Age-related loss of skeletal muscle function; impairment of gene expression

Mechano Growth Factor (MGF) is derived from the insulin-like growth factor (IGF-I) but its sequence differs from the systemic IGF-I produced by the liver. MGF is expressed by mechanically overloaded muscle and is involved in tissue repair and adaptation. It is expressed as a pulse following muscle damage and involved in the activation of muscle satellite (stem) cells. These donate nuclei to the muscle fibers that are required for repair and for the hypertrophy processes which may have similar regulatory mechanisms. Muscles in the elderly are unable to upregulate MGF in response to exercise. This is also true in certain diseases and this helps to explain muscle loss in those conditions. There is evidence that MGF is a local tissue repair factor as well as a growth factor and that it has an important role in damage limitation and inducing repair in other post-mitotic tissues. As there is no cell replacement in these tissues there has to be an effective local cellular repair mechanism. With advancing years this seems to become deficient and there is an increased chance that the damaged cells will undergo cell death leading to progressive loss of tissue function.     

The IGF-I splice variant MGF increases progenitor cells in ALS, dystrophic, and normal muscle

The effects of muscle splice variants of insulin-like growth factor I (IGF-I) on proliferation and differentiation were studied in human primary muscle cell cultures from healthy subjects as well as from muscular dystrophy and ALS patients. Although the initial numbers of mononucleated progenitor cells expressing desmin were lower in diseased muscle, the E domain peptide of IGF-IEc (MGF) significantly increased the numbers of progenitor cells in healthy and diseased muscle. IGF-I significantly enhances myogenic differentiation whereas MGF E peptide blocks this pathway, resulting in an increased progenitor (stem) cell pool and thus potentially facilitating repair and maintenance of this postmitotic tissue.     

Insulin-Like Growth Factor I (IGF-1) Ec/Mechano Growth Factor – A Splice Variant of IGF-1 within the Growth Plate

Human insulin-like growth factor 1 Ec (IGF-1Ec), also called mechano growth factor (MGF), is a splice variant of insulin-like growth factor 1 (IGF-1), which has been shown in vitro as well as in vivo to induce growth and hypertrophy in mechanically stimulated or damaged muscle. Growth, hypertrophy and responses to mechanical stimulation are important reactions of cartilaginous tissues, especially those in growth plates. Therefore, we wanted to ascertain if MGF is expressed in growth plate cartilage and if it influences proliferation of chondrocytes, as it does in musculoskeletal tissues. MGF expression was analyzed in growth plate and control tissue samples from piglets aged 3 to 6 weeks. Furthermore, growth plate chondrocyte cell culture was used to evaluate the effects of the MGF peptide on proliferation. We showed that MGF is expressed in considerable amounts in the tissues evaluated. We found the MGF peptide to be primarily located in the cytoplasm, and in some instances, it was also found in the nucleus of the cells. Addition of MGF peptides was not associated with growth plate chondrocyte proliferation.     

The structure-function relationships of insulin-like growth factor 1 Ec in C2C12 cells

Insulin-like growth factor 1 (IGF1) is a crucial growth factor, that regulates skeletal muscles development during cell growth and repair. Recently, its alternative splicing variant, named IGF1Ec, also named mechano-growth factor (MGF), has gained attentions as a new damage repair factor. However, the structure-function relationships of IGF1Ec have not been fully clarified due to contradictory reports. In this study, we systematically investigated physiologic responses of C2C12 muscle cells to IGF1Ec, IGF1 and MGF E peptide. Our data indicate that while the N-terminal sequence of IGF1Ec, which is homolog in part with IGF1, promotes proliferation; the C-terminal sequence of IGF1Ec, which is identical to MGF E, promotes differentiation and migration of C2C12 cells. Our results suggest that MGF E cannot completely replace all the functions of IGF1Ec on muscle repair and regeneration, and elucidate the relationships between structure and function of IGF1Ec.     

