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. 2011;6(9):e24714.
doi: 10.1371/journal.pone.0024714. Epub 2011 Sep 13.

CREB Is Activated by Muscle Injury and Promotes Muscle Regeneration

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Free PMC article

CREB Is Activated by Muscle Injury and Promotes Muscle Regeneration

Randi Stewart et al. PLoS One. .
Free PMC article

Abstract

The cAMP response element binding protein (CREB) plays key roles in differentiation of embryonic skeletal muscle progenitors and survival of adult skeletal muscle. However, little is known about the physiologic signals that activate CREB in normal muscle. Here we show that CREB phosphorylation and target genes are induced after acute muscle injury and during regeneration due to genetic mutation. Activated CREB localizes to both myogenic precursor cells and newly regenerating myofibers within regenerating areas. Moreover, we found that signals from damaged skeletal muscle tissue induce CREB phosphorylation and target gene expression in primary mouse myoblasts. An activated CREB mutant (CREBY134F) potentiates myoblast proliferation as well as expression of early myogenic transcription factors in cultured primary myocytes. Consistently, activated CREB-YF promotes myoblast proliferation after acute muscle injury in vivo and enhances muscle regeneration in dystrophic mdx mice. Our findings reveal a new physiologic function for CREB in contributing to skeletal muscle regeneration.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. CREB is activated by muscle injury.
A) Immunohistochemistry with anti-pCREB(S133) antibody shown on uninjured and injured contralateral muscles 3 days after cardiotoxin (CTX) injection; inset, no primary antibody control (npc). B) CREB target gene mRNAs Sik1 and Nr4a2 in mouse gastrocnemius muscle 3 days after CTX injection. Relative mRNA amounts to 18 S rRNA, normalized to naïve muscle. n = 2 naïve, 3 CTX. * p<.05, ** p<.01. Similar data were obtained on an independent cohort. C) In situ hybridization with Nr4a2 antisense riboprobe on uninjured and 3 day CTX-injected contralateral legs; no riboprobe control (blank, inset). D) Phospho-CREB immunohistochemistry and npc on gastrocnemius muscle from a 4-week old Dmdmdx mouse. E) Sik1 mRNA in wild-type and Dmdmdx gastrocnemius tissue from 4-week old mice. mRNA normalized to Gapdh, averaged by genotype (±stdev); n = 6 WT, 6 Dmdmdx; ** p<.01. F) In situ hybridization with antisense Nr4a2 riboprobe or blank (control) on Dmdmdx gastrocnemius at 4 weeks of age. G) Immunofluorescence staining of Dmdmdx gastrocnemius (4 wk) showing pCREB (green), Pax7 (red) and merge. Filled arrowheads, co-localized Pax7 and pCREB; open arrowheads, Pax7-positive nuclei without high pCREB; dotted circles, centrally nucleated myofibers. Bars, 50 µm (A, C, D, F), 10 µm (G).
Figure 2
Figure 2. CREB is activated by crushed muscle extract in primary myoblasts.
A) Phospho-CREB (pCREBS133), pATF1, total CREB and HSP90 in primary skeletal myoblasts treated with FSK/IBMX (cAMP) or bFGF for 10 min. B) pCREB, total CREB and HSP90 in primary skeletal myoblasts treated with crushed muscle extract (CME) for 0, 5 and 10 min. C) Amounts of Sik1 and Nr4a2 mRNA in primary skeletal myoblasts left untreated or incubated 1 h with PBS or CME. D) A-CREB expression in primary skeletal myoblasts blocks Sik1 and Nr4a2 induction by CME (1 h). In C and D, mRNA is normalized to Gapdh, represented as fold change above unstimulated (± stdev). Representative of 3 experiments. For D, treatments were normalized to GFP, unstimulated for each target gene.
Figure 3
Figure 3. CREB-YF enhances myoblast response to cAMP.
A) Amino acid sequence of CREB PKA phosphorylation site (Ser133) showing Y134F mutation. B) Targeting strategy for CREB-YF knock-in strain. Mouse Creb1 locus with Y134 in exon 5 (top); targeting construct inserts neo cassette in intron 4 and Y134F/StuI mutation (middle); targeted locus contains Y134F mutation. S, B: StuI, BamHI restriction sites. Southern probes indicated. C) Phospho-CREB, CREB and HSP90 protein in gastrocnemius muscle from Creb+/+ and CrebYF/YF mice. n = 4 per genotype. D) Nr4a2 mRNA in primary skeletal myoblasts from wild-type and CREB-YF mice, stimulated with FSK/IBMX for indicated times. Nr4a2 normalized to Gapdh, expressed in arbitrary units (A.U.). **, p<.01; *, p<.05 between genotypes. Average of 3 experiments ± stdev.
Figure 4
Figure 4. Activated CREB promotes myoblast proliferation and differentiation.
A) Growth curve assay of Creb+/+ and CrebYF/YF myoblasts over 5 days. Arrows, medium added. **, p<.01 between genotypes. B) Percent of BrdU-positive nuclei in asynchronous myoblast cultures. Means of 10 fields among 3 replicates. *, p<.05. C) CyclinA and HSP90 protein in asynchronous myoblasts. D) Myf5 and GAPDH control in differentiating Creb+/+ and CrebYF/YF myocytes. E) Muscle-specific proteins in Creb+/+ and CrebYF/YF myocytes after the indicated times in differentiation medium (DM). MYOG, myogenin; MHC, myosin heavy chain; GAPDH loading control. F) Phase contrast images of 2 day differentiated Creb+/+ and CrebYF/YF myotubes. Bar, 100 µm.
Figure 5
Figure 5. CREB promotes proliferation and muscle regeneration in mice.
A) EdU-positive nuclei (magenta) and all nuclei (DAPI, blue) in injured region (below dotted line) of adult male Creb+/+ and CrebYF/YF mice 5 days after CTX injection with EdU delivery on days 2–4 after cardiotoxin. B) Average number of EdU-positive nuclei per injured area (mm2) in 5 fields per mouse. n = 5 mice per genotype, *, p<0.05 by 2-tailed paired t-test. C) Anti-dystrophin immunohistochemistry in gastrocnemius sections from 4-week old CREB-YF-mdx mice of indicated genotypes. Dystrophin, magenta; DAPI, green. D) Confocal micrographs of regenerating myofibers in cross-sections of CREB-YF-mdx gastrocnemius tissue from 4-week old mice visualized by embryonic myosin heavy chain (eMHC, magenta, broken outlines) and central nuclei (DAPI, green, arrow). E) Number of regenerating myofibers per area (±stdev) in fields representing an entire cross-section of the gastrocnemius muscle from animals of the indicated genotypes (4-week old). n = 6 DmdmdxCreb+/ +, 5 DmdmdxCrebYF/YF; *, p<.05. F) Percent regenerating myofiber area (± stdev) in gastrocnemius tissue. n = 3 DmdmdxCreb+/ +, 4 DmdmdxCrebYF/YF; *, p<.05. Bars, 50 µm.

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