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Insulin-like Growth Factor-II Delays Early but Enhances Late Regeneration of Skeletal Muscle

Sonnie P. Kirk, Jenny M. Oldham, Ferenc Jeanplong and John J. Bass

Functional Muscle Genomics, AgResearch, Ruakura Agricultural Research Centre, Hamilton, New Zealand

Correspondence to: Dr. Jenny Oldham, Functional Muscle Genomics, AgResearch, Ruakura Agricultural Research Centre, Private Bag 3123, Hamilton, New Zealand. E-mail: jenny.oldham{at}agresearch.co.nz

	Summary

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This study tested whether administration of insulin-like growth factor-II (IGF-II) enhances muscle regeneration. Rat biceps femoris muscle was damaged with notexin and then IGF-II was administered for up to 7 days. Results show that the proportion of nuclei containing or surrounded by immunoreactivity to MyoD, myogenin, and developmental myosin heavy chain (dMHC) is less in the IGF-II treatment group relative to the control group on days 1 (p=0.057), 2 (p=0.034), and 3 (p=0.047), respectively. This indicates a delay in muscle precursor cell (MPC) proliferation and differentiation with IGF-II administration. This effect was not associated with decreased binding capacity of the type 1 IGF receptor, as determined by receptor autoradiography in day 1 muscle sections (NS), but was associated with inhibition of phagocytic processes. The cross-sectional area of regenerating muscle fibers was significantly greater in the IGF-II treatment group than in the control group by day 7 (p=0.0092). The enhancing effect of IGF-II on late muscle regeneration, when the main process taking place is fiber enlargement, coincides with the period in which IGF-II is normally expressed by regenerating muscle, indicating that greater endogenous production of IGF-II would be associated with improved regeneration.

(J Histochem Cytochem 51:1611–1620, 2003)

Key Words: developmental myosin heavy • chain • proliferation • myogenin • differentiation • MyoD • satellite cell • damage • type 1 IGF receptor • rat

	Introduction

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MUSCLE FIBERS are the basic unit of skeletal muscle. Myonuclei in muscle fibers are postmitotic, whereas satellite cells, which are located between the plasmalemma of the muscle fiber and the surrounding basal lamina, can undergo mitosis to form the muscle precursor cells (MPCs) necessary for growth and repair. During regeneration after muscle damage, muscle fibers undergo phagocytosis to remove necrotic debris, after which MPCs proliferate, differentiate, and fuse to form myotubes (Hansen–Smith and Carlson 1979; Grounds 1991), a period referred to as early regeneration. In contrast to early regeneration, late regeneration consists of muscle fiber enlargement, complete with reinnervation and maturation, to form mature muscle fibers.

The myogenic regulatory factors (MRFs) play an important role in the commitment of cells to the myogenic lineage and in their ensuing differentiation to form mature skeletal muscle (Rudnicki and Jaenisch 1995). MRFs have a coordinated and sequential pattern of expression during MPC activation, proliferation, and differentiation. MyoD and myf-5 are expressed in a mutually exclusive manner in proliferating cells. However, only cells expressing MyoD undergo subsequent differentiation (Kitzmann et al. 1998). Expression of myogenin and MRF-4 mRNA occurs after the expression of MyoD and myf-5 mRNAs (Cornelison and Wold 1997), with the expression of myogenin mRNA coincident with early differentiation (Smith et al. 1994; Yoshida et al. 1998). In recent years, MyoD expression has been routinely used in studies of muscle regeneration as a marker for MPC proliferation and myogenin expression as a marker for the entry of MPCs into the differentiation pathway (Floss et al. 1997; Merly et al. 1999; Jin et al. 2000).

