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. 2012 Jun;26(6):2363-73.
doi: 10.1096/fj.11-190728. Epub 2012 Feb 23.

Thrombospondin-4 regulates fibrosis and remodeling of the myocardium in response to pressure overload

Ella G Frolova  1 , Nikolai Sopko, Lauren Blech, Zoran B Popovic, Jianbo Li, Amit Vasanji, Carla Drumm, Irene Krukovets, Mukesh K Jain, Marc S Penn, Edward F Plow, Olga I Stenina
Affiliations

Thrombospondin-4 regulates fibrosis and remodeling of the myocardium in response to pressure overload

Ella G Frolova et al. FASEB J. 2012 Jun.

Abstract

Thrombospondin-4 (TSP-4) expression increases dramatically in hypertrophic and failing hearts in rodent models and in humans. The aim of this study was to address the function of TSP-4 in the heart. TSP-4-knockout (Thbs4(-/-)) and wild-type (WT) mice were subjected to transverse aortic constriction (TAC) to increase left ventricle load. After 2 wk, Thbs4(-/-) mice had a significantly higher heart weight/body weight ratio than WT mice. The additional increase in the heart weight in TAC Thbs4(-/-) mice was due to increased deposition of extracellular matrix (ECM). The levels of interstitial collagens were higher in the knockout mice, but the size of cardiomyocytes and apoptosis in the myocardium was unaffected by TSP-4 deficiency, suggesting that increased reactive fibrosis was the primary cause of the higher heart weight. The increased ECM deposition in Thbs4(-/-) mice was accompanied by changes in functional parameters of the heart and decreased vessel density. The expression of inflammatory and fibrotic genes known to be influential in myocardial remodeling changed as a result of TSP-4 deficiency in vivo and as a result of incubation of cells with recombinant TSP-4 in vitro. Thus, TSP-4 is involved in regulating the adaptive responses of the heart to pressure overload, suggesting its important role in myocardial remodeling. Our study showed a direct influence of TSP-4 on heart function and to identify the mechanism of its effects on heart remodeling.

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Figures

Figure 1.
Figure 1.
Expression of TSP-4 in the heart. A) TSP-4 (red; arrows indicate valves, membranous septum, interventricular septum) in the heart (blue denotes nuclei); composite figure from 4 images taken at ×20. B) TSP-4 in perimysium. C) TSP-4 in the matrix between cardiomyocytes. D) TSP-4 in the blood vessels. E, F) TSP-4 in two representative blood vessels: endothelial cells (anti-vWF, magenta); smooth muscle actin (anti-α-actin, green). Arrows (A–F) indicate TSP-4 staining. G, H) Expression of TSP-4 in the heart of sham-operated (G) and TAC-operated WT mouse (H), 2 wk after TAC.
Figure 2.
Figure 2.
TSP-4 is localized in the extracellular matrix of myocardium. A) Costaining of myocardium tissue from TAC-operated WT mice (2 wk after TAC) with anti-collagen I (red; a), anti-collagen IV (red; d), and anti-TSP-4 (green; b, e) antibodies, and overlay images (c, f). B) In situ TSP-4 mRNA hybridization. a, b) Heart of a sham-operated WT mouse. c, d) Heart of a TAC-operated WT mouse. a, c) TSP-4 mRNA (red). b, d) Masson trichrome staining (blue, ECM; red, cardiomyocytes and smooth muscle cells).
Figure 3.
Figure 3.
Increased HW in Thbs4−/− mice. A) HW/BW (mg/g) in sham- and TAC-operated mice of 58 wk of age (2 wk after TAC); n = 5. B) HW/BW (mg/g) in nonoperated mice of 80 wk of age; WT: n = 7; Thbs4−/−: n = 8. C) HW (mg) in nonoperated mice of 80 wk of age; WT: n = 7, Thbs4−/−: n = 8. Values are means ± se.
Figure 4.
Figure 4.
Microvessels in hearts of Thbs4−/− mice subjected to TAC. Endothelial cells were stained by injection of fluorescein-labeled lectin; percentage of stained area (n=5, 2 sections/mouse), 2 wk after TAC.
Figure 5.
Figure 5.
Extracellular matrix deposition in Thbs4−/− mice subjected to TAC. Masson trichrome-stained heart sections of age-matched TAC-operated WT (A, C, E) and Thbs4−/− (B, D, F) mice, 2 wk after banding (scale bar=400 m). Extracellular matrix is stained blue.
Figure 6.
Figure 6.
Interstitial fibrosis and the expression of collagens and hypertrophy markers in Thbs4−/− mice 2 wk after TAC. A) Fibrosis in sham-and TAC-operated mice, percentage fibrosis of the tissue section (total area=100%); n = 5. B) Fibrosis in nonoperated 80-wk-old mice, percentage fibrosis of the tissue section (total area=100%); n = 5. C) Expression of mRNA of hypertrophy markers and collagens in hearts of TAC-operated mice. D) Expression of collagen II mRNA. Shaded bars, WT mice; solid bars, Thbs4−/− mice. Values are means ± se.
Figure 7.
Figure 7.
Collagens in hearts of Thbs4−/− mice subjected to TAC. Collagens I–V visualized in the ECM of myocardium tissue sections of sham- and TAC-operated WT and Thbs4−/− mice (2 wk after TAC). Red, collagen; blue, nuclei. Scale bars = 40 μm.
Figure 8.
Figure 8.
Regulation of inflammatory and fibrosis-related genes in vivo and of collagen production in vitro by TSP-4. A) Decreased levels of inflammatory markers in hearts of Thbs4−/− mice subjected to TAC. Real-time quantitative RT-PCR (qRT-PCR); levels of mRNA in TAC-operated WT mice = 100%; mean ± se, n = 5, 2 wk after TAC. Levels are normalized to corresponding values in sham-operated mice. B) Levels of MMPs in hearts of Thbs4−/− mice subjected to TAC. For real-time qRT-PCR, levels are normalized to corresponding values in sham-operated mice; means ± se, n = 5, 2 wk after TAC. C) Human foreskin fibroblasts (HFFs) were incubated with 50 μg/ml recombinant TSP-4 or gelatin for 24–48 h. Solubilized cells and their matrices were tested in dot-blot assays (n=3) using anti-collagen I-V, anti-collagen I, anti-collagen II, anti-collagen III, anti-collagen IV, anti-collagen V, or anti-β-actin (loading control) antibodies. Representative blot. D) Quantification of the results of experiment in C, n = 3. E) Cells [human umbilical vein endothelial cells (HUVECs), RF/6A microvascular endothelial cells, and HFFs] were treated as in C. Representative blot. F) Quantification of the results of experiment in E; n = 3.
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