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Tristetraprolin and LPS-inducible CXC chemokine are rapidly induced in presumptive satellite cells in response to skeletal muscle injury

Center for Cellular and Molecular Biology, Hyderabad 500 007 India

View larger version (37K): [in a new window]   Fig. 1. (A) DNA synthesis in reversibly arrested C2C12 myoblasts. Immunodetection of BrdU in asynchronous myoblasts (Mb), cells synchronized by 48 hours in suspension (S48), activated cells 28 hours after replating (R28) and 3 day myotube cultures (Mt). Arrowheads in R28 point to a labeled mitosis (telophase). In differentiated cultures (Mt), residual cycling myoblasts incorporate label, whereas myotube nuclei do not. (B) All arrested myoblasts progress to S phase upon reactivation. Cumulative DNA synthesis in synchronized C2C12 myoblasts after labeling with BrdU for 2-48 hours of reactivation (R2-R48) shows that 98% of arrested cells re-enter the cell cycle. The extended G1 phase is consistent with inclusion of a G0-G1 transition phase. For comparison, note that asynchronous cells (Mb) labeled for 2 hours show high levels of DNA synthesis, whereas suspension-arrested cells (S) show <2% S phase cells despite labeling for 12 hours. Data represent the means±s.e.m. of duplicate samples per time point. Similar results were obtained with two independent experiments. (C) Synchronous activation of G0 myoblasts requires both adhesion and mitogens. FACS analysis of adherent C2C12 myoblasts (Mb) reveals a DNA content profile typical of an asynchronous population. 48 hours after suspension (S48), most cells show a G1 DNA content consistent with arrest in G0. Replating for 6 hours in GM (R6) or for 24 hours in DM (R24D) does not alter the profile, whereas by 24 hours in GM, adhesion- and mitogen-dependent signals synergize to return arrested cells to S phase (R24G). Data represent the means±s.d. of four independent experiments.

View larger version (99K): [in a new window]   Fig. 2. (A) Myogenic specification factors are suppressed in G0 and activated in G1. Western blot analysis of total protein shows that both MyoD and Myf5 are present in asynchronous adherent cultures of C2C12 myoblasts (Mb) but not in C3H fibroblasts (Fb) as expected. 72 hour myotube cultures (Mt) strongly express MyoD but not Myf5. Neither MRF is expressed after 12 or 48 hours of suspension culture (S12, S48). Replating for 2 to 30 hours (R2-R30) causes induction of MyoD earlier (at R6) than Myf5 (at R18). Data are representative of three independent experiments. (B) Synchronization in G0 does not induce differentiation. Northern blot analysis of RNA isolated from asynchronous, arrested, reactivated or differentiated C2C12 cells. The differentiation marker MCK is only detected in myotubes (Mt), but not in asynchronous myoblasts (Mb), at 12 or 48 hours in suspension (S12, S48) or at 2-30 hours after replating (R2-R30). Histone mRNA indicates the extent of DNA synthesis. MyoD and Myf5 mRNAs correlate with the protein data in Fig. 2A. Data are representative of three independent experiments.

View larger version (45K): [in a new window]   Fig. 3. (A) Cell cycle dependent expression in cultured C2C12 myoblasts of genes involved in muscle regeneration. (A) RNA analysis of adherent myoblasts (Mb), suspension cultures at 12, 24 or 48 hours (S12, S24, S48), replated cultures at 2-30 hours (R2-R30) and myotubes (Mt). c-met transcripts are expressed throughout the cell cycle but are downregulated during differentiation. HGF transcripts are undetectable in asynchronous or differentiated cultures but expressed in synchronized cells during arrest and early activation. M-cad and PEA-3 mRNAs show classic cell cycle dependence: suppression in G0 and activation in G1 (PEA-3) and G1-S (M-Cad). Ethidium bromide staining of rRNA [28S] indicates equal loading. Data are representative of three independent experiments. (B) Semi-quantitative RT-PCR analysis of CD34 expression. Compared with asynchronous myoblasts (Mb), relative levels of CD34 mRNA rise in suspension-arrested cells (S12, S60) and decline during cell cycle activation upon replating (R1, R12, R30). Primers used detect a region common to both splice variants of CD34 mRNA. Values (upper panel) represent the means±s.e.m. of duplicate assays for CD34 RNA normalized with respect to L7 control RNA for each sample, shown in the Southern blot (lower panel). Similar results were obtained with two independent time course experiments.