Insulin-like growth factor (IGF)-I and IGF-II regulate growth and development, as demonstrated by igf1 and igf2 null mice, both of which are 60% of normal weight at birth (DeChiara et al. 1990; Liu et al. 1993; Powell–Braxton et al. 1993). IGFs stimulate muscle cell proliferation and differentiation in culture through interaction with the type 1 IGF receptor (Ewton et al. 1987; Duclos et al. 1991). IGF binding to the type 1 IGF receptor stimulates tyrosine kinase activity and activation of the mitogen-activated protein (MAP) kinase signaling pathway during proliferation (Coolican et al. 1997) and differentiation (Gredinger et al. 1998; Zetser et al. 1999; Wu et al. 2000), in addition to activation of the phosphoinositide 3'-kinase (PI3K) pathway during differentiation (Coolican et al. 1997). Stimulation of muscle cell differentiation by IGFs is biphasic: low levels of IGF facilitate differentiation, whereas high levels of IGF inhibit differentiation (Florini et al. 1986).

The IGF axis, which includes not only the growth factors IGF-I and IGF-II but also receptors, binding proteins, and binding protein-related proteins, is tightly regulated via positive and negative feedback loops among the various components. IGF-I, IGF-II, and high concentrations of insulin downregulate IGF-II at the transcriptional level (Magri et al. 1994), while IGF-II downregulates the type 1 IGF receptor via transcriptional downregulation and increased receptor degradation (Rosenthal et al. 1991; Rosenthal and Brown 1994). Effects of IGF-II on the type 1 IGF receptor may influence the timing of differentiation in myogenic cells, as suggested by studies showing advanced differentiation with overexpression of the type 1 IGF receptor (Quinn et al. 1994; Quinn and Haugk 1996) or a delay in differentiation with functional inactivation of the type 1 IGF receptor (Cheng et al. 2000).

Previous studies by our group have shown specific temporal regulation of IGF-II during skeletal muscle regeneration, in which IGF-II gene expression occurred concurrently with MPC differentiation and myotube formation (Kirk et al. 1996). When overexpressed in cultured C2 myoblasts, autocrine IGF-II advances the onset of differentiation (Stewart et al. 1996). Although the expression of IGF-II mRNA in our earlier regeneration studies was not associated with MPC proliferation, studies by others (McFarland et al. 1993; Bach et al. 1995; Minniti et al. 1995) clearly show an enhancement of myoblast proliferation in culture with administration of IGF-II. Therefore, the aim of the present study was to test the hypothesis that administration of IGF-II peptide during regeneration of skeletal muscle enhances MPC proliferation and differentiation. A histological approach was used to determine the receptor binding capacity, muscle fiber cross-sectional area, and the proportion of nuclei expressing muscle-specific proteins in areas of comparable damage.

	Materials and Methods

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IGF-II Peptide Administration
Alzet miniosmotic pumps (Model 1007D; Alza, Mountain View, CA) were used to continuously administer IGF-II peptide during the course of muscle regeneration. Seven-day pumps were fitted with single-lumen vinyl catheters (SV55 tubing, inner diameter 0.80 mm, outer diameter 1.20 mm; Dural Plastics and Engineering, Dural, NSW, Australia) so that they could be implanted distant to the regenerating area. Catheter and pump assemblies were filled with a 0.29 µg/µl solution of recombinant human IGF-II (OM-001, lot EJI-O01; GroPep Pty, Adelaide, Australia; reconstituted to 1 mg/ml in 10 mM acetic acid) diluted in RPMI 1640 medium, thus giving a delivery rate of 0.145 µg IGF-II/hr, or 3.48 µg IGF-II/day. Alzet pumps for control animals contained diluent only.

Surgical Procedures
A total of 75 male Sprague–Dawley rats were housed in the Small Animal Colony of Ruakura Agricultural Research Centre. At approximately 7 weeks of age and weighing 290 g, 70 rats were injected with the myotoxin notexin (Venom Supplies Pty, Tanunda, South Australia), and implanted with the primed Alzet pumps. The anesthetic used for this procedure was 5 mg/ml Rompun (xylazine), 37.5 mg/ml ketamine HCL in sterile water (0.2 ml/100 g bw). The end of the catheter was positioned over the right biceps femoris muscle and the notexin (2 µg in 10 µl) injected deep to the site of IGF-II administration. Rats were walking normally and bearing weight on both legs without favoritism within 2.5 hr of surgery. This study had approval from the AgResearch Animal Ethics Committee and was conducted in accordance with New Zealand animal welfare legislation.

