Chemokine expression and control of muscle cell migration during myogenesis

Abstract

Adult regenerative myogenesis is vital for restoring normal tissue structure after muscle injury. Muscle regeneration is dependent on progenitor satellite cells, which proliferate in response to injury, and their progeny differentiate and undergo cell-cell fusion to form regenerating myofibers. Myogenic progenitor cells must be precisely regulated and positioned for proper cell fusion to occur. Chemokines are secreted proteins that share both leukocyte chemoattractant and cytokine-like behavior and affect the physiology of a number of cell types. We investigated the steady-state mRNA levels of 84 chemokines, chemokine receptors and signaling molecules, to obtain a comprehensive view of chemokine expression by muscle cells during myogenesis in vitro. A large number of chemokines and chemokine receptors were expressed by primary mouse muscle cells, especially during times of extensive cell-cell fusion. Furthermore, muscle cells exhibited different migratory behavior throughout myogenesis in vitro. One receptor-ligand pair, CXCR4-SDF-1alpha (CXCL12), regulated migration of both proliferating and terminally differentiated muscle cells, and was necessary for proper fusion of muscle cells. Given the large number of chemokines and chemokine receptors directly expressed by muscle cells, these proteins might have a greater role in myogenesis than previously appreciated.

Fig. 1.

Chemokines and their receptors are expressed during in vitro myogenesis. (A) During myotube formation, the majority of myoblasts (red) terminally differentiate into myocytes (green) which migrate, adhere and fuse with one another to form small nascent myotubes with few nuclei (blue). Subsequently, nascent myotubes fuse with myocytes to form large mature myotubes with many nuclei (blue). (B) Primary mouse muscle cells were immunostained for eMyHC at different times in DM and the percentage of nuclei within eMyHC⁺ cells (differentiation index) was determined. By 16 hours in DM, most nuclei were in eMyHC⁺ cells. (C) The fusion index, or percentage of nuclei in myotubes, increased with time, and by 48 hours the majority of nuclei were within myotubes. (D) A real-time RT-PCR array was used to analyze the time-course of expression in vitro for 84 genes pertaining to chemokines. Positive results were obtained for 80 genes. Three patterns of expression were observed with mRNA steady state levels peaking at 16, 24 or 36 hours in DM, times of extensive differentiation and fusion. The number of genes with peak expression levels at each time point is shown. (E) Time course of expression for three representative genes peaking at 16 (CCR3), 24 (CXCR4) or 36 hours (IL13) in DM. Data are means ± s.e.m., n=3.

Fig. 2.

Changes in migratory behavior with muscle cell differentiation. (A) Migratory paths of mononucleated primary mouse muscle cells at 0, 6, 16, 24, 36 and 48 hours in DM. Tracks were taken from 3 hours of time-lapse microscopy with pictures every 5 minutes. Representative graphs are shown from one of three independent isolates with 20 cells each. (B) Frequency distribution of cell velocity at different times in myogenesis. A total of 60 cells were analyzed. Data are n=3.

Fig. 3.

Myocytes do not migrate to canonical myoblast migratory factors. Primary mouse myoblasts (Mb) and myocytes (Mc, 24 hours in DM) were allowed to migrate in Boyden chambers to control medium (C) or medium containing 100 ng/ml HGF or PDGF for 5 hours. Myocyte migration to conditioned medium (CM) from cultures in DM for 24 hours was also tested. Data are mean ± s.e.m., n=3–5 (*P<0.05 compared with control; **P<0.05 compared with myoblasts).

Fig. 4.

Myocytes exist during muscle regeneration. (A) Mononucleated cells were isolated from gastrocnemius muscles at days 3, 5 and 7 after injury and immunostained with antibodies against CD31 (APC), CD45 (APC) and α7 integrin (PE): CD31⁺ CD45⁺, to identify endothelial and immune cells and α7-integrin⁺ CD31⁻ CD45⁻ for myogenic cells. Myogenic cells constituted ~8% of the total mononucleated cells at day 3. Isotype controls were used to determine proper gating (left panel). (B) The percentage of myogenic cells remained stable during muscle regeneration. (C) Mononucleated cells were isolated from gastrocnemius muscles at indicated days after injury and immunostained with antibodies against CD31 (APC), CD45 (APC), α7 integrin (PE) and p21 (FITC) to identify terminally differentiated muscle cells. Myogenic α7-integrin⁺ CD31⁻ CD45⁻ cells were analyzed for p21. Isotype control was used to determine proper gating (left panel). (D) The percentage of p21⁺ myogenic cells was highest at day 5 after injury. (E) Mononucleated α7-integrin⁺ CD31⁻ CD45⁻ myogenic cells isolated from gastrocnemius muscles 5 days after injury were plated in vitro and immunostained for differentiation markers, myogenin (top left) and eMyHC (top right) or appropriate IgG controls (bottom). Scale bar: 10 μm. (F) The percentage of myogenin⁺ and eMyHC⁺ cells in E; ~60% of cells were myogenin⁺ a marker for earlier stages of differentiation, and ~20% were eMyHC⁺, a marker for later stages of differentiation. Data are from a pool of ten mice.