Expression of muscle anabolic and metabolic factors in mechanically loaded MLO-Y4 osteocytes

Lack of physical activity results in muscle atrophy and bone loss, which can be counteracted by mechanical loading. Similar molecular signaling pathways are involved in the adaptation of muscle and bone mass to mechanical loading. Whether anabolic and metabolic factors regulating muscle mass, i.e., insulin-like growth factor-I isoforms (IGF-I Ea), mechano growth factor (MGF), myostatin, vascular endothelial growth factor (VEGF), or hepatocyte growth factor (HGF), are also produced by osteocytes in bone in response to mechanical loading is largely unknown. Therefore, we investigated whether mechanical loading by pulsating fluid flow (PFF) modulates the mRNA and/or protein levels of muscle anabolic and metabolic factors in MLO-Y4 osteocytes. Unloaded MLO-Y4 osteocytes expressed mRNA of VEGF, HGF, IGF-I Ea, and MGF, but not myostatin. PFF increased mRNA levels of IGF-I Ea (2.1-fold) and MGF (2.0-fold) at a peak shear stress rate of 44Pa/s, but not at 22Pa/s. PFF at 22 Pa/s increased VEGF mRNA levels (1.8- to 2.5-fold) and VEGF protein release (2.0- to 2.9-fold). Inhibition of nitric oxide production decreased (2.0-fold) PFF-induced VEGF protein release. PFF at 22 Pa/s decreased HGF mRNA levels (1.5-fold) but increased HGF protein release (2.3-fold). PFF-induced HGF protein release was nitric oxide dependent. Our data show that mechanically loaded MLO-Y4 osteocytes differentially express anabolic and metabolic factors involved in the adaptive response of muscle to mechanical loading (i.e., IGF-I Ea, MGF, VEGF, and HGF). Similarly to muscle fibers, mechanical loading enhanced expression levels of these growth factors in MLO-Y4 osteocytes. Although in MLO-Y4 osteocytes expression levels of IGF-I Ea and MGF of myostatin were very low or absent, it is known that the activity of osteoblasts and osteoclasts is strongly affected by them. The abundant expression levels of these factors in muscle cells, in combination with low expression in MLO-Y4 osteocytes, provide a possibility that growth factors expressed in muscle could affect signaling in bone cells.     

Expression of insulin-like growth factor I, insulin-like growth factor binding proteins, and collagen mRNA in mechanically loaded plantaris tendon.

Insulin-like growth factor I (IGF-I) is known to exert an anabolic effect on tendon fibroblast production of collagen. IGF-I's regulation is complex and involves six different IGF binding proteins (IGFBPs). Of these, IGFBP-4 and -5 could potentially influence the effect of IGF-I in the tendon because they both are produced in fibroblast; however, the response of IGFBP-4 and -5 to mechanical loading and their role in IGF-I regulation in tendinous tissue are unknown. A splice variant of IGF-I, mechano-growth factor (MGF) is upregulated and known to be important for adaptation in loaded muscle. However, it is not known whether MGF is expressed and upregulated in mechanically loaded tendon. This study examined the effect of mechanical load on tendon collagen mRNA in relation to changes in the IGF-I systems mRNA expression. Data were collected at 2, 4, 8 and 16 days after surgical removal of synergistic muscle to the plantaris muscle of the rat, thus increasing the load to plantaris muscle and tendon. Nearly a doubling of the tendon mass was observed after 16 days of loading. A rapid rise in tendon procollagen III mRNA was seen after 2 days whereas the increase in procollagen I mRNA was significant from day 8. MGF was expressed and upregulated in loaded tendon tissue with a faster response than IGF-I, which was increased from day 8. Finally, IGFBP-4 mRNA was increased with a time pattern similar to procollagen III, whereas IGFBP-5 decreased at day 8. In conclusion, loading of tendon tissue results in an upregulation of IGF-I, IGFBP-4, and procollagen and is associated with an increase in tendon mass. Also, MGF is expressed with an early upregulation in loaded tendon tissue. We suggest that the IGF-I system could be involved in collagen synthesis in tendon in response to mechanical loading.     

A novel mechanical loading model for studying the distributions of strain and mechano-growth factor expression

Mechano-growth factor (MGF), an insulin-like growth factor-I (IGF-I) splice variant, often serves as an important local tissue repair factor in response to the mechanical environment. However, there is no model for exhibiting the MGF expression in a series of strain distribution up to now. In this study, a novel mechanical loading model containing different stresses and strains simultaneously was developed to examine the MGF expression. The strain distributions were predicted by finite element modeling. The MC3T3-E1 cells on a silicone membrane with a central circular hole were exposed to a variable strain environment through stretching. The finite element analysis showed that, when the strain reached the magnitude of 10%, the strain concentration near the circular hole displayed along with the vertical stretch direction, while the minimum strain appeared in the parallel stretch direction. Furthermore, the results showed that MGF expression decreased gradually from high to low strain regions by immunocytochemistry. Meanwhile, the proliferation of osteoblasts increased significantly in the high strain region. In conclusion, this mechanical loading model can present the different distributions of the strain of osteoblasts in vitro. MGF expression and osteoblast proliferation have a high correlation with the levels of strain.     

