Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction (original) (raw)

Animals and surgery. We used the progeny of heterozygous breeding pairs of mice with targeted disruption of MMP-9, as described by Vu et al. (11). MMP-9–deficient mice have delayed long-bone growth and development due to delayed angiogenesis and ossification; however, by adulthood, these changes result in only a 10% shortening in the long bones. Animals with an FVB background were backcrossed; our studies used the homozygous MMP-9-deficient and sibling WT offspring of generation six or higher. Offspring were ear-tagged and coded, with tail DNA samples harvested for genotyping using PCR. For MMP-9, we used a sense oligonucleotide primer (5′-GCA TAC TTG TAC CGC TAT GG -3′) and an antisense primer (5′-TAA CCG GAG GTC CAA ACT GG-3′). For the neomycin cassette, we also used a sense oligonucleotide primer (5′-GAA GGG ACT GGC TGC TAT TG-3′) and an antisense primer (5′-AAT ATC ACG GGT AGC CAA CG-3′). Again, all procedures were performed without knowledge of genotype.

Males with deletion of MMP-9 and WT males, ranging in age from 8 weeks to 10 weeks, and in weight from 25 grams to 30 grams, underwent coronary artery ligation for the production of MI. Surgical procedures have been described in detail elsewhere (9, 12). Briefly, after anesthesia with pentobarbital (25–30 μg/g intraperitoneally) and intubation with a polyethylene tube (size 60), animals were ventilated with a volume-cycled rodent respirator (Harvard Apparatus Co., South Natick, Massachusetts, USA) with a 2–3 mL/cycle at a respiratory rate of 115 cycles/min. After thoracotomy, ligation of the left coronary artery was performed with a 7-0 silk suture, 3–4 mm from the tip of the left auricle. Pallor, regional hypokinesia, and enlargement of the left ventricle confirmed the presence of an infarction. The chest wall was closed with a continuous 6-0 prolene suture, and the skin was closed with 4-0 polyester sutures. The animals were then extubated and allowed to recover from surgery under a heating lamp for 1 hour. Antibiotic prophylaxis was not given, but no apparent infection developed in any animal during the course of the study or at the time of autopsy. All mice were housed under identical conditions, and were given food and water ad libitum. The Harvard Medical School Standing Committee on Animal Research approved the study protocol.

Echocardiographic imaging. Echocardiographic studies were performed under light anesthesia with spontaneous respiration using intraperitoneal 2,2,2-tribromoethanol (Avertin; Aldrich, Milwaukee, Wisconsin, USA) in a 2.5% wt/vol solution (8 μL/g of mouse) as previously described (9). An ultrasonographer experienced in rodent imaging performed the echocardiographic studies, using commercially available equipment (Sonos 5500; Hewlett Packard Medical Products, Andover, Massachusetts, USA) and an 8- to 12-MHz transducer. Dynamically focused annular array and fusion frequency technologies were used, allowing ultrasound frequencies of up to 18 MHz. A standoff was used, depth was set at 4 cm, and the zoom mode was used to optimize resolution and penetration. Frame acquisition rates using the loop mode reached up to 120 MHz, allowing excellent temporal resolution. Identical zoom size and depth settings were used between examinations to facilitate calibration for offline analysis.

M-mode images were obtained at a sweep speed of 100 mm/s. Two-dimensional image-guided M-mode recordings were made at the midpapillary level. Apical image-guided M-mode recordings were obtained at the greatest area/diameter in the lower third of the left ventricle. The images were recorded on S-VHS tapes for offline analysis. Echocardiographic studies were performed at baseline (18–24 hours after the surgical procedure), at 4 days, at 8 days, and at 15 days after the surgery, immediately before the sacrifice of the animals. We allowed an 18- to 24-hour recovery period to minimize the residual negative inotropic and chronotropic effects that may be encountered after general anesthesia (furthermore, quality of echocardiographic imaging is not optimal immediately after a thoracotomy). Echocardiographic acquisition and analysis was performed by a single echocardiographer blinded to mouse genotype. End-diastolic and end-systolic diameters were measured, and fractional shortening was calculated. For each measurement, three consecutive cardiac cycles were measured and averaged.

Tissue collection. Mice were sacrificed after the last echocardiographic study, on day 15. Hearts were excised, and the right and left ventricles were separated. A transverse section (5–7 mm) was obtained at the midventricular level. Tissue sections were embedded in OCT compound (Sakura, Torrance, California, USA) and frozen in 2-methylbutane prechilled with liquid nitrogen. Tissue blocks were stored at –80°C until sectioning. In a subset of eight animals (4 MMP-9 KO and 4 WT), the left ventricle was divided between the apical and papillary regions and frozen in liquid nitrogen for immunoblot analysis.

Northern and Western blots. Total cellular RNA was isolated by a modification of the acid guanidinium thiocyanate and phenol/chloroform extraction method, using TRIzol LS Reagent (Life Technologies Inc., Gaithersburg, Maryland, USA) according to the manufacturer’s protocol. RNA was size fractionated on 1.0% agarose, transferred to a nylon membrane, and cross-linked with ultraviolet radiation. A murine collagen type Iα cDNA was radiolabeled using a random primer labeling kit (Life Technologies Inc.). Normalization of RNA for equal loading was carried out by rehybridizing with a murine GAPDH cDNA probe. Autoradiographs were scanned using version 1.62 of the Image program from the National Institutes of Health.