Muscle Histology
Animals were sacrificed by CO₂ gas followed by cervical dislocation on days 0–7, five rats per treatment per day. Pump and catheter assemblies were removed and both right (damaged) and left (undamaged) biceps femoris muscles dissected. Muscle tissue was frozen for immunohistochemistry, in vitro autoradiography, and fiber area analyses by immersion in melting isopentane (BDH; Poole, Dorset, UK), then stored at -80C. Muscle for histological staining (hematoxylin and eosin) was fixed with a 4% solution of formaldehyde in 0.9% PBS (Oxoid; Basingstoke, Hampshire, UK). The core of damage, i.e., the site at which notexin-induced damage was most abundant and virtually all muscle fibers were damaged by notexin, was used for all analyses of regenerating tissue. Muscle fibers that were undamaged by notexin injection were eliminated from the analyses of MyoD, myogenin, dMHC, receptor autoradiography, and regenerating muscle fiber size. The site of notexin injection was intentionally located just below (deep to) the site of peptide infusion to optimize for peptide effects in the core of damage.

Immunohistochemistry
MyoD, myogenin, and developmental myosin heavy chain (dMHC) proteins were used as markers for proliferation and differentiation in muscle sections undergoing regeneration. IHC and quantitative analysis of these proteins allowed a comparison of the regeneration time courses between the IGF-II and control groups. For myogenin IHC, frozen tissue samples were sectioned (8 µm), then fixed for 10 min in formalin (1.3% formaldehyde in PBS). Fixative solution was washed off in a bath of Tris-buffered saline (TBS; 0.05 M Tris, pH 7.4, 0.1 M NaCl; 5 min). Then endogenous peroxidase was quenched with a 5 min incubation in 3% H₂O₂ in TBS containing 0.1% Tween-20 (TBST). Sections were rinsed for 5 min with TBS, then nonspecific binding of the antibody inhibited by a 20-min incubation in a blocking solution consisting of 10% normal donkey serum (NDS) in TBST with 0.2% (w/v) BSA (TBSTB). Excess blocking solution was removed by gentle tapping and sections were incubated for 1 hr with rabbit anti-rat myogenin antibody (Santa Cruz Biotechnology; Santa Cruz, CA) diluted 1:200 in blocking solution. Sections were rinsed twice for 5 min each in TBST. Then biotinylated donkey anti-rabbit Ig (Amersham Pharmacia Biotech; Auckland, New Zealand) diluted 1:200 in TBSTB containing 1.5% NDS was placed on sections for 30 min. Sections were rinsed twice for 5 min each in TBST baths, then incubated for 30 min with streptavidin–biotin–horseradish peroxidase (SA-B-HRP; Amersham Pharmacia Biotech) diluted 1:200 in TBSTB. Sections were then rinsed in two baths of TBS for 5 min each. Bound antibody was visualized as a brown precipitate by incubating sections with 3,3-diaminobenzidine (DAB) solution (DAB tablet set; Sigma-Aldrich, St Louis, MO) for 6 min. Sections were rinsed in distilled water for 2 min, counterstained with Mayer's hematoxylin for 1.3 min, rinsed in tapwater for 4 min, and then sections were dehydrated, cleared in xylene, and mounted.

The protocol for MyoD IHC used a mouse anti-recombinant mouse MyoD antibody (Pharmingen clone MAb 5.8A; BD Biosciences, Franklin Lakes, NJ) diluted 1:80, and was followed by a biotinylated sheep anti-mouse Ig (Amersham Pharmacia Biotech) secondary antibody. The MyoD antigen was very sensitive to fixation because poor results were obtained with sections fixed with formalin, cold acetone, or ethanol. However, a 15-min incubation in 1% paraformaldehyde yielded excellent results. The intensity of the MyoD signal was compromised by the presence of BSA and serum in the first and secondary antibody incubations, so these were omitted. The omission of BSA and serum from the primary and secondary antibody solutions increased nonspecific binding to necrotic tissue (data not shown). However, this did not interfere with interpretation or signal quantification. The IHC signal for MyoD was less intense than that for both myogenin and dMHC.

