The role of growth factors in the regeneration of skeletal muscle
The overall aim of this thesis was to examine myostatin and components of the insulin-like growth factor (IGF) axis during regeneration, to determine whether these factors were temporally regulated during regeneration, whether the absence or presence of growth hormone (GH) affected their levels, and lastly, whether the administration of exogenous IGF-II enhanced muscle regeneration. A histological approach was utilised, to determine specific effects on individual tissue types (regenerating muscle fibres, survivor muscle fibres, undamaged muscle fibres, and connective tissue) within damaged muscle. Regeneration was induced by injection of notexin, a myotoxin, into muscle. The first experiment tested the hypothesis that the IGFs and their receptors are regulated during muscle regeneration, and that the levels of these components are regulated by GH. The experimental model used was the GH-deficient dw/dw rat in which damage and subsequent regeneration were induced by a single intramuscular injection of notexin, then either GH- or saline-administered during the regeneration period. IGF-I and -II mRNA were assessed by in situ hybridisation, and binding determined by in vitro incubations with ¹²⁵I-IGF competed with unlabelled homologous IGF (specific binding). The presence of IGF binding proteins (IGFBPs) was determined by comparison of the specific binding of ¹²⁵I-IGF-I with the residual binding of ¹²⁵I-IGF-I following competition with des(l-3)IGF-I (which has a greatly reduced affinity for IGFBPs relative to IGF-I). Results of the localisation studies revealed up-regulation of IGF-I and -II specific binding in regenerating fibres at the time of myotube formation, and indicated the presence of IGFBPs in damaged muscle tissues at the same timepoint. IGF-I and -II mRNAs were significantly up-regulated in regenerating muscle fibres concurrent with myotube formation and enlargement, while IGF-I mRNA was also elevated in regenerating muscle fibres at the time of muscle precursor cell proliferation. IGF-I mRNA was elevated in connective tissue of damaged, relative to undamaged, muscle during early regeneration. GH administration increased bodyweight, and the weights of damaged and undamaged muscles. GH administration did not affect the level of specific binding when ¹²⁵I-IGFI was used as the ligand, or the level of IGFBPs as determined by competition of ¹²⁵I-IGF-I binding with unlabelled IGF-I versus unlabelled des(l-3)IGF-I, however GH administration did result in increased specific binding of ¹²⁵I-IGF-II to all damaged muscle tissues, relative to muscles from saline-treated animals. GH did not affect IGF-I or -II mRNA levels in damaged muscle tissues. In summary, this trial showed that all components of the IGF axis examined showed temporal regulation following muscle damage, and that GH administration significantly increased the binding of ¹²⁵I-IGF-II to damaged, but not undamaged, muscle tissues. The second hypothesis tested in this thesis is that the negative regulator of muscle growth, myostatin, is regulated during muscle regeneration, and that its levels are decreased in muscles undergoing enhanced growth due to the administration of GH. The temporal regulation of myostatin protein was assessed by immunohistochemical staining of regenerating muscle sections of Sprague-Dawley rats, and of GH-deficient dw/dw rats. The effect of GH on myostatin protein levels was determined by comparing myostatin protein levels in saline- versus OH-treated dw/dw rats. Myostatin immunostaining is present in the cytoplasm of fast muscle fibres, and is absent from the connective tissue of undamaged muscles, however following notexin injection, abundant myostatin immunostaining was observed at early timepoints in connective tissue, and high intensity immunostaining was observed in both fast and slow necrotic muscle fibres. Myostatin protein was absent from muscle precursor cells at the time of proliferation, and fusion to form new myotubes. Myostatin then gradually appeared in the muscle fibres undergoing enlargement. GH administration did not affect the temporal regulation, or level, or myostatin immunostaining observed. These findings suggest a role for myostatin in the regulation of muscle regeneration, including possible effects on connective tissue deposition. The third hypothesis tested was that administration of IGF-II peptide during muscle regeneration would advance the onset of muscle precursor cell proliferation and differentiation. This was tested by implanting miniosmotic pumps fitted with catheters and filled with either IGF-II (to deliver 3.48 ug IGF-II/day) or vehicle (equal volume), in the subcutaneous compartment of Sprague-Dawley rats, then inducing damage and regeneration adjacent to the site of peptide of release. Results of the immunohistochemical analysis of MyoD, myogenin and developmental myosin heavy chain proteins in damaged muscle sections showed that IGF-II administration resulted in a delay in the onset of muscle precursor cell proliferation and differentiation, as compared to vehicle only controls. In vitro incubations using ¹²⁵I-IGF-I as the ligand showed no difference in the IGF binding capacity of day 1 tissues, indicating that the delay in early regeneration was not caused by down-regulation of the Type 1 IGF receptor in response to administered IGF-II. Cross-sectional areas of regenerating muscle fibres on day 7 showed that late regeneration of muscle fibres was enhanced relative to the control, vehicle-only group. The period in which the administration of IGF-II enhanced muscle regeneration coincides with the time that IGF-II mRNA is elevated in regenerating muscle fibres, as shown in the first trial, suggesting that a greater endogenous production of IGF-II is associated with enhanced regeneration. In conclusion, the results of these studies indicate that changes in myostatin and components of the IGF axis are associated with muscle regeneration, and that the levels of these components are differentially regulated depending on tissue type. These studies suggest that IGF-II may be an effective therapeutic agent in regenerating skeletal muscle, pending refinement of the administration protocol.
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