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. 2015 Nov 12;1(1):1-7.
doi: 10.15436/2741-0598.15.005.

Mice lacking MKP-1 and MKP-5 Reveal Hierarchical Regulation of Regenerative Myogenesis

Hao Shi  1 , Florian Gatzke  2 , Julia M Molle  1 , Han Bin Lee  1 , Emma T Helm  1 , Jessie J Oldham  1 , Lei Zhang  2 , David E Gerrard  1 , Anton M Bennett  3
Affiliations

Mice lacking MKP-1 and MKP-5 Reveal Hierarchical Regulation of Regenerative Myogenesis

Hao Shi et al. J Stem Cell Regen Biol. .

Abstract

The relative contribution of the MAP kinase phosphatases (MKPs) in the integration of MAP kinase-dependent signaling during regenerative myogenesis has yet to be fully investigated. MKP-1 and MKP-5 maintain skeletal muscle homeostasis by providing positive and negative effects on regenerative myogenesis, respectively. In order to define the hierarchical contributions of MKP-1 and MKP-5 in the regulation of regenerative myogenesis we genetically ablated both MKPs in mice. MKP-1/MKP 5-deficient double-knockout (MKP1/5- DKO) mice were viable, and upon skeletal muscle injury, were severely impaired in their capacity to regenerate skeletal muscle. Satellite cells were fewer in number in MKP1/5-DKO mice and displayed a reduced proliferative capacity as compared with those derived from wild-type mice. MKP1/5-DKO mice exhibited increased inflammation and the macrophage M1 to M2 transition during the resolution of inflammation was impaired following injury. These results demonstrate that the actions of MKP-1 to positively regulate myogenesis predominate over those of MKP-5, which negatively regulates myogenesis. Hence, MKP-1 and MKP-5 function to maintain skeletal muscle homeostasis through non-overlapping and opposing signaling pathways.

Keywords: MAP kinase phosphatases; Macrophage function; Mitogen-activated protein kinase; Regenerative myogenesis; Signal transduction pathways.

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Figures

Figure 1
Figure 1
Skeletal muscle regeneration is impaired in Mkp1/5-DKO mice. (A) Mkp1 and Mkp5 mRNA expression in WT and DKO muscles. (B–C) Mouse body weight (B) and weights of gastrocnemius (GA) and tibialis anteria (TA) muscle (C). (D) H&E staining of non- damaged GA muscle. (E–F) Histogram (E) and quantification (F) of the cross sectional area (CSA) of non-damaged GA muscle. (G) H&E staining of WT and DKO GA muscles 11 days post cardiotoxin-induced injury. (H–I) Histogram (H) and quantification (I) of the CSA of the newborn fibers in (G). (J) Quantification of the fibers containing 1, 2, or 3 and more nuclei in (G). Scar bars in (D and G), 100 μm. Data represent means ± SEM from n=6 eight-week-old males in each genotype. *** P < 0.001 compared to WT.
Figure 2
Figure 2
MKP1/5-DKO mice have reduced muscle stem cell content. (A) Pooled hindlimb muscles were digested to isolate single cells for flow cytometry. SCs were sorted based on the surface markers Vcam+CD45−CD31−Sca1−. Calcein Blue AM was used to identify live cells, whereas propidium iodide for dead cells. (B) SC content in WT and DKO muscles. Values on the gates represent percent of muscle stem cells of the live cells (Calcein Blue AM+PI− population). (C) Muscle cryosections were stained for Pax7, and the number of Pax7+ cells was counted. Data represent means ± SEM from n=6 mice in each genotype. *** P < 0.001 compared to the WT mice.
Figure 3
Figure 3
MKP1/5-DKO SCs have impaired prolifertation in vitro. (A–B) Histogram (A) and quantification (B) clone size of the isolated SCs. SCs were isolated from skeletal muscles from WT and DKO mice, and cultured in collagen-coated plate for 7d. (C–D) Representative photo micrographs (C) and quantification (D) of BrdU incorporation into SCs. SCs were isolated and cultured as in (A–B). BrdU (10 μM) was added to the culturte medium for 1h before fixation and staining. (E–F) Representative photo micrographs (E) and quantification (F) of SC differentiation in vitro. SCs were isolated and cultured in Matrigel-coated plates. Equal number of cells were plated and induced to differentiate for 3d. Scar bars in (C and E), 200 μm. Data represent means ± SEM from three independent experiments. *** P < 0.001 compared to WT.
Figure 4
Figure 4
MKP1/5-DKO muscle exhibits enhanced inflammatory responses after damage. GA muscle from eight-week-old mice was damaged by cardiotoxin injection. (A) 42h post injury, muscles were harvested, muscle sections were stained with Ly6b.2 antibody. (B–D) 11d post injury, muscles were stained for CD11b and Ly6b.2 respectively (B) and quantified (C and D). Data represent means ± SEM from n=6 males in each genotype. * P < 0.05;*** P < 0.001 compared to WT. Scale bars, 100 μm.
Figure 5
Figure 5
MKP1/5-DKO differentially affects M1 and M2 macrophage marker gene expression. 3 days post injury, TA muscle was harvested, RNA was extracted and quantitative RT-PCR was performed to detect gene expression of M1 and M2 macrophage markers. (A–C) Relative mRNA abundance of M1 macrophage markers Tnf (A), Il1b (B), and Nos2 (C). (D–F) Relative mRNA abundance of M2 macrophage markers Il10 (D), Retnla (E), and Vcam1 (F). Data represent means ± SEM from n=5 in each genotype. ** P < 0.01;*** P < 0.001 compared to WT.
Figure 6
Figure 6
MKP1/5-DKO impairs macrophage skewing during muscle regeneration. Tibialis anterior muscle was injected with cardiotoxin, 3d (A–F) or 6d (G) later, muscles were harvested, digested, and underwent FACS analysis. (A) % CD45+ cells in damaged muscle. (B) % F4/80+ cells of CD45+ cells. (C) % Neutrophil (NF) (F4/80− Ly6G/Chi) of CD45+ cells. (D) Neutrophil (NF) (F4/80− Ly6G/Chi), M1 macrophage (F4/80loLy6G/Chi) and M2 macrophage (F4/80hiLy6G/Clo) populations of CD45+ cells. (E) M1 and M2 subpopulations of F4/80+ cells. (F) CD206 expression in F4/80+ cells from 3d post injury muscles. Solid line, WT; Dotted line, DKO. (G) CD206 expression in F4/80+ cells from 6d post injury muscles. Solid line, WT; Dotted line, DKO. (F–G) Insets: values represent relative median fluorescence intensity (MFI) of PE. Data represent means ± SEM from n=6 four-month-old males in each genotype. ** P < 0.01; *** P < 0.001 compared to WT.
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