IHC to detect dMHC in tissue sections was performed using a mouse anti-rat dMHC antibody (mouse anti-rat; Novocastra Laboratories, Newcastle upon Tyne, UK) diluted 1:60, with normal swine serum used in place of NDS. Muscle sections that were immunostained with dMHC had a more intense signal when fixed with cold acetone (2 min, then air-dried) than when fixed with formalin, paraformaldehyde, or ethanol. However, the tissue morphology after cold acetone fixation was poor. To improve the tissue morphology after fixation with cold acetone, a subsequent formalin fixation step (1.3% formalin for 10 min, followed by two five-min washes in TBST) was inserted after the primary antibody/TBST washing steps.

The specificity of staining for all antibodies was determined by immunostaining with (a) diluent only for the primary antibody step, and (b) a matched concentration of normal immunoglobulin (DAKO; Carpinteria, CA) from the same species as the primary antibody. All negative control sections were devoid of staining with the exception of MyoD controls, which showed the same low-level diffuse background staining over necrotic debris that the positive section showed, due to the omission of BSA and serum from the staining solutions (see above).

For quantitation of immunostained sections, the numbers of marker-positive nuclei were determined and then expressed as a proportion of total nuclei (myogenic and non-myogenic) in an area. All MyoD and virtually all myogenin immunoreactivity was contained in nuclei. Occasional immature myotubes that showed cytoplasmic localization of myogenin were not included in quantitation of the myogenin signal. Nuclei in dMHC-immunostained sections were considered positive if they were surrounded by dMHC(+) cytoplasm.

Receptor Autoradiography
Receptor autoradiography to determine type 1 IGF receptor levels in all day 1 damaged muscles was carried out according to the method of Elliott et al. (1992), with radiolabeled peptide prepared according to the iodogen method (Salacinski et al. 1981) as previously applied to IGF (Hodgkinson et al. 1987), except that the radionuclide used was ¹²⁵I. In brief, serial sections (8 µm) were incubated with [¹²⁵I]-recombinant human IGF-I to determine total binding and with radiolabeled IGF-I solution containing excess (1 µg/ml) unlabeled recombinant human IGF-I to determine nonspecific binding. Binding of IGF-I was characterized by competing radiolabeled IGF-I binding to serial sections with 1 µg/ml des(1–3)IGF-I, 1 µg/ml IGF-II, or 10 ng/ml insulin in place of the unlabeled IGF-I. After incubation, the sections were apposed to X-OMAT-AR5 film (Eastman Kodak; Rochester, NY) for 8 days to generate macroautoradiograms, analysis of which showed no difference in the displacement of [¹²⁵I]-IGF-I binding by unlabeled des(1–3)-IGF-I vs unlabeled IGF-I. These results indicate that the binding observed was to receptor and not binding protein. Furthermore, competition with unlabeled IGF-II and insulin did not indicate binding to either the type 2 IGF or insulin receptors, respectively. Microautoradiograms were generated by coating the sections with liquid photographic emulsion (LM-1; Amersham Pharmacia Biotech), exposing them for 14 days, then developing the slides and staining the sections with hematoxylin and eosin.

The resultant silver grains of the microautoradiograms were manually counted using the ScionImage quantitation system described below. Results are expressed as the specific binding (the difference between total and nonspecific binding) in grains/µm².

Quantitative Analyses
Chromogenic signals resulting from IHC, counts of total nuclei, and radiographic grains arising from receptor autoradiography experiments were analyzed by capturing digital images of the tissue sections with a CMS700 image analysis system (Scion; Frederick, MD). Images were saved as TIFF files and then manually counted in ScionImage. The cross-sectional areas of regenerated and undamaged muscle fibers were determined by capturing images from muscle sections that had been immunostained with dMHC antibody. Crosssections of individual fibers were outlined and the number of pixels per fiber calculated by the ScionImage software contained in the CMS700 image analysis system. The number of regenerating fibers analyzed per animal ranged from 31 to 149.

Statistical Analysis
Fiber area and receptor autoradiography data were analyzed by Student's t-test and the values given are the mean ± SEM. Data for MyoD, myogenin, dMHC, and the total number of nuclei per unit area were log-transformed, then analyzed by Student's t-test for differences at individual time points and by ANOVA to determine overall effects. Day 1 values were omitted from the ANOVA for myogenin and dMHC due to high zero components in the datasets. Values shown for MyoD, myogenin, and dMHC are the mean ± SEM.