机械生长因子E肽的功能及研究展望

机械生长因子(MGF)E肽是胰岛素样生长因子Ⅰ(IGF-Ⅰ)基因剪接后的一段长40个氨基酸残基的延伸肽,其编码基因由IGF-Ⅰ基因的外显子5、6及部分外显子4组成。近年来的实验证明,MGFE肽能独立发挥促进肌肉肥大、修复肌肉损伤、保护神经元、提高心脏功能等多种重要的生理作用,有望对肌肉萎缩、肌营养不良、神经退行性疾病及大脑局部缺血等相关病症的新型药物开发产生重大推动,引起了国内外学者的广泛关注。     

Mechanical signals and IGF-I gene splicing in vitro in relation to development of skeletal muscle

It has been shown that the insulin-like growth factor (IGF-I) gene is spliced in response to mechanical signals producing forms of IGF-I which have different actions. In order to study how mechanical signals influence this gene splicing in developing muscle, C2C12 cells were grown in three-dimensional (3D) culture and subjected to different regimens of mechanical strain. IGF-IEa which initiates the fusion of myoblasts to form myotubes was found to be constitutively expressed in myoblasts and myotubes (held under endogenous tension) and its expression upregulated by a single ramp stretch of 1-h duration but reduced by repeated cyclical stretch. In contrast, mechano growth factor (MGF), which is involved in the proliferation of mononucleated myoblasts that are required for secondary myotube formation and to establish the muscle satellite (stem) cell pool, showed no significant constitutive expression in static cultures, but was upregulated by a single ramp stretch and by cycling loading. The latter types of force simulate those generated in myoblasts by the first contractions of myotubes. These data indicate the importance of seeking to understand the physiological signals that determine the ratios of splice variants of some growth factor/tissue factor genes in the early stages of development of skeletal muscle.     

Stimulation of mechano-growth factor expression by myofibrillar proteins in murine myoblasts and myotubes

Mechano-growth factor (MGF) is a product of alternative splicing of the insulin-like growth factor 1 (IGF-1) mRNA. MGF is known to stimulate myoblast proliferation and to protect neurons and cardiomyocytes from apoptosis. MGF expression is dramatically increased in response to mechanical stimuli and tissue damage. The mechanisms of induction of MGF expression are as yet imperfectly understood. There is certain evidence that some protein factors able to stimulate MGF synthesis in normal myoblasts are released from damaged muscle. This study was undertaken to explore the nature of these protein inductors of MGF expression and to investigate the mechanism of their action. We report here that myofibrillar fraction of skeletal muscle homogenate activated MGF expression in murine myoblasts and myotubes in culture. The expression of another splice form of IGF-1 gene, IGF-1Ea, was also stimulated by myofibrils. Three myofibrillar proteins able to stimulate MGF synthesis were isolated. These proteins were identified by MALDI and immunoblotting as myomesin, myosin-binding protein C, and titin. The activation of MGF expression was associated with the increase of cAMP level in the cells. Inhibitor of adenylyl cyclase dideoxyadenosine arrested stimulation of MGF synthesis by all three myofibrillar proteins.     

Cells migrating to sites of tissue damage in response to the danger signal HMGB1 require NF-kappaB activation

Tissue damage is usually followed by healing, as both differentiated and stem cells migrate to replace dead or damaged cells. Mesoangioblasts (vessel-associated stem cells that can repair muscles) and fibroblasts migrate toward soluble factors released by damaged tissue. Two such factors are high mobility group box 1 (HMGB1), a nuclear protein that is released by cells undergoing unscheduled death (necrosis) but not by apoptotic cells, and stromal derived factor (SDF)-1/CXCL12. We find that HMGB1 activates the canonical nuclear factor kappaB (NF-kappaB) pathway via extracellular signal-regulated kinase phosphorylation. NF-kappaB signaling is necessary for chemotaxis toward HMGB1 and SDF-1/CXCL12, but not toward growth factor platelet-derived growth factor, formyl-met-leu-phe (a peptide that mimics bacterial invasion), or the archetypal NF-kappaB-activating signal tumor necrosis factor alpha. In dystrophic mice, mesoangioblasts injected into the general circulation ingress inefficiently into muscles if their NF-kappaB signaling pathway is disabled. These findings suggest that NF-kappaB signaling controls tissue regeneration in addition to early events in inflammation.     

