Functional muscle hypertrophy by increased insulin-like growth factor 1 does not require dysferlin.

IntroductionDysferlin loss-of-function mutations cause muscular dystrophy, accompanied by impaired membrane repair and muscle weakness. Growth promoting strategies including insulin-like growth factor 1 (IGF-1) could provide benefit but may cause strength loss or be ineffective. The objective of this study was to determine whether locally increased IGF-1 promotes functional muscle hypertrophy in dysferlin-null (Dysf-/- ) mice.MethodsMuscle-specific transgenic expression and postnatal viral delivery of Igf1 were used in Dysf-/- and control mice. Increased IGF-1 levels were confirmed by enzyme-linked immunosorbent assay. Testing for skeletal muscle mass and function was performed in male and female mice.ResultsMuscle hypertrophy occurred in response to increased IGF-1 in mice with and without dysferlin. Male mice showed a more robust response compared with females. Increased IGF-1 did not cause loss of force per cross-sectional area in Dysf-/- muscles.DiscussionWe conclude that increased local IGF-1 promotes functional hypertrophy when dysferlin is absent and reestablishes IGF-1 as a potential therapeutic for dysferlinopathies

Type III collagen modulates fracture callus bone formation and early remodeling

Type III collagen (Col3) has been proposed to play a key role in tissue repair based upon its temporospatial expression during the healing process of many tissues, including bone. Given our previous finding that Col3 regulates the quality of cutaneous repair, as well as our recent data supporting its role in regulating osteoblast differentiation and trabecular bone quantity, we hypothesized that mice with diminished Col3 expression would exhibit altered long‐bone fracture healing. To determine the role of Col3 in bone repair, young adult wild‐type (Col3+/+) and haploinsufficent (Col3+/−) mice underwent bilateral tibial fractures. Healing was assessed 7, 14, 21, and 28 days following fracture utilizing microcomputed tomography (microCT), immunohistochemistry, and histomorphometry. MicroCT analysis revealed a small but significant increase in bone volume fraction in Col3+/− mice at day 21. However, histological analysis revealed that Col3+/− mice have less bone within the callus at days 21 and 28, which is consistent with the established role for Col3 in osteogenesis. Finally, a reduction in fracture callus osteoclastic activity in Col3+/− mice suggests Col3 also modulates callus remodeling. Although Col3 haploinsufficiency affected biological aspects of bone repair, it did not affect the regain of mechanical function in the young mice that were evaluated in this study. These findings provide evidence for a modulatory role for Col3 in fracture repair and support further investigations into its role in impaired bone healing. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:675–684, 2015.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/111249/1/jor22838.pd

Insulin-like growth factor-I E-peptide activity is dependent on the IGF-I receptor.

Insulin-like growth factor-I (IGF-I) is an essential growth factor that regulates the processes necessary for cell proliferation, differentiation, and survival. The Igf1 gene encodes mature IGF-I and a carboxy-terminal extension called the E-peptide. In rodents, alternative splicing and post-translational processing produce two E-peptides (EA and EB). EB has been studied extensively and has been reported to promote cell proliferation and migration independently of IGF-I and its receptor (IGF-IR), but the mechanism by which EB causes these actions has not been identified. Further, the properties of EA have not been evaluated. Therefore, the goals of this study were to determine if EA and EB possessed similar activity and if these actions were IGF-IR independent. We utilized synthetic peptides for EA, EB, and a scrambled control to examine cellular responses. Both E-peptides increased MAPK signaling, which was blocked by pharmacologic IGF-IR inhibition. Although the E-peptides did not directly induce IGF-IR phosphorylation, the presence of either E-peptide increased IGF-IR activation by IGF-I, and this was achieved through enhanced cell surface bioavailability of the receptor. To determine if E-peptide biological actions required the IGF-IR, we took advantage of the murine C2C12 cell line as a platform to examine the key steps of skeletal muscle proliferation, migration and differentiation. EB increased myoblast proliferation and migration while EA delayed differentiation. The proliferation and migration effects were inhibited by MAPK or IGF-IR signaling blockade. Thus, in contrast to previous studies, we find that E-peptide signaling, mitogenic, and motogenic effects are dependent upon IGF-IR. We propose that the E-peptides have little independent activity, but instead affect growth via modulating IGF-I signaling, thereby increasing the complexity of IGF-I biological activity

E-peptides inhibit myoblast differentiation.

<p><b>A–C</b>. C2C12 cells were grown to confluency and switched to differentiation media (Day 0). Media was changed every day and synthetic peptides (100 nM) were added to the fresh media. Quantitative RT-PCR was used to measure expression of differentiation markers: MyoD (<i>Myod</i>, A), Myogenin (<i>Myog</i>, B), Embryonic Myosin (<i>Myh3</i>, C). Expression of the markers at Days 1, 2, and 3 were compared to Day 0 to obtain fold change. Bars represent fold change means ± s.e.m. of N = 3 replicates. *, p<0.05 for fold change expression comparisons via 2-way ANOVA followed by a Bonferroni post-test. <b>D</b>. Synthetic EA and EB peptides were incubated with growth media and aliquots were taken at times indicated for immunoblotting analysis. <b>E–F</b>. Quantification of (D) and analysis of peptide half-life. Bars represent percent of original intensity ± s.e.m. of N = 3 replicates.</p

E-peptides augment IGF-IR activation and cell surface localization.

<p><b>A</b>. P6 cells overexpressing IGF-IR were treated with synthetic E-peptides with and without recombinant IGF-I for 15 minutes, and cell lysates were utilized for KIRA assays. Level of absorbance indicates the extent of IGF-IR phosphorylation. Bars represent means ± s.e.m. of N = 6 wells. <b>B</b>. OD 450 from A were compared to No Peptide for each IGF-I concentration, and the % change is graphed. <b>C</b>. P6 cells were treated as in A for a localization assay for times indicated, and biotin labeled before lysis. The optimal concentrations of E-peptides and IGF-I from the A were used (E-peptides 100 nM, IGF-I 10 nM). Surface IGF-IR was normalized to Total IGF-IR and compared to NoTx at t0 to get % IGF-IR on cell surface. Bars represent means ± s.e.m. of N = 6 wells. Samples were compared to no peptide (A and B,*), NoTx (C,*) or IGF-I (C,†) via 2-way ANOVA followed by a Bonferroni post-test. * or †, p<0.05; ***, p<0.001.</p

EA and EB increase MAPK signaling in C2C12 cells.

<p><b>A</b>. Cells were starved in media without serum, and treated with synthetic E-peptides at concentrations indicated for 20 minutes. Protein lysates were separated via SDS-PAGE and immunoblotted for Phosphorylated ERK 1 and 2 (P-ERK1/2), stripped, and blotted for Total ERK 1 and 2 (T-ERK1/2). <b>B–C</b>. Quantification of A. <b>D</b>. Cells were treated as above at optimal doses (EA and Scr 1 µM, EB 10 nM) for times indicated. <b>E–F</b> Quantification of C. NoTx at 30 minutes was included in each experiment for normalization between blots. For B–C and E–F, bars represent means ± s.e.m. of N = 3 replicates. *, p<0.05; ***, p<0.001, for comparisons to NoTx via 2-way ANOVA followed by a Bonferroni post-test.</p

E-peptides affect IGF-IR downstream signaling.

<p><b>A–C</b>. C2C12 cells were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045588#pone-0045588-g002" target="_blank">Figure 2</a>, with 0 nM or 2 nM IGF-I and optimal doses of the E-peptides (EA 1 µM, EB 10 nM) for 20 minutes. <b>B–C</b>. Quantification of Akt and ERK1/2 phosphorylation after EA (B) or EB (C) treatment. EA and EB alone are compared to NoTx, while IGF-I with E-peptides are compared to 2 nM IGF-I alone. Data are presented as the effect on phosphorylation after E-peptide treatment compared to NoTx or NoPeptide plus IGF-I. Bars represent means ± s.e.m. of N = 3–4 replicates. †, p<0.05 for comparisons of 0 nM IGF samples to their 2 nM IGF-I counterparts via student t-tests.</p

Synthetic E-peptide sequences.

<p><b>A</b>. Rodent <i>Igf1</i> 3′ splicing leads to two mRNA isoforms. While mature IGF-I is encoded by exons 3 and 4, the E-peptides are encoded by exons 4, 5, and/or 6. EA isoforms exclude exon 5, while EB isoforms retain exon 5, leading to an altered reading frame and earlier stop codon in exon 6. Exons not drawn to scale <b>B</b>. Synthetic E-peptide amino acid sequences. EA and EB are less than 50% identical. Scr = Scrambled peptide. * = potential glycosylation sites in EA. The portion of EB that corresponds to MGF is <u>underlined</u>.</p

EB increases in myoblast proliferation and migration are MAPK and IGF-IR signaling dependent.

<p><b>A</b>. C2C12 cells were plated in 96 well plates, starved for 6 hours, and treated with synthetic E-peptides. A BrdU plate assay was used to quantify proliferating cells, where increased absorbance is correlated with increased proliferation. Bars represent means ± s.e.m. of N = 10 wells. <b>B</b>. A similar BrdU assay was used to visualize the proliferating cells on slides. Cells were treated as above (EA and Scr 100 nM, EB and IGF 10 nM), fixed, and stained with BrdU and DAPI. Total cells and proliferating (BrdU positive) cells were counted from 3 10× fields for each slide, and bars represent means ± s.e.m. of N = 5 slides. For A and B, *, p<0.05; ***, p<0.001 for comparisons to NoTx via 1-way ANOVA followed by a Tukey post-hoc test. <b>C</b>. C2C12 cells were tested as in A, except an inhibitor of MEK, PD 098059 (PD, 50 µM) or IGF-IR (NVP, 100 nM) was included in the cell media. Bars represent means ± s.e.m. of N = 18 wells for No Inhibitor (No Inh) and N = 8 for with inhibitors. <b>D</b>. C2C12 cells were plated in the upper chamber of 24-well plate trans-well migration inserts in 0% serum media. Cells were allowed to migrate for 5 hours and stained with DAPI, imaged and counted. Synthetic E-peptides (100 nM) were added to upper and lower chambers with or without inhibitors (PD 50 µM, NVP 100 nM). Images were taken as in B, and bars represent means ± s.e.m. of N = 4 slides. For C and D, *, p<0.05; ***, p<0.001 for comparisons to NoTx via 2-way ANOVA followed by a Bonferroni post-test. †, p<0.05 for comparisons to No Inh via 2-way ANOVA followed by a Bonferroni post-test.</p