	Results

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Histological Assessment of Regeneration
The time course of muscle regeneration in the core of damage in animals that were not treated with IGF-II is shown in Figure 1 . In undamaged muscles (day 0, Figure 1A), fibers were whole and there were low numbers of mononucleate cells. However, by day 1 in notexin-injected control muscle (Figure 1B), substantial numbers of infiltrating phagocytic and inflammatory cells were observed both in and outside of muscle fibers. Approximately 50% of damaged muscle fibers in control animals showed signs of phagocytic infiltration, and considerable necrotic fiber cytoplasm fragmentation was observed. Day 2 muscles from control animals (Figure 1E) contained large accumulations of mononucleate cells, with low to moderate amounts of necrotic debris. Fusion was first observed on day 3 after notexin injection (Figure 1C), with high levels of myotube formation occurring on both days 3 and 4 such that the bulk of the regenerated myofibers had been formed by day 4. Myotube enlargement occurred primarily between days 5 and 7, with the myotube diameter on day 7 still less than that of survivor fibers (Figure 1D).

View larger version (161K): [in this window] [in a new window]   Figure 1 Time course of muscle damage and regeneration after notexin injection on day 0. (A–D) Regeneration in control animals. Fibers are whole in undamaged muscle (A, day 0), but by day 1 after notexin injection (B) muscle fibers are both infiltrated (B, white arrow) and non-infiltrated (B, black arrow) by phagocytic cells. By day 3 (C), myotubes are evident (C, black arrows) and by day 7 (D) these regenerating myotubes (D, black arrows) have enlarged but are still smaller in diameter than the survivor fibers (D, white arrow). (E,F) Histological difference between regenerating muscle of control (E) and IGF-II-treated (F) animals, as observed on day 2. Muscle sections from IGF-II-treated rats have abundant necrotic debris (arrowhead), a higher proportion of cells with the morphological appearance of phagocytes (white arrows), and a lower proportion of mononucleate cells with the morphological appearance of MPCs (black arrows) compared to regenerating muscle sections of control rats. Hematoxylin and eosin stain, Bar = 100 µm.

Proliferation and Differentiation of MPCs
MyoD, myogenin, and dMHC levels were determined on days 1–4 because this period included the main proliferation and differentiation events during regeneration. Results are expressed as the proportion of marker-positive nuclei per total nuclei within the damaged region. Throughout days 1–4 there was no significant difference in the total number of nuclei (myogenic and non-myogenic) per unit area between the IGF-II treatment and control groups in an overall analysis (0.00084 ± 0.000078 vs 0.00095 ± 0.000078 nuclei per pixel, respectively; p=0.168) or on specific days.

MyoD.
The proportion of MyoD-positive nuclei changed significantly over time (p<0.001), with an increase up to day 2 in the percentage of nuclei that contain MyoD protein and then a decrease (Figure 2) . The IGF-II treatment group had a lower proportion of MyoD-positive nuclei relative to the control group on day 1 (p=0.057; Figures 2 and 3) .

View larger version (11K): [in this window] [in a new window]   Figure 2 Early regeneration is delayed in muscles receiving exogenous IGF-II. Sections of regenerating muscle tissue from control () and IGF-II-treated () animals were immunostained with antibodies to MyoD, myogenin, or dMHC. Then the proportion of nuclei containing either MyoD or myogenin, or surrounded by dMHC-positive cytoplasm, was determined by quantitative analysis. Results show that the proportion of nuclei containing or surrounded by immunostaining from IGF-II-treated animals is decreased relative to control animals on days 1 (p=0.057), 2 (*p<0.05), and 3 (*p<0.05), respectively. Values shown are the means ± SEM (n=3–5 per treatment per time point) plotted on a log scale.

