Expression profiling of cytokines and related genes in regenerating skeletal muscle after cardiotoxin injection: a role for osteopontin

Abstract

To examine the roles of cytokines in muscle regeneration, we injected cardiotoxin into mouse tibialis anterior muscle and examined the expression profiles of cytokines and related genes in the regeneration process. Expression of 40, 64, and 7 genes among 522 genes spotted on a cytokine expression array were increased more than fivefold at 48 hours, 96 hours, and 7 days after toxin injection, respectively, when compared with those of the control muscle. Especially the levels of mRNA for chemokines and chemokine receptors, many of which are potent regulators of macrophages, were highly elevated 48 hours after injury. The expression of osteopontin (OPN), a versatile regulator of inflammation and tissue repair, was up-regulated more than 118-fold in regenerating muscle at 48 hours after injury. Northern blotting confirmed that the expression of OPN was highest at 48 hours after cardiotoxin injection and declined sharply thereafter. Immunohistochemistry showed that OPN was detected both in the cytoplasm of macrophages and in necrotic muscle infiltrated with macrophages. Our studies suggest OPN may serve as an adhesion molecule that promotes macrophage binding to necrotic fibers and may be an important mediator in the early phase of muscle regeneration.

Figure 1.

Histological changes of TA muscle after cardiotoxin injection. H&E sections of normal (A) and injected TA muscles of 8- to 9-week-old ICR mice at 48 hours (B), 96 hours (C), and 7 days (D) after cardiotoxin injection. Arrows in B show infiltration of mononucleated cells into necrotic muscle fibers. Arrows in C indicate immature myotubes with central nuclei and basophilic cytoplasm. At 7 days after treatment, the regenerating muscle fibers are larger caliber and the number of inflammatory cells is greatly reduced (D). Scale bar, 100 μm.

Figure 2.

Cytokine cDNA array analysis. A: Partial image of cytokine expression array of untreated (control side) and cardiotoxin-injected (injection side) TA muscles 48 hours after treatment. Arrows indicate the spots of OPN cDNA. B: The scattergraphs show the expression levels (pixel/mm²) of each gene 48 hours and 96 hours after cardiotoxin injection. The transverse axis represents the normalized signal intensities obtained from control muscles, and the longitudinal axis represents those from injected muscles. The data are averages of three independent experiments. One dot corresponds to one gene. C: The scattergraph analysis of representative cytokines and related gene families. The axes are as in B. Blue dots and pink dots are the results at 48 hours and 96 hours, respectively.

Figure 3.

OPN transcripts are abundant in cardiotoxin-injured muscle. A: Top: Northern blotting of OPN mRNA in normal (lane 1) and cardiotoxin-injected TA muscle at 8 hours (lane 2), 24 hours (lane 3), 48 hours (lane 4), 96 hours (lane 5), and 7 days (lane 6) after treatment. The level of OPN mRNA is considerably up-regulated at 48 hours after cardiotoxin injection. Bottom: The expression of 18s RNA. B: Time course of the relative expression of OPN mRNA during the skeletal muscle regeneration is shown.

Figure 4.

Immunohistochemical analysis of OPN in cardiotoxin-treated muscle. Transverse sections at 24 hours (A, C), 48 hours (B, D), 96 hours (E), and 7 days (F) after cardiotoxin injection were stained with anti-OPN antibody. C and D are high-power magnifications of the areas indicated by rectangles in A and B, respectively. The immunoreactivity is observed in mononucleated cells (inset in D) and necrotic fibers. The intact area, which escaped cardiotoxin injury, is indicated in B. Note that necrotic areas where mononucleated cells have not infiltrated yet were negative for OPN staining (asterisks in B). At 96 hours after treatment, OPN expression was considerably decreased when compared to that at 48 hours, but some OPN-positive mononucleated cells were observed (arrows in E). F: OPN staining was not detected at 7 days after the treatment. G: Positive control section from kidney of ICR mice. OPN was expressed in the long loop of Henle and in the distal tubule of the kidney. H: The negative control section of muscle at 2 days after cardiotoxin treatment showed no apparent staining. All sections were stained by the immunoperoxidase (ABC) method with methyl green (A-F) or hematoxylin (G and H). Scale bars: 400 μm (A, B), 200 μm (C, D, E, and B), 100 μm (G and H).

Figure 5.

F4/80-positive cells produce OPN. A: H&E sections of TA muscles at 48 hours after injection. After immunofluorescence observation, the section was washed and restained with H&E. A and B are identical fields. B: Sections of cardiotoxin-injected muscle were immunostained for OPN (green) and F4/80, a macrophage marker (red). Nuclei were stained with TOTO-3 (blue). C: High-power magnification of indicated area in B. Arrows show doubly stained cells. Immunofluorescence with TOTO-3. Scale bars: 40 μm (B); 16 μm (C).