Matrix metalloproteinase 13 is a new contributor to skeletal muscle regeneration and critical for myoblast migration

Abstract

Efficient skeletal muscle repair and regeneration require coordinated remodeling of the extracellular matrix (ECM). Previous reports have indicated that matrix metalloproteinases (MMPs) play the pivotal role in ECM remodeling during muscle regeneration. The goal of the current study was to determine if the interstitial collagenase MMP-13 was involved in the muscle repair process. Using intramuscular cardiotoxin injections to induce acute muscle injury, we found that MMP-13 expression and activity transiently increased during the regeneration process. In addition, in muscles from mdx mice, which exhibit chronic injury, MMP-13 expression and protein levels were elevated. In differentiating C2C12 cells, a murine myoblast cell line, Mmp13 expression was most pronounced after myoblast fusion and during myotube formation. Using pharmacological inhibition of MMP-13 to test whether MMP-13 activity is necessary for the proliferation, differentiation, migration, and fusion of C2C12 cells, we found a dramatic blockade of myoblast migration, as well as a delay in differentiation. In contrast, C2C12 cells with stable overexpression of MMP-13 showed enhanced migration, without affecting myoblast maturation. Taken together, these results support a primary role for MMP-13 in myoblast migration that leads to secondary effects on differentiation.

Keywords: collagenase; matrix metalloproteinase; muscle repair; myoblast maturation.

Fig. 1.

Matrix metalloproteinase (MMP) levels in acute and chronic muscle damage. A–C: results from quantitative RT-PCR show different patterns of Mmp2, Mmp9, and Mmp13 expression during repair following cardiotoxin (CTX) injection. *Significantly different (P < 0.05, by 1-way ANOVA followed by Bonferroni's post hoc analysis) from no damage (day 0). D: elevation of Mmp2, Mmp9, and Mmp13 expression in muscles from mdx compared with C57BL/6 (C57) mice (dashed line). *P < 0.05, mdx vs. C57 (by unpaired t-test). E: collagen zymography showing activity of MMP-2, MMP-9, and MMP-13. Activity follows a time course similar to that of transcriptional changes during repair following CTX injection. Samples consist of 15 μg of protein from zymogen extracts of CTX-injected and noninjected (ND) muscles. F: immunoblots of MMP-13 in zymogen extracts of regenerating muscle. Results further confirm that the 52-kDa band is MMP-13. G: immunoblots of MMP-2, MMP-9, and MMP-13 in muscles from mdx and C57 mice. H: results from G showing significantly higher MMP-9 and MMP-13 levels in mdx muscles than in C57 (dashed line). *P < 0.05, mdx vs. C57 (by unpaired t-test).

Fig. 2.

Change in MMP-13 in differentiating C2C12 cells. A: progressive increase in Mmp13 expression during differentiation. Values are means ± SE of 3 separate experiments. *P < 0.05 (by 1-way ANOVA followed by Bonferroni's post hoc analysis) vs. day 0 (D0). B: increase in MMP-13 protein levels by day 1 of differentiation and elevation of MMP-13 in proliferating cells [growth medium (GM) condition]. Serum-free medium collected every 24 h from differentiating myoblasts shows secreted MMP-13 by day 1 of differentiation. C: increased MMP-13 activity in zymogen extracts of cell pellets as myoblasts differentiate. Values are means ± SE of 3 separate experiments. *P < 0.05 vs. D0 (by 1-way ANOVA followed by Bonferroni's post hoc analysis).

Fig. 3.

Immunocytochemistry for MMP-13 in proliferating and differentiating C2C12 cells. Top: MMP-13 staining alone (red). Bottom: merged image with phalloidin-Alexa 488 (staining actin, green) and 4′,6-diaminido-2-phenylindole (DAPI, blue) to reveal nuclei. Note increased MMP-13 in the daughter nuclei upon cell division (Proliferation, left). MMP-13 is not evident until day 2 of differentiation, after which myotubes exhibit positive cytoplasmic staining. Scale bars, 50 μm.