View larger version (138K): [in this window] [in a new window]   Figure 3 Photomicrographs of immunostaining obtained with antibodies directed against MyoD (A,B), myogenin (C,D), and dMHC (E,F), in regenerating muscle sections from control (A,C,E) and IGF-II-treated (B,D,F) rats. Sections are from day 1 (A,B), day 2 (C,D), and day 3 (E,F) after injection of notexin. Arrows indicate nuclei that contain positive immunostaining for MyoD or myogenin, or are surrounded by cytoplasmic immunostaining for dMHC. Sections were counterstained with hematoxylin. Bar = 40 µm.

dMHC.
No dMHC protein was observed in day 1 damaged muscles. However, from days 2 and 3 onward dMHC was observed in the cytoplasm of mononucleated cells and myotubes in regenerating areas (Figure 3). As regeneration proceeded, dMHC was observed primarily in regenerating myotubes, and less frequently in mononucleated cells (Figure 3). Immunostaining with dMHC antibody identified a flattened morphology in the early regenerated myotubes. These early myotubes stained particularly intensely for dMHC protein. In day 4 muscle sections immunostained with dMHC antibody, newly regenerated myotubes were often closely juxtaposed around the circumference of earlier myotubes, suggesting that the earlier myotubes may be functioning as a scaffold for additional newly regenerated myotubes (data not shown).

Results show that the proportion of nuclei in dMHC-positive cells changed significantly over time (p<0.001; Figure 2), with a sharp increase in dMHC immunoreactivity up to day 3, followed by a more gradual increase to day 4. The IGF-II treatment group had significantly fewer nuclei in dMHC-positive cells relative to the control group on day 3 (p=0.047; Figures 2 and 3).

Binding to the Type 1 IGF Receptor
The initial delay in muscle regeneration in response to IGF-II administration suggested that there may have been a downregulation of the type 1 IGF receptor in response to the administered IGF-II. Therefore, receptor autoradiography was performed to determine levels of the type 1 IGF receptor in regenerating muscles from treated and control groups 1 day after damage. This time point was chosen because the earliest differences in the expression of myogenic markers in response to IGF-II were noted on day 1, suggesting a possible difference in receptor levels at or before this time. The results of this analysis show that the relative levels of IGF-binding were not significantly different between IGF-II-treated and control muscles (p=0.311; 0.137 ± 0.063 grains/µm² vs 0.247 ± 0.073 grains/µm², respectively).

Late Regeneration
Muscle fiber size was measured at the final sampling point to determine whether the delay in regeneration with IGF-II treatment, as previously noted on days 1 (MyoD), 2 (myogenin), and 3 (dMHC), was still apparent at day 7. Quantitation of the cross-sectional area of regenerating muscle fibers showed that treatment with IGF-II resulted in larger regenerated muscle fiber areas relative to control muscles on day 7 (p=0.0092), as shown in Figure 4 . The cross-sectional area of adjacent undamaged muscle fibers was also analyzed to determine whether the enhancement of fiber size was specific to the regenerating fibers only or whether it was an overall effect on fiber size. Results showed that there was no significant difference in the cross-sectional areas of IGF-II-treated and control undamaged muscle fibers on day 7 (p=0.543; 0.00224 ± 0.00030 mm² vs 0.001978 ± 0.00026 mm², respectively). This indicates that the enhancement of fiber size was specific to the regenerating fibers only.

View larger version (64K): [in this window] [in a new window]   Figure 4 IGF-II treatment increases muscle fiber size during late regeneration. Photomicrographs of muscle 7 days after notexin injection from control (A) and IGF-II-treated (B) animals. Hematoxylin and eosin stain. Bar = 100 µm. (C) Analysis of cross-sectional fiber areas shows that IGF-II treatment during regeneration significantly (**p=0.0092) increases muscle fiber size by day 7 after notexin injection. Values shown are the means (n=3/4) and the error bars show 1 SEM.