View larger version (116K): [in a new window]   Fig. 4. Identification of genes expressed by arrested undifferentiated C2C12 muscle cells. (A) Differential display PCR analysis. Expression profiles of RNA from asynchronous (Mb) suspension-arrested (S) and differentiated (Mt) C2C12 cell cultures displayed by differential display PCR. (Mb and S were analyzed in duplicate.) The arrowhead indicates a fragment representing a cDNA expressed in arrested myoblasts but not in either asynchronous or differentiated cultures (see Fig. 4E). (B-E) Transcripts detected by differential display PCR fragments are enriched in synchronized C2C12 cells. Fragments excised from differential display PCR gels were labeled and used to probe 10 µg of total RNA from asynchronous myoblasts (Mb), arrested myoblasts (S) and myotubes (Mt). Each fragment (CF1-4) detected a unique transcript; all were expressed preferentially in synchronized cells. The sizes of the transcripts (B-E) are 3.5 kb (CF1/matrilin2), 2.2 kb (CF2/Znf216), 1.7 kb (CF3/TTP) and 1.4 kb (CF4/LIX). Positions of 28 and 18S rRNA are marked (-). L7 demonstrates equal loading of RNA.

View larger version (26K): [in a new window]   Fig. 5. LIX and TTP are induced during C2C12 myoblast activation in culture. RNA isolated from a time course of cell cycle activation was probed with each fragment described in Fig. 4. Mb, asynchronous myoblasts; S12, S48, S60, cells held in suspension for 12, 48 or 60 hours, respectively; R 0.5-30, cells replated for 0.5 to 30 hours after arrest in suspension; Mt, myotubes. All four transcripts are detected in G0 synchronized cells and further induced transiently in newly activated cells. Data are representative of three independent experiments.

View larger version (126K): [in a new window]   Fig. 6. Time course of muscle regeneration induced by focal freeze injury. Transverse cryosections from TA muscles of 3-month-old C57 mice subjected to focal damage in vivo were analyzed by HE staining. Uninjured muscle (U) shows a typical pattern of evenly sized myofibers with peripheral nuclei. 30 minutes after injury no histological changes are evident, whereas hyper-contracted fibers (arrowhead) and infiltrating mononuclear cells (arrow) appear by 2 and 6 hours, respectively. Mononucleated cells (inflammatory cells and myoblasts) increase in number in the lesion over the next 3 days. By 7 days post injury, myoblast fusion results in centrally nucleated regenerating fibers (arrowhead) of a smaller caliber than the adjacent uninjured fibers. The cross-sectional area of regenerating fibers matures to that of undamaged fibers over the next week (14d).

View larger version (102K): [in a new window]   Fig. 7. LIX and TTP are rapidly induced in response to muscle injury. RNA was isolated from uninjured adult mouse skeletal muscle (0) and at different times post injury (PI) and probed for TTP and LIX expression. LIX is induced at 6 hours and peaks prior to the peak of proliferation (assessed by Histone H2B mRNA). TTP was activated by 30 minutes after injury, well before the activation of MyoD. Each lane represents pooled RNA (20 µg) from TA muscles of two to three mice. Data are representative of three independent experiments.

View larger version (95K): [in a new window]   Fig. 8. LIX and TTP transcripts are distributed in a pattern similar to MyoD in injured skeletal muscle. RNA in situ hybridization to frozen sections (20 µm) of injured TA muscle using digoxigenin-labeled antisense probes to LIX, MyoD and TTP transcripts (A, B and C, respectively). Absence of staining in the myofiber interior suggests sequestration of the transcripts in mononuclear cells at the myofiber periphery. The same probes show no hybridization to uninjured muscle (a, b and c). LIX and MyoD were detected 6 hours post injury and TTP at 3 hours post injury. (D) shows TTP-positive cells located in the lesion; an adjacent uninjured area is devoid of TTP transcripts. Data are representative of three independent experiments each involving multiple cryosections from two mice.

View larger version (117K): [in a new window]   Fig. 9. TTP transcripts colocalize with a subset of nuclei in injured muscle. TTP transcripts were visualized by RNA in situ hybridization at 2 hours post injury (A) and nuclei counter-stained with Hoechst 33342 (B). There is a close correlation of TTP transcripts with a small proportion of the nuclei in the damaged area (arrowheads). The arrow indicates the occasional intense digoxigenin signal that quenched the Hoechst fluorescence of the underlying nucleus.

View larger version (101K): [in a new window]   Fig. 10. TTP is expressed in mononucleated cells that lie beneath the myofiber basal lamina. Co-detection of laminin with Pax7 (A,C,E) or TTP (B,D,F) transcripts in cryosections of TA muscle 1 hour after injury using combined immunofluorescence (E,F) and in situ hybridization (A,B); nuclei are counterstained with Hoechst 33342 (C,D). Pax 7 RNA (A) and TTP RNA (B) are associated with nuclei that lie below a laminin sheath (arrowheads in E,F). The same sublaminar cell (A,C,E or B,D,F, respectively) is indicated by arrows. Interstitial cells that do not express Pax7 or TTP RNA are indicated by `V' arrowhead.