Insulin-like growth factor-I extends in vitro replicative life span of skeletal muscle satellite cells by enhancing G1/S cell cycle progression via the activation of phosphatidylinositol 3'-kinase/Akt signaling pathway

Interest is growing in methods to extend replicative life span of non-immortalized stem cells. Using the insulin-like growth factor I (IGF-I) transgenic mouse in which the IGF-I transgene is expressed during skeletal muscle development and maturation prior to isolation and during culture of satellite cells (the myogenic stem cells of mature skeletal muscle fibers) as a model system, we elucidated the underlying molecular mechanisms of IGF-I-mediated enhancement of proliferative potential of these cells. Satellite cells from IGF-I transgenic muscles achieved at least five additional population doublings above the maximum that was attained by wild type satellite cells. This IGF-I-induced increase in proliferative potential was mediated via activation of the phosphatidylinositol 3'-kinase/Akt pathway, independent of mitogen-activated protein kinase activity, facilitating G(1)/S cell cycle progression via a down-regulation of p27(Kip1). Adenovirally mediated ectopic overexpression of p27(Kip1) in exponentially growing IGF-I transgenic satellite cells reversed the increase in cyclin E-cdk2 kinase activity, pRb phosphorylation, and cyclin A protein abundance, thereby implicating an important role for p27(Kip1) in promoting satellite cell senescence. These observations provide a more complete dissection of molecular events by which increased local expression of a growth factor in mature skeletal muscle fibers extends replicative life span of primary stem cells than previously known.     

Viral expression of insulin-like growth factor-I isoforms promotes different responses in skeletal muscle.

Insulin-like growth factor I (IGF-I) is a critical protein for skeletal muscle development and regeneration. Its ability to promote skeletal muscle hypertrophy has been demonstrated by several methods. Alternative splicing of the Igf-1 gene does not affect the mature IGF-I protein but does produce different E peptide extensions, which have been reported to modify the potency of IGF-I. Viral-mediated delivery of murine IGF-IA and IGF-IB into skeletal muscle of 2-wk-old and 6-mo-old mice was utilized to compare the effects of the isoforms on muscle mass. In young mice, tissue content of IGF-I protein was significantly higher in rAAV-treated muscles than control muscles at 1, 2, and 4 mo postinjection. Viral injection of IGF-IB produced two- to sevenfold more IGF-I than rAAVIGF-IA. Hypertrophy was observed 2 and 4 mo postinjection, where both rAAVIGF-IA and rAAVIGF-IB were equally effective in increasing muscle mass. These results suggest that there is a threshold of IGF-I production necessary to promote muscle hypertrophy in young growing animals regardless of isoform. In 6-mo-old animals, only rAAVIGF-IA produced significant increases in muscle size, even though increased IGF-I content was observed after injection of both isoforms. Therefore, the ability for IGF-IB to promote muscle hypertrophy is only effective in growing animals, suggesting that the bioavailability of this isoform or its receptor affinity diminishes with age.     

MGF E peptide improves anterior cruciate ligament repair by inhibiting hypoxia-induced cell apoptosis and accelerating angiogenesis

Severe hypoxic microenvironment endangers cell survival of anterior cruciate ligament (ACL) fibroblasts and is harmful to ACL repair and regeneration. In the current study, we explored the effects of mechanogrowth factor (MGF) E peptide on the hypoxia-induced apoptosis of ACL fibroblasts and relevant mechanisms. It demonstrated that severe hypoxia promoted hypoxia-inducible factor-1α (HIF-1α) expression and caused cell apoptosis of ACL fibroblasts through increasing caspase 3/7/9 messenger RNA (mRNA), cleaved caspase 3 and proapoptotic proteins expression levels but decreasing antiapoptotic proteins expression levels. Fortunately, MGF E peptide effectively protected ACL fibroblasts against hypoxia-induced apoptosis through regulating caspase 3/7/9 mRNA, cleaved caspase 3 and apoptosis-relevant proteins expression levels. Simultaneously, mitochondrial, @@@MEK-ERK1/2 (extracellular-signal-regulated kinase 1/2), and phosphoinositide-3-kinase-protein kinase B (PI3K-Akt) pathways were involved in MGF E peptide regulating hypoxia-induced apoptosis of ACL fibroblasts. In rabbit ACL rupture model, MGF E peptide also decreased HIF-1α expression levels, cell apoptosis, and facilitated cell proliferation. In addition, MGF could accelerate angiogenesis after ACL injury probably owing to its recruitment of proangiogenesis cells by stromal cell-derived factor 1α/CXCR4 axis and stimulation of vascular endothelial growth factor α expression level. In conclusion, our findings suggested that MGF E peptide could be utilized for ACL repair and regeneration and supplied experimental support for its application in clinical ACL treatment as a potential strategy.     