For Western analysis, frozen tissue was homogenized in buffer containing 1% nonylphenol ethoxylate, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors in PBS. Equal amounts of the denatured protein from the supernatant were loaded per lane for SDS-PAGE. After running the gel, proteins were transferred to a PVDF membrane. After blocking with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20, membranes were incubated with antibody against MMP-2, MMP-3, TIMP-1 (Oncogene; Cambridge, Massachusetts, USA), or MMP-13 (Chemicon; Temecula, California, USA) for 60 minutes, followed by washing and incubation with a matching secondary antibody (Bio-Rad Laboratories Inc., Hercules, California, USA). Membranes were then incubated with a chemiluminescent agent (Renaissance; DuPont NEN, Boston, Massachusetts, USA) and autoradiographed.

Immunocytochemistry. Fresh-frozen sections (6 μm) were fixed with fresh 4% paraformaldehyde for 10 minutes and rinsed in PBS. Tissue sections were preincubated with 0.3% hydrogen peroxide in PBS to inhibit endogenous peroxidase activity, and then incubated with primary antibodies diluted in PBS, supplemented with 4% of the species-respective normal serum for 30 minutes at room temperature. After washing with PBS, species-appropriate biotinylated secondary antibodies were applied, followed by avidin-peroxidase complexes (Vector Laboratories, Burlingame, California, USA). The reaction was visualized with 3-amino-9-ethyl carbazole as substrate (AEC; Sigma Chemical Co., St. Louis, Missouri, USA), and counterstained with Gill’s hematoxylin solution. The following primary antibodies were used: anti-mouse neutrophils (Cedarlane Laboratories, Hornby, Ontario, Canada) and antibodies against human α-smooth muscle actin (DAKO Corp., Carpinteria, California, USA), mouse macrophage Mac-3, and human endothelial cell CD31, CD4, and CD8 (PharMingen, San Diego, California, USA). For each section, cells positive for CD4, CD8, and CD31 were counted in the infarcted area of mouse heart in at least 7–10 random high-power fields. To analyze cells that stained positive for Mac3 and α-smooth muscle actin, a computer-based image analysis was used (see below). Cell counting and quantitative morphometry were done by two observers who were unaware of genotype.

Collagen content. Picrosirius red polarization microscopy was performed for detection of interstitial collagen according to Junqueira’s method with our modifications (13, 14). Birefringence under illumination with polarized light identifies collagen, including types I and III (13). Sections from a subset of 15 mice (7 MMP-9 KO and 8 WT) that survived the 15-day protocol were chosen; MI sizes were qualitatively similar by Masson’s trichrome and picrosirius red staining. Fresh-frozen sections (6 μm) were fixed in 10% neutral buffered formalin, rinsed with distilled water, and incubated with 0.1% picrosirius red F3BA (Polysciences Inc., Warren, Pennsylvania, USA) in saturated picric acid for 90 minutes. Sections were rinsed twice with 0.01 N HCl for 1 minute, and then immersed in distilled water. After dehydration with 70% ethanol for 30 seconds, sections were visualized under polarized light and photographed with the same exposure time for each section as previously described (14). Two predefined regions (infarcted and noninfarcted) were chosen and photographed at low power (×40). A microplate reader–based quantitation of collagens was performed as described by others (15, 16). Briefly, collagen standards and lysates were plated on microtiter wells in triplicate, and dried onto the plates. The wells were stained with 0.1% Sirius red F3BA in saturated picric acid for 1 hour, and washed with 10 mM HCl and 0.1 mM NaOH. Absorbance was read at 540 nm. Previously, this test has been shown to be comparable to the colorimetric hydroxyproline assay (16).

Quantitative analysis for histology. Analysis of immunohistochemistry for macrophages, neutrophils, α-actin, and picrosirius red staining was performed with a computer-based quantitative 24-bit (16.2 million unique combinations) color image analysis system (Optimas 5.2; Optimas Corp., Bothell, Washington, USA) (17). Micrographs were scanned into a 1,000 × 1,000 image buffer. A color threshold mask for immunostaining was defined to detect the red color by sampling, and the same threshold was applied to all specimens. The percentage of the total area with positive color for each section was recorded. For picrosirius red staining, a negative background (black) was chosen for thresholding, and the positive area was calculated by subtraction. Collagen volume fraction was calculated as the sum of stained tissue divided by the sum of muscle area and connective tissue in the visual field of the section. This approach predicts the proportion of myocardium occupied by fibrillar collagen, and correlates closely with the hydroxyproline concentration of the tissue (18).

Statistical analysis. In a previous study, we found that some mice that underwent surgical ligation of the left coronary artery, despite paleness and bulging of the left ventricle at the time of surgery, did not develop either wall-motion abnormalities that were visible by echocardiography or histologic evidence of infarction. Not surprisingly, these animals do not demonstrate a progressive increase in LV dimensions (9). Based on these observations, our study was designed to include in the statistical analysis only the animals that revealed a wall-motion abnormality during the baseline study, within 24 hours of the surgical procedure. This determination was made at the time of the initial echocardiogram without knowledge of mouse genotype.

The mortality data (deaths occurring soon after surgery and during the 15-day protocol, including causes of death) were analyzed with the Pearson χ2 test. Two-way repeated-measures mixed-model ANOVA was used to test differences between groups regarding the evolution across time of ventricular dilation. When a possible interaction was found (P < 0.25), slice effects (also known as simple effects) were analyzed, i.e., time effect was analyzed for each genotype group, and differences between the groups were analyzed for each level of time. To keep the overall statistical significance at 5%, the levels of significance for testing simple time effects were adjusted according to the Bonferroni rule. For comparisons over time after MI, repeated measures ANOVA was used. To account for the three within-group comparisons, P < 0.0167 was considered significant. For between-group comparisons at each timepoint, a less conservative level was used; P < 0.05 was considered significant. All the analyses were performed with the MIXED procedure of SAS 6.12 (SAS Institute Inc., Cary, North Carolina, USA) to handle missing data. All values are expressed as mean ± SD.