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Chemokine receptor CCR2 involvement in skeletal muscle regeneration

Gordon L. Warren^*, Tracy Hulderman, Dawn Mishra, Xin Gao, Lyndell Millecchia, Laura O’Farrell^*, William A. Kuziel and Petia P. Simeonova^,1

^* Department of Physical Therapy, Georgia State University, Atlanta, Georgia;
Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia; and
Autoimmune and Inflammatory Diseases, Protein Design Labs, Fremont, California, USA

¹Correspondence: E-mail: psimeonova{at}cdc.gov

SPECIFIC AIMS

We hypothesized that signaling through CCR2, a major receptor for monocyte chemoattractant protein-1 MCP-1 (CCL2), affects degeneration/regeneration after skeletal muscle injury via indirect and direct effects on muscle precursor cells. We characterized localization of CCR2 in regenerating muscle [freeze-injured mouse tibialis anterior (TA) muscle] and compared cellular/molecular characteristics of skeletal muscle regeneration in CCR2-deficient vs. wild-type (WT) mice.

PRINCIPAL FINDINGS

1. CCR2 cellular localization in injured skeletal muscle
To determine whether expression of CCR2 is localized to macrophages and/or muscle precursor cells in injured muscle, we conducted double immunostaining for CCR2 and Mac-3, a marker of activated monocytes/macrophages, or myogenin, a marker of activated and differentiating muscle precursor cells. CCR2 was found to be localized to the membrane of cells expressing cytoplasmic localized Mac-3 (Fig. 1 A) or nuclear localized myogenin (Fig. 1B ), demonstrating that macrophages and muscle precursor cells are both targets for MCP-1 signaling.

View larger version (50K): [in this window] [in a new window]   Figure 1. Immunofluorescent staining for CCR2, Mac-1, and myogenin in TA muscle 2 days after freeze injury. Images are representative of those obtained on muscles from 4 WT mice. Nuclei were stained with DAPI and appear blue. x640. Bars = 10 µm. Control experiments without primary antibody demonstrated that immunostaining signals observed were specific. A) CCR2-Mac-3 localization: a) CCR2 staining (green); b) Mac-3 staining (red); c) DAPI staining; d) merged image showing colocalization of CCR2 and Mac-3, which produced an orange color. B) CCR2-myogenin localization: a) CCR2 staining (green); b) myogenin staining (red); c) DAPI staining; d) merged image of membrane CCR2 (green), nuclear myogenin (red), and DAPI stainings.

2. CCR2-deficient mice show a regeneration defect with enhanced adipogenesis and fibrosis
We compared CCR2-deficient and WT mice on the histological characteristics of TA muscle regeneration after freeze injury. Analysis of H&E-stained sections demonstrated no visible differences between the two types of mice regarding the histology of control TA muscle (data not shown) and in the extent of tissue damage and inflammatory response observed 3 days postinjury (Fig. 2 A). Two weeks after injury, virtually no sign of previous damage (except for regenerated myofibers with centrally located nuclei) was detectable in WT mice, indicating complete regeneration (Fig. 2B ). In CCR2–/– mice, the picture was different and included increased interstitial space, high numbers of inflammatory cells, large rounded (swollen) myofibers, and small centrally nucleated myofibers, demonstrating impaired regeneration (Fig. 2B ). To quantify the impaired regeneration in the CCR2–/– mice, we compared regenerating myofiber CSA at day 14 postinjury in 7 CCR2–/– mice with that in 5 WT mice. The mean regenerating myofiber CSA was 60% smaller in CCR2–/– mice than in the WT; there was no significant difference between the two types of mice in the CSA of uninjured myofibers (Fig. 2C ). To evaluate whether the enlarged interstitial space in injured muscle from CCR2–/– mice was associated with fibroblast accumulation, we conducted immunostaining for ER-TR7, a marker of reticular fibroblasts. At 14 days after injury, positive staining was found to surround regenerated myofibers in WT mice, but staining was expanded markedly in the interstitial space of injured muscles from CCR2–/– mice (Fig. 2D ). Three weeks after injury, lesions were no longer noticeable in muscle from WT mice (Fig. 2E ). In contrast, fat infiltration was apparent in injured muscle of CCR2–/– mice as shown by H&E-staining (Fig. 2E ). Collagen, as demonstrated by blue staining in Masson’s trichrome-stained sections, was distributed around fat deposits in injured muscles of CCR2–/– mice (Fig. 2F ). The fibrosis was accompanied by calcium deposits in injured muscles of CCR2–/– mice (Fig. 2G ). Histopathological grading of fat infiltration, fibrosis, and calcium deposition 21 days postinjury averaged a 3 (moderate-to-severe) in CCR2–/– mice compared with a 1 (minimal alterations) in WT mice.

View larger version (55K): [in this window] [in a new window]   Figure 2. Microscopic appearance of TA muscle of WT and CCR2–/– mice 3, 14, and 21 days after freeze injury. Images are representative of those obtained on muscles from at least 4 mice from each of the 2 groups. Bars = 100 µm. A) H&E staining of frozen transverse sections of TA muscle 3 days after injury. x400. B) H&E staining of frozen transverse sections of TA muscle 14 days after injury. x200. C) Average myofiber cross-sectional areas quantified for both regenerating (open bar) and uninjured myofibers (black bar) using H&E-stained histological sections obtained from muscles excised 14 days after injury (WT, n=5; CCR2–/–, n=7). D) ER-TR7 immunostaining on frozen transverse sections of TA muscle 14 days after injury.: x400. E) H&E staining of longitudinal sections of TA muscle 21 days postinjury. x100. Arrow indicates fat accumulation. F) Masson’s trichrome staining of longitudinal sections of TA muscle 21 days postinjury. x200. Arrow: collagen (blue). G) Van Kossa’s staining of longitudinal sections of TA muscle 21 days postinjury. x200. Calcium deposits are stained dark gray.

