Mitigation of Fibrosis after Myocardial Infarction in Rats by Using a Porcine Cholecyst Extracellular Matrix

Abstract

Fibrosis that occurs after nonfatal myocardial infarction (MI) is an irreversible reparative cardiac tissue remodeling process characterized by progressive deposition of highly cross-linked type I collagen. No currently available therapeutic strategy prevents or reverses MI-associated fibrotic scarring of myocardium. In this study, we used an epicardial graft prepared of porcine cholecystic extracellular matrix to treat experimental nonfatal MI in rats. Graft-assisted healing was characterized by reduced fibrosis, with scanty deposition of type I collagen. Histologically, the tissue response was associated with a favorable regenerative reaction predominated by CD4-positive helper T lymphocytes, enhanced angiogenesis, and infiltration of proliferating cells. These observations indicate that porcine cholecystic extracellular matrix delayed the fibrotic reaction and support its use as a potential biomaterial for mitigating fibrosis after MI. Delaying the progression of cardiac tissue remodeling may widen the therapeutic window for management of scarring after MI.

Figure 1.

Diagram of rat heart (sternal view) showing the site of left coronary artery ligation made for inducing myocardial infarction.

Figure 2.

Clinical monitoring of myocardial infarction (MI) in rats. (A) Serum biochemical values of rats that underwent ligation of the left anterior descending coronary artery are significantly higher at 24 h after surgery than at baseline (−2 d), indicative of MI. Control, black; C-ECM, red. (B) Representative electrocardiograms of the control and C-ECM-grafted group recorded at baseline (−2 d) and at 1 and 4 wk after MI, indicating functional damage in the heart. (C) Box plot showing the ST elevation, QT interval, RR interval, and QRS amplitude of all rats in both groups, suggesting uniformity in the extent of MI between the 2 experimental groups (control, black; C-ECM, gray).

Figure 3.

Echocardiographic monitoring of myocardial infarction (MI) in rats. (A) Representative echocardiographs of rats with MI. (B) Bar diagram showing the quantitative variation in the end-systolic septal thickness (IVSs), end-diastolic septal thickness (IVSd), and fractional shortening and ejection fraction at 1 and 4 wk, confirming the uniformity in the induction of MI between groups (control, solid bars; C-ECM, hatched bars). Data were analyzed by 2-way ANOVA followed by the Sidak multiple-comparisons test; *P < 0.05.

Figure 4.

Confirmation of myocardial infarction (MI) based on necropsy. Gross photographs of cross sections of heart specimens of control rats (A₁–A₃) with their sketches (B₁–B₃) and rats with C-ECM–grafted hearts (C₁–C₃) with their sketches (D₁–D₃); samples were collected at necropsy, 4 wk after ligation of the left coronary artery. Samples show the extent of thinning and necrosis (appreciated as whitish areas) of the left ventricular free wall as well as differing degrees of concentric hypertrophy among rats. The subscripts 1–3 indicate cross-sections of the heart made at 3 planes posterior to the ligature. (E) The extent of the myocardial infarction was quantified and did not differ between groups.

Figure 5.

Quantification of histology images. Bar graph showing A, the number of myocardiocytes and B, their diameter (Dmaj, major cell diameter and Dmin, minor cell diameter) in the interventricular septum (IVS) counted from H&E images of control animals and cholecystic extracellular matrix (C-ECM)-grafted animals, *P < 0.05 indicated significant difference between the groups. C, Bar graph showing the area of right ventricular (RV), left ventricular free wall (LVFW), infarct, interventricular septum (IVS) and the total area quantified from the Masson’s trichrome stained images showing hypertrophy in the control group (control, solid bars; C-ECM, hatched bars).

Figure 6.

Histomorphologic evaluation of the heart (low magnification). Photomicrographs of representative histology sections stained with (A and B) hematoxylin and eosin (H&E), (C and D) picrosirius red (PSR), Masson trichrome (MT), and (G and H) Herovici stains of samples from the control rats (A, C, E, G) and C-ECM–grafted rats (B, D, F, H) at magnifications of 1.25× (subscript 1) and 4× (subscript 2). The extent of necrosis in these samples was evident at all magnifications, corroborating the observations at gross morphology (refer to Figure 8 for quantitative data). The grafted C-ECM (arrow) appeared as refractile material at the epicardial aspect of the myocardial wall. Note that panels A₂, C₂, E₂, and G₂ are stitched images, for improved illustration.