	Discussion

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The related peptide IGF-I is reported to increase satellite cell proliferation and differentiation after nerve crush injury in IGF-I transgenic mice (Rabinovsky et al. 2003) and to cause marked hyperplasia in diaphragms of transgenic mdx mice expressing IGF-I (Barton et al. 2002). IGF-II has been firmly established as a proliferation- and differentiation-enhancing peptide, with the evidence for this coming from a number of in vitro studies (Minniti et al. 1995; Stewart et al. 1996; Prelle et al. 2000). Given the body of evidence for positive effects of IGFs on proliferation and differentiation, the observation of an opposite effect in this regeneration experiment was surprising. Here we have reported induction times of less than 1 day for MyoD, 1–2 days for myogenin, and 2 days for dMHC protein in rat muscle. Peak values for the proportion of nuclei either containing (MyoD, myogenin) or surrounded by (dMHC) immunostaining were obtained on days 2, 3, and 4, respectively. This pattern of immunostaining is similar to the pattern of MyoD, myogenin, and dMHC immunostaining reported by Yablonka–Reuveni and Rivera (1994) for satellite cells on cultured rat muscle fibers. The effect of IGF-II administration on the markers of proliferation and differentiation were of a highly consistent sequential nature, with reductions from control levels observed by IHC on day 1 for MyoD, day 2 for myogenin, and day 3 for dMHC. In all cases this occurred before peak expression of these markers. This pattern, taken together with the fact that the effect of IGF-II treatment occurred as early as day 1, suggests that IGF-II may have affected a process up to or on day 1 after damage and that this initial setback of the regeneration process by IGF-II was carried on until at least the stage of myotube fusion. We therefore considered that the initial inhibition of regeneration by IGF-II may have been due to downregulation of the type 1 IGF receptor in response to the administered IGF.

IGF-II can downregulate the type 1 IGF receptor in muscle cells via transcriptional downregulation and/or increased receptor degradation (Rosenthal and Brown 1994; Rosenthal et al. 1994). IGFs bind to and exert effects through the type 1 IGF receptor during stimulation of MPC proliferation and differentiation (Ewton et al. 1987; Duclos et al. 1991). Functional inactivation of the type 1 IGF receptor also leads to delayed differentiation in mouse C2C12 cells (Cheng et al. 2000). In the present trial there was no difference in IGF binding capacity between IGF-II-treated and control muscles at day 1.

A further explanation of our results might be that the degree of damage by notexin differed between treatment groups. However, the amount of notexin injected into the biceps femoris muscle was carefully controlled and animals from the two treatment groups were operated on at the same time during surgery. Therefore, we consider this highly unlikely. Furthermore, there was no significant difference in the size of undamaged fibers on day 1 or in the total number of nuclei per unit area (overall or on days 1, 2, 3, or 4) in damaged areas between the IGF-II and control group muscles. Finally, if there was a lesser degree of damage in the IGF-II treatment group, a smaller absolute number of activated satellite cells would be expected, but if their activities are being measured per total nuclei, as they are in this study, the time course of their proliferation and differentiation would probably be earlier, not later, owing to a reduction in the time for phagocytic events to occur.

Our results suggest that, rather than affecting type 1 IGF receptor levels, IGF-II treatment affected another early process, such as phagocytosis and/or MPC activation. The rate of phagocytosis in damaged muscle can affect subsequent phases of regeneration (Grounds 1991), and in this study we observed histological evidence of delayed phagocytosis by way of increased necrotic debris on day 2, resulting from IGF-II treatment. A role for the IGF system in the modulation of inflammatory and phagocytic responses is supported by studies in which immune neutralization of endogenous IGF-I during muscle repair resulted in enhanced macrophage infiltration (Lefaucheur et al. 1996). In the present trial, however, we did not observe inhibition of phagocytosis until 1 day later than the observed decrease in the proportion of nuclei containing MyoD. The means by which macrophages contribute to the regeneration process is not limited merely to the removal of necrotic tissue but rather includes the production of growth factors that are capable of affecting MPCs (Grounds 1991; Layne and Farmer 1999). One such growth factor is tumor necrosis factor-alpha (TNF-) (Renier et al. 1996), a growth factor believed to be responsible for muscle wasting in a number of pathological conditions, including cancer (Meadows et al. 2000). Monocytes and macrophages upregulate TNF- in response to exogenous IGF-I in vitro (Renier et al. 1996) and TNF- inhibits IGF-I-induced stimulation of myogenesis in cultured C2C12 cells (Layne and Farmer 1999). These findings therefore lend support to the interaction of IGFs with phagocytic processes. A detailed examination of the different phagocytic cell populations within the 0–24-hr time period after notexin injection and IGF-II administration in a future study is warranted.