2. Prolonged inflammatory response occurs in injured skeletal muscle of CCR2–/– mice
Inflammatory response was evaluated by immunostaining for Mac-1 and Mac-3, markers of monocytes/macrophages, and Gr-1, a marker of neutrophils. Three days after injury, infiltrating inflammatory cells in the damaged muscle regions from C57BL/6 and CCR2–/– mice were positive for Mac-1 and Mac-3. The number of monocytes/macrophages that accumulated in injured muscle was not visibly reduced in CCR2–/– vs. WT mice. At 14 days after injury, whereas injured muscles of WT mice showed minimal Mac-1 or Mac-3 immunostaining, those of CCR2–/– mice were characterized by extensive immunostaining, indicating a prolonged period of macrophage accumulation. There was minimal Gr-1 immunostaining 3 days after injury in muscles from both types of mice and no staining was present in either 14 days postinjury. Immunohistochemistry for a vascular endothelial cell marker, CD31/PECAM-1, was conducted to assess whether angiogenesis was impaired in regenerating muscle of CCR2–/– mice. We observed no differences in the number of CD31-positive cells in CCR2–/– and WT groups.

Consistent with histological findings, the expression of genes coding for inflammatory mediators (Mac-1 and TNF-) in the injured muscle was altered in the CCR2–/– mice compared to the WT mice.

3. NCAM, but not MyoD and myogenin, expression is altered in injured muscle of CCR2–/– mice
To evaluate the extent of activation and maturation of muscle precursor cells, real-time PCR was used to determine expression of MyoD, myogenin, and NCAM (markers of proliferating, differentiating, and fusing muscle precursor cells, respectively). Myogenin expression was increased by injury to the same extent in muscles of WT and CCR2–/– mice 1, 3, or 14 days postinjury. Similarly, no difference between the two types of mice was observed in expression of MyoD. In contrast, at 3 and 14 days postinjury, real-time PCR demonstrated greater NCAM mRNA transcript levels in muscles of CCR2–/– mice compared with those of WT mice. Consistent with PCR findings, immunostaining demonstrated increased NCAM protein levels in injured muscle of CCR2–/– mice compared with WT mice at 14 days postinjury. In contrast to the minimal NCAM immunostaining observed in a few centrally nucleated myofibers of WT injured muscle, strong positive immunostaining of small mononucleated cells resembling small centrally nucleated myofibers was observed in injured muscle of CCR2–/– mice. Since NCAM expression is not unique to regenerating muscle cells, immunostaining for desmin was performed on serial sections to determine if cells expressing NCAM were muscle precursor cells. Positive immunostaining for NCAM coincided with positive staining for desmin.

4. Impaired time-course of functional recovery of injured muscle of CCR2–/– mice
To evaluate the time course of functional recovery from injury, maximal isometric torque production of the anterior crural muscles was measured before and at several time points after injury. Both types of mice demonstrated similar muscle strengths before injury and similar decrements immediately after injury (59%). Recovery of strength was apparent in WT mice by 7 days postinjury but not in CCR2–/– mice (P=0.008). The slower rate of strength recovery for CCR2–/– mice was maintained during the 2nd wk of recovery. By 14 days postinjury, strength deficits for WT and CCR2–/– mice were 28% and 42%, respectively (P=0.001), and the difference was maintained until at least 28 days postinjury.

CONCLUSIONS AND SIGNIFICANCE

In the current studies, we demonstrated that a deficiency of CCR2 leads to histopathological and functional impairments during skeletal muscle regeneration after experimentally induced injury. A delay in the regenerative process was accompanied with prolonged inflammation, increased fat accumulation, and a fibrotic response in injured skeletal muscle of CCR2–/– mice. These alterations may be related to impaired activities of macrophages and/or muscle precursor cells, both of which express the CCR2 receptor after injury.

Recovery of muscle strength was significantly delayed after injury in CCR2–/– mice compared with WT mice. However, replication and differentiation processes of muscle precursor cells are probably not dependent on signaling through the CCR2 receptor since gene expression of MyoD and myogenin was not significantly affected by CCR2 deficiency. Gene and protein expression of NCAM was accelerated in injured CCR2–/– muscle. Altered NCAM expression might be a compensatory response to impaired fusion activities of CCR2-deficient cells. Thus, CCR2 activation in muscle precursor cells may contribute to fusion/maturation processes occurring during muscle regeneration. An increased deposition of adipose tissue was observed in regenerating CCR2–/– muscle. It is possible that a MCP-1-CCR2 signaling axis directly modulates adipogenesis or that fat deposition occurs as a compensatory event for impaired myogenesis. This study suggests for the first time that CCR2-dependent signals are involved in regulating muscle precursor cells during regeneration (Fig. 3 ). Future in vitro studies would clarify the role of CCR2 in myoblast migration, fusion, and/or adipogenesis.

View larger version (13K): [in this window] [in a new window]   Figure 3. Schematic diagram. CCR2 is expressed not only on macrophages, but activated muscle precursor cells. Signaling through this receptor may contribute to migration and fusion activities of activated muscle precursor cells during regeneration. Deficiency of the CCR2 is associated with delayed muscle regeneration and shifting toward fibrosis and adipogenesis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2421fje;

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