Fig. 5.

CXCR4 and SDF-1α are expressed during myogenesis in vitro and in vivo. (A) Primary mouse myoblasts and myocytes were immunostained with antibodies against CXCR4 (APC) in vitro. (B) The percentage of CXCR4⁺ cells was quantified; a significantly higher percentage of myocytes were CXCR4⁺. (C) Representative histogram; the level of CXCR4 per cell was also increased between myoblasts and myocytes. (D) Mean fluorescence intensity of CXCR4 per cell; myocytes contained almost twice as much CXCR4 per cell. (E) Myocytes were 18% larger than myoblasts. (F) Mononucleated cells were isolated from gastrocnemius muscles at days 3 and 5 after injury and immunostained with antibodies against CD31 (FITC), CD45 (FITC), α7 integrin (PE) and CXCR4 (APC). Cells were analyzed with the following criteria: CD31⁺ CD45⁺, to identify endothelial and immune cells and α7-integrin⁺ CD31⁻ CD45⁻ for myogenic cells. Day 5 is shown. (G) Myogenic α7-integrin⁺ CD31⁻ CD45⁻ cells were analyzed for CXCR4 and a representative histogram is shown. (H) The percentage of CXCR4⁺ myogenic cells; a higher percentage of myogenic cells were CXCR4⁺ at day 5. (I) The mean fluorescence intensity of CXCR4 per cell was also increased between day 3 and 5; myocytes contained almost twice as much CXCR4 per cell. (J) The level of SDF-1α secreted by primary mouse muscle cells in vitro during myogenesis (24 hours CM) was determined by ELISA. (K) The level of SDF-1α in crushed muscle extract determined by ELISA. The level of SDF-1α was increased at day 3. In all flow cytometry experiments: propidium iodide (PI) was used to remove dead cells from analysis; representative flow plots are shown and isotype controls were used to determine proper gating. Data are means ± s.e.m., n=3 (*P<0.05 compared with Mb, control or 0 days as appropriate).

Fig. 6.

CXCR4 and SDF-1α regulate migration of myoblasts and myocytes, and are necessary for myogenesis. (A) Boyden chamber experiments were performed with primary mouse myoblasts (Mb) and myocytes (Mc) with varying concentrations of SDF-1α. Myoblasts exhibited peak migration to 200 ng/ml, whereas myocytes migrated to 10–50 ng/ml. (B) AMD3100 or vehicle (V) was added to cultures with differentiation media (DM). Cultures were fixed and immunostained for embryonic myosin heavy chain (eMyHC) at 24 hours in DM. Scale bar: 50 μm. (C) Fusion index calculated as the number of nuclei in myotubes divided by the total number of nuclei. Addition of AMD decreased fusion at 24 hours in DM. (D) CXCR4 protein levels were decreased by Cxcr4 siRNA by ~45%. Tubulin was used a loading control. (E) Cells treated with control or Cxcr4 siRNA were placed in differentiation media (DM), fixed and immunostained for embryonic myosin heavy chain (eMyHC) at 48 hours in DM. (F) The total number of nuclei in each field was calculated. No difference between control and Cxcr4 siRNA cultures was observed at 24 hours, indicating that cell survival during differentiation was not affected by Cxcr4 siRNA. (G) Differentiation index calculated as the number of nuclei in eMyHC⁺ cells divided by the total number of nuclei. No difference was observed, suggesting that terminal differentiation was not affected by Cxcr4 siRNA. (H) Fusion index calculated as the number of nuclei in myotubes divided by the total number of nuclei. Cxcr4 siRNA decreased fusion at both 24 and 48 hours in DM. Data are means ± s.e.m., n=3 (*P<0.05 compared with control or Mb, **P<0.05 compared with Mb at same concentration).