Fig. 4.

Modulation of MMP-13 in myoblasts. A: increasing concentrations of MMP-13 inhibitor were added to samples of recombinant MMP-13 or MMP-2 (rMMP-13 and rMMP-2), and enzyme activity was measured for each MMP. MMP-13 activity was blocked in a dose-dependent manner, with significant decreases in activity at 1 and 10 μM inhibitor. MMP-2 activity was not affected by the inhibitor at any concentration. *P < 0.05 vs. 0 μM (by 1-way ANOVA followed by Bonferroni's post hoc analysis). B: immunoblot of stable C2C12 cells expressing MMP-13 and/or green fluorescent protein (GFP). Note heightened levels of MMP-13 in cell extracts from proliferating myoblasts (Cell Pellet). Secretion of MMP-13 is increased in serum-free medium (Medium) following 24 h of incubation with cells. GFP blotting is a control for transfection efficiency, and GAPDH is a loading control. Blot represents 1 of 3 clonal lines.

Fig. 5.

Effect of MMP-13 on myoblast proliferation measured by 5-bromo-2P-deoxyuridine (BrdU) incorporation. A: MMP-13 inhibitor does not impair proliferation in C2C12 myoblasts in GM. B: stable expression of MMP-13 does not alter proliferation in C2C12 cells in the absence of serum compared with cells stably expressing GFP vector only. Values are means ± SE for 3 separate experiments.

Fig. 6.

Effect of MMP-13 activity on myoblast migration measured by Transwell assays. A: MMP-13 or MMP-2 inhibition in GM significantly impairs movement of C2C12 myoblasts allowed to migrate for 6 h. The combination of both inhibitors further impairs migration of C2C12 myoblasts. Serum is a primary source for MMPs, because migration is significantly reduced in its absence. Values are means ± SE for 7 separate experiments. B: stable expression of MMP-13 in the absence of serum significantly increases migration of C2C12 myoblasts allowed to migrate for 6 h compared with cells stably expressing GFP vector only. Note values for cells stably expressing GFP vector only are equivalent to values for native C2C12 myoblasts in serum-free medium. Values are means ± SE for 3 separate experiments. C: MMP-13 inhibition significantly impairs migration of C2C12 myoblasts in GM allowed to migrate for 12 h. Values are means ± SE for 3 separate experiments. For each experiment, the average number of cells passing the Transwell filter from 5 random fields was determined. *P < 0.05, **P < 0.001 (by Bonferroni's post hoc tests and unpaired t-tests).

Fig. 7.

Effect of MMP-13 activity on C2C12 differentiation. A: MMP-13 inhibition significantly impairs expression of myogenin in differentiating C2C12 cells. B: increased production of MMP-13 in stable C2C12 myoblasts undergoing differentiation does not alter myogenin expression. C: embryonic myosin heavy chain expression is reduced, evident at day 2 of differentiation in the presence of MMP-13 inhibitor. D: embryonic myosin heavy chain expression is not significantly altered in C2C12 cells with stable expression of MMP-13 compared with GFP vector controls. Values are means ± SE for 3 separate experiments. *P < 0.05 vs. D0 for each condition (by 2-way ANOVA followed by Bonferroni's post hoc analysis). †P < 0.05 vs. control (or GFP) at each time point(by 2-way ANOVA followed by Bonferroni's post hoc analysis).

Fig. 8.

Effect of MMP-13 activity on myoblast fusion. Myotubes expressing embryonic myosin and with ≥3 nuclei were counted. A: MMP-13 inhibition does not impair myoblast fusion during differentiation. B: stable expression of MMP-13 does not alter C2C12 myoblast fusion compared with cells stably expressing GFP vector only. Values are means ± SE for 3 separate experiments for each condition. *P < 0.05 vs. D0 for each condition (by 2-way ANOVA followed by Bonferroni's post hoc analysis).