Figure 7.

Histomorphologic evaluation of the heart (high magnification). Photomicrographs of representative histology sections stained with (A and B) hematoxylin and eosin (H&E), (C and D) picrosirius red (PSR), (E and F) Masson trichrome (MT), and (G and H) Herovici stains of samples from the control rats (A, C, E, and G) and C-ECM-grafted rats (B, D, F, H); magnification, 40×. Myocardial degeneration and necrosis, moderate chronic inflammation, and moderate fibrosis were apparent at higher magnifications. Note that panels A, B, C, D, and F are stitched images, for improved illustration.

Figure 8.

Quantification of infarct area and collagen deposition. Bar diagram representing stereology data A) the proportion of the infarct area, estimated by image analysis from images of tissue sections stained with (A) picrosirius red (PSR) and (B) Masson trichrome (MT). Infarct area was not different between groups, further indicating the uniformity in MI induction across the 2 experimental groups. Bar graphs showing significant (*P < 0.05; unpaired t-test) reduction in the (C) percentage of fibrosis in the presence of the C-ECM graft and in the (D) percentage area occupied by type I collagen and type III collagen, indicating a significant (*P < 0.05; unpaired t-test) decrease in the type I collagen content in the C-ECM group (control, black; C-ECM, gray). (E) Bar graph showing the ratio of type I to III collagen in the control group compared with the C-ECM group.

Figure 9.

Immunohistochemistry for specific proteins. Light micrographs of histology sections collected from (A) control and (B) C-ECM–grafted rats after immunostaining with antibodies against proliferating cell nuclear antigen (PCNA; subscript 1), CD68 (macrophages; 2), CD4 (helper T-lymphocytes; 3), and CD8 (cytotoxic T-lymphocytes; 4). Scale bars, 50 µm. (C) Bar diagrams showing the quantitative information of the positive cells in these 2 groups. *P < 0.05 (unpaired t-test).

Figure 10.

Immunohistochemistry for myofibroblasts. A, Light micrographs of histology sections collected from control and B, cholecystic extracellular matrix (C-ECM)-grafted animals after immunostaining for α-smooth muscle actin (ASMA) positive myofibroblasts, confirming similar fibrogenesis in both the groups as evident from C, the representative graph indicating no difference between the groups. Scale bar, 100 μm.

Figure 11.

Expression of Connexin 43. Photomicrographs showing the difference in the number of Connexin 43 cells demonstrated by immunohistochemistry to CN43 epitope, in A, control B, C-ECM group and C, bar graph showing the quantitative data (scale bar, 50 μm). The results were analysed by unpaired t-test (*P < 0.05).

Figure 12.

Differential distribution of proliferating cells proximal and distal the xenograft. Light micrograph of a cholecystic extracellular matrix (C-ECM, lakes of brown coloured biomaterial, marked by black dotted lines) graft-assisted healing reaction in myocardium following myocardial infarction demonstrating the abundance of proliferating cell nuclear antigen (PCNA) positive brown coloured nucleated cells proximal (black asterisk) compared to distal (white asterisk) areas of the infarct. (* a composite of 8 stitched images is presented for better illustration) Scale bar, 100 μm. Please see the Fig. 9 C₁ for the quantitative data.

Figure 13.

Anti-inflammatory response of lymphocytes to the xenograft. A pictogram representing the ratio of CD4 (helper T lymphocytes) to CD8 (cytotoxic T lymphocytes) positive cells in the infarct areas of both the groups, deducted from the data presented in Fig. 9: A3-C3, A4-C4. The ratio was higher in the C-ECM group indicating an anti-inflammatory graft acceptance reaction compared to a pro-inflammatory reaction in unassisted healing response.

Figure 14.

Immunohistochemistry for CD31 and ASMA. Light micrographs of histology sections collected from (A) control and (B) C-ECM–grafted rats after immunostaining with antibodies against CD31 and α smooth muscle actin (ASMA); scale bar, 50 µm. (C) Bar diagrams showing the quantitative information of the positive cells in these 2 groups. *P < 0.05 (unpaired t-test).