Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle

Abstract

Injured muscle can initiate regeneration promptly by activating myogenic cells that proliferate and differentiate into myotubes and myofibers. However, the recovery of the injured skeletal muscle often is hindered by the development of fibrosis. We hypothesized that the early-appearing myogenic cells in the injured area differentiate into myofibroblasts and eventually contribute to the development of fibrosis. To investigate this, we transplanted a genetically engineered clonal population of muscle-derived stem cells (MC13 cells) into the skeletal muscle of immunodeficient SCID mice, which were lacerated 4 weeks after transplantation. The MC13 cells regenerated numerous myofibers in the nonlacerated muscle and these myogenic cells were gradually replaced by myofibroblastic cells in the injured muscle. Our results suggest that the release of local environmental stimuli after muscle injury triggers the differentiation of myogenic cells (including MC13 cells) into fibrotic cells. These results demonstrate the potential of muscle-derived stem cells to differentiate into different lineages and illustrate the importance of controlling the local environment within the injured tissue to optimize tissue regeneration via the transplantation of stem cells.

Figures

Figure 1.

Schematic representation of the experimental design.

Figure 2.

Illustration of the isolation of primary muscle cells (pp1-pp6) via the preplate technique (A). MC13 cells purified from pp6 are positive for β-galactosidase (B, C) and desmin (E). Even after differentiation, the MC13 cells remain positive for LacZ (D). The MC13 cells are negative for α-SMA and vimentin (F, G). These results were confirmed by RT-PCR (H). The pp1/pp2 cell population was positive for both vimentin and α-SMA, but the NIH/3T3 cell population was positive only for vimentin. MC13 cells and pp6 cell populations were both negative for vimentin and α-SMA. β-Actin was used as an external control for the RT-PCR experiment.

Figure 3.

Many MC13 cells had fused into myotubes and myofibers at 6 weeks after transplantation in the nonlacerated muscle (A–D). The injected sites were positive for α-SMA (E, F), but negative for vimentin (G, H). At 9 weeks after transplantation, most of the β-galactosidase-expressing myofibers had matured and become larger than at 6 weeks after transplantation (I–L). However, the injected sites were negative for both α-SMA (M, N) and vimentin (O, P).

Figure 4.

The MC13 cells had differentiated into myotubes and myofibers in the injured skeletal muscle at 2 weeks after injury (A–D). However, the injected sites were highly positive for both α-SMA (E, F) and vimentin (G, H) at 2 weeks after injury. At 5 weeks after injury, the regenerating myofibers present at 2 weeks after injury had disappeared and been replaced by scar tissue (I–L). Indeed, most of the β-galactosidase-expressing injected cells were now found in the scar tissue (I–L) because the injected sites were highly positive for both α-SMA (M, N) and vimentin (O, P).

Figure 5.

Co-localization of β-galactosidase-expressing cells with α-SMA-positive cells in the injured skeletal muscle. α-SMA was expressed in β-galactosidase-expressing myofibers at 1 week after laceration (A–C, asterisks). The expression of α-SMA also was found in the interstitial tissue between the myofibers (A–C, arrows). The vast majority of β-galactosidase-expressing myofibers was negative for α-SMA (A–C, arrowheads). However, the expression of α-SMA was mainly observed in interstitial tissue at 3 weeks after laceration (D–F, arrows). There were a few small myofibers in the lacerated area co-expressing β-galactosidase and α-SMA (D–F, asterisks). At 5 weeks after laceration, a complete absence of β-galactosidase-expressing myofibers was observed in the injected site, and the expression of α-SMA was found exclusively in interstitial tissue containing numerous β-galactosidase-expressing cells (G–I, arrows).

Figure 6.

The quantitation of β-galactosidase-expressing myofibers in both injured and nonlacerated skeletal muscle revealed that: 1) similar numbers of LacZ-expressing myofibers were found in injured (1 week after injury) and nonlacerated muscle at 5 weeks after transplantation; and 2) a significant decrease in LacZ-expressing myofibers was found in the injured muscle (3 and 5 weeks after injury) when compared to nonlacerated muscle at 7 and 9 weeks after transplantation. At 5 weeks after injury (9 weeks after transplantation), there was a near complete absence of LacZ-expressing myofibers in the injured skeletal muscle.

Figure 7.

The LacZ-positive cells isolated within the pp1/pp2 fraction from the injured muscle at 2 weeks after laceration (A, C) were also positive for α-SMA (B) and vimentin (D). The percentage of LacZ-positive cells isolated within the pp1/pp2 and pp4/pp5 fractions from the injured and control skeletal muscles at 5, 7, and 9 weeks after implantation revealed that: 1) a significantly higher percentage of LacZ-positive cells was found in the pp4/pp5 fraction than in the pp1/pp2 fraction from the injured muscle at 1 week after injury; 2) a similar percentage of pp1/pp2 and pp4/pp5 cells were LacZ-positive at 3 weeks after injury; and 3) the percentage of LacZ-positive cells was significantly higher in the pp1/pp2 fraction than in the pp4/pp5 fraction at 5 weeks after injury. In contrast, a significantly higher percentage of LacZ-positive cells were consistently found in the pp4/pp5 fraction than in the pp1/pp2 fraction at all time points tested in the control muscle (5, 7, and 9 weeks after transplantation). **, P < 0.01.

Figure 8.

Implication of TGF-β1 in the development of scar tissue within the injured skeletal muscle. There is expression of TGF-β1 in injured skeletal muscle (asterisks indicate myofibers and arrows indicate interstitial tissue) at 3 days after injury (cardiotoxin, A; laceration, B). This TGF-β1 expression decreased in the lacerated injured skeletal muscle at 7 days (C) and 14 days (D). RT-PCR (E) and Western blot (F) indicated an induction of α-SMA and vimentin expression in MC13 cells stimulated with TGF-β1 in a dose-dependent manner. For both RT-PCR and Western blot, lane 1 represents nonstimulated MC13 cells and lanes 2 to 5 represent MC13 cells stimulated with 0.01, 0.1, 1, and 5 ng/ml of TGF-β1, respectively. In E, the M indicates marker DNA ladder control.