A biphasic effect of IGFs on myoblast differentiation in culture has been shown for both IGF-I and IGF-II, whereby high concentrations of IGFs inhibit differentiation and low concentrations of IGF promote differentiation (Florini et al. 1986). In cultured L6 myoblasts, inhibition of differentiation at high concentrations of IGF-I is dependent on the presence of high levels of type 1 IGF receptor (Quinn and Roh 1993). In the present study, in which MPC proliferation and differentiation were delayed, we administered 3.48 µg IGF-II per day, an amount chosen because it approximated the amount of IGF-II (on a per unit muscle basis) that was used in an earlier study showing an effect of systemic IGF-II on body weight gain (Conlon et al. 1995). In this study it was the only dose tested. Therefore, it is unknown whether administration of a lesser dose of IGF-II might have altered the outcome.

A beneficial effect of IGF-II during late regeneration was found in this study, whereby IGF-II treated fibers were larger in cross-sectional area at day 7 than control group fibers. This finding was precisely the opposite to what might have been anticipated given that early regeneration was delayed by the administration of IGF-II. This suggests that the administered IGF-II had a pronounced effect on the regenerated muscle between days 4 and 7, when one of the key processes occurring was fiber enlargement. Likewise, an increase in muscle fiber diameter has been reported for laceration-damaged muscle treated with IGF-I peptide (Menetrey et al. 2000) and a decrease in fiber diameter observed after treatment of damaged muscle with anti-IGF-I antibodies (Lefaucheur and Sébille 1995). In contrast to the increased fiber size observed in the regenerating muscle fibers from the IGF-II treatment group relative to the control group, the adjacent undamaged muscle fibers did not show a similar enlargement, indicating that the response to IGF-II was specific to the regenerating muscle fibers alone during that period rather than being a generalized hypertrophic-type response to IGF-II, such as has been documented for IGF-I (Adams and McCue 1998; Barton–Davis et al. 1999; Barton et al. 2002). A likely explanation for these results is that the administered IGF-II had an enhancing effect on reinnervation of the regenerating muscle fibers. IGF-II has positive effects on neural tissue, as illustrated by studies in which IGF-II administration to damaged nerves results in increased motorneuron survival and enhanced nerve regeneration (Near et al. 1992; Pu et al. 1999). Notexin injection causes denervation of 70% of the fibers in rat soleus muscle (Harris et al. 2000), and the ensuing functional innervation is complete by 7 days (Whalen et al. 1990). Sesodia and Cullen (1991) report that the regeneration of denervated and non-denervated rat soleus muscle is identical up to 3–4 days after notexin injection. However, after that time non-denervated muscles grow more rapidly than denervated muscles. Therefore, in the current study IGF-II administration may have enhanced reinnervation of the regenerating muscle, thus allowing more rapid growth of the regenerating muscle fibers between days 4 and 7 after damage. The positive effect of IGF-II on muscle regeneration during this period is of particular interest because this is the time in which endogenous IGF-II is normally expressed during notexin-induced regeneration in rats (Kirk et al. 1996). This suggests that a greater induction of endogenous IGF-II expression during this time could have a positive effect on muscle regeneration.

In summary, this study clearly shows that local administration of IGF-II to regenerating skeletal muscle results in delayed MPC proliferation and differentiation events, most likely due to an inhibition of phagocytic processes. Furthermore, muscle fiber enlargement during late regeneration is enhanced by the presence of exogenous IGF-II, possibly due to improved muscle fiber reinnervation. These findings indicate that IGF-II has pleiotropic effects in regenerating skeletal muscle, probably as a function of the unique environment present during the sequential steps that make up the whole of muscle regeneration.

	Acknowledgments

 
Supported by the Foundation for Research, Science and Technology of New Zealand.

We wish to thank S. Stuart, R. Broadhurst, G. Smith, and B. Smith for assistance with animal work, D. McGlynn for assistance with trial work and slide preparation, and Dr N. Cox for statistical analysis.

	Footnotes

 
Received for publication December 2, 2002; accepted July 30, 2003

	Literature Cited

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