Mechanical signal transduction in skeletal muscle growth and adaptation.

The adaptability of skeletal muscle to changes in the mechanical environment has been well characterized at the tissue and system levels, but the mechanisms through which mechanical signals are transduced to chemical signals that influence muscle growth and metabolism remain largely unidentified. However, several findings have suggested that mechanical signal transduction in muscle may occur through signaling pathways that are shared with insulin-like growth factor (IGF)-I. The involvement of IGF-I-mediated signaling for mechanical signal transduction in muscle was originally suggested by the observations that muscle releases IGF-I on mechanical stimulation, that IGF-I is a potent agent for promoting muscle growth and affecting phenotype, and that IGF-I can function as an autocrine hormone in muscle. Accumulating evidence shows that at least two signaling pathways downstream of IGF-I binding can influence muscle growth and adaptation. Signaling via the calcineurin/nuclear factor of activated T-cell pathway has been shown to have a powerful influence on promoting the slow/type I phenotype in muscle but can also increase muscle mass. Neural stimulation of muscle can activate this pathway, although whether neural activation of the pathway can occur independent of mechanical activation or independent of IGF-I-mediated signaling remains to be explored. Signaling via the Akt/mammalian target of rapamycin pathway can also increase muscle growth, and recent findings show that activation of this pathway can occur as a response to mechanical stimulation applied directly to muscle cells, independent of signals derived from other cells. In addition, mechanical activation of mammalian target of rapamycin, Akt, and other downstream signals is apparently independent of autocrine factors, which suggests that activation of the mechanical pathway occurs independent of muscle-mediated IGF-I release.     

Pretreatment with mechano-growth factor E peptide protects bone marrow mesenchymal cells against damage by fluid shear stress

Improper fluid shear stress (FSS) can cause serious damages to bone marrow mesenchymal stem cells (MSCs). Mechano-growth factor (MGF) E peptide pretreatment was proposed to protect MSCs against FSS damage in this study. MSCs were exposed to FSS for 30 min after they were pretreated with MGF E peptide for 24 h. Then, the effects of MGF E peptide on the viability, proliferation and cell apoptosis of MSCs were investigated. MGF E peptide pretreatment could recover the cellular metabolic activity of MSCs reduced by 72 dyne cm^?2 FSS and had a synergistic effect with FSS on the cellular metabolic viability of MSCs under 24 and 72 dyne cm^?2 FSS. These results suggested that MGF E peptide pretreatment could be an effective method for the protection of FSS damage in bone tissue engineering.     

Actions of insulin-like growth factor-I on the B104 neuronal cell line: effects on cell replication, receptor characteristics, and influence of secreted binding protein on ligand binding

Several peptide growth factors influence the growth and differentiation of neural cells. To investigate further the growth-promoting effects of the somatomedins on cells of neural origin, the authors characterized the binding and mitogenic effects of insulin-like growth factor-I (IGF-I) on a functionally differentiated rat neuronal cell line (B104). Specific, high-affinity (Kd approximately equal to 10(-9) M) receptors for IGF-I were abundant (approximately 124,000 binding sites/B104 cell). These IGF-I receptors were similar to those of non-neural tissue in that they contained 135,000 dalton binding subunits (demonstrated by affinity labeling and autoradiography) and recognized insulin at high concentrations. IGF-I was more potent than insulin at stimulating B104 cell replication in serum-free medium and, at an initial concentration of 100 ng/ml, was the only exogenous growth factor needed to maintain growth through several cell divisions. Furthermore, cells of later passage were found to secrete specific IGF binding proteins that produced an unusual, biphasic binding curve in radioligand displacement studies. These binding proteins apparently sequester