased in old vs. young mice, indicating a more profound LV remodeling and dysfunction following MI. In fact, the extent of LV remodeling measured at day 7 in old infarcted mice was underestimated considering that the 40% of old mice that died of rupture had the most severe infarct expansion. Clinical studies have also shown that elderly patients (>75 yr) had a worse LV dilation and dysfunction compared with a younger age group (<75 yr) at 1 wk after acute MI (14).
LV function was markedly impaired in infarcted mice, evidenced by depressed hemodynamic indexes. Surprisingly, old mice with MI had higher systolic and diastolic blood pressure than young mice, although there was no such difference between the two age groups with sham operation. Clinical studies have reported that systolic hypertension after acute MI is associated with increased risk of cardiac rupture (4). Higher blood pressure represents an elevated afterload leading to an increased ventricular wall stress (23). The combination of morphological destruction of the infarcted wall and increased wall stress due to higher blood pressure and LVSP would make the LV more vulnerable to rupture. However, it remains to be determined whether a higher blood pressure in old mice is attributable to an enhanced sympathetic tone or activation of the ANG II system after MI.
The total collagen content in many tissue has been found to be augmented with aging (3). We observed a higher total and type III collagen content in old than in young mice with sham operation. Further biochemical assay revealed increased insoluble and decreased soluble collagen fractions in old mice, indicating an increase in collagen cross-linking with aging. Following MI, there was a time-dependent and comparable increase in total collagen in the noninfarct and more dramatically in the infarct regions. The increase in collagen type I and III at the infarcted tissue was faster and more pronounced in old than in young mice, together with markedly elevated MMP-9 in old infarcted mice, suggesting a faster collagen turnover in aged hearts. We previously reported a high collagen content in a transgenic strain overexpressing β2-adrenergic receptor in the heart that was associated with a lower incidence of rupture (7). A recent study reported by Ichihara et al. (11) showed that deletion of ANG II type 2 receptor inhibits collagen deposition and causes post-MI cardiac rupture in mice. However, a paradoxic observation in the present study is that a higher collagen content and cross-linking in the aged heart was accompanied with a higher risk of rupture. The possible explanation is that the extent of collagen degradation following MI is far greater in old than young hearts; therefore, a preexisting higher content of collagen is not enough to prevent rupture in aged hearts.
Acute MI results in irreversible cell death and tissue necrosis, which trigger the release of proinflammatory cytokines and promote inflammatory cell infiltration in the infarcted tissue. Following the initiation of the inflammatory response, a rapid activation of MMPs occurs (18). In the present study, a greater macrophages and neutrophils infiltration was in accordance with a multifold increase in the expression of inflammatory cytokines in old mice following MI, indicating a higher degree of inflammatory response in aged hearts. Furthermore, a stepwise increase of MMP activity was detected during week 1 after MI. In particular, the level of MMP-9 increased sharply and reached a peak level at day 3 in both age groups. A significant higher MMP-9 level at day 3 in old than in young mice was accompanied with a higher incidence of rupture, suggesting more severe collagen matrix degradation in aged hearts. The association between inflammatory cell accumulation and MMP-9 activation in the aged heart indicates that inflammatory cells, particularly macrophages, are the major source of MMP-9 in the infarcted myocardium. Importantly, recent clinical studies have documented that 1) higher plasma levels of C-reactive protein and IL-6 after acute MI were associated with a worsened LV remodeling and 2) similar temporal changes in the plasma level of MMP-2 and MMP-9 after acute MI (26, 29), as seen in mouse studies (27), and an elevated MMP-9 activity were closely correlated with the extent of LV remodeling (26, 29). Taken together, our data suggest that an enhanced inflammatory response and MMP activation, especially MMP-9, are important mechanisms responsible for a higher incidence of rupture and more severe LV remodeling in aged mice.
In conclusion, our study demonstrates a higher risk of cardiac rupture, more severe infarct expansion, and LV remodeling and dysfunction in old than in young mice after MI despite a higher collagen content and increased cross-linking in aged hearts. The underlying mechanism is a greater inflammatory cell infiltration in aged hearts that is associated with significantly enhanced expression of proinflammatory cytokines and elevated MMP-9 activity, implying a greater extent of matrix collagen degradation. In addition, a higher blood pressure, which may increase peripheral resistance and LV wall stress, may also contribute to a higher risk of rupture and more profound acute cardiac remodeling in aged hearts following MI.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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P < 0.05.
) and the no. of positively stained cells counted (x40 objective) from 3- and 12-mo-old mice at day 3 after MI (n = 10/group). Images from sham-operated hearts were stained for macrophages as a reference. P < 0.05 vs. 3-mo-old group (*) and vs. neutrophils (#). B: mRNA expression of tumor necrosis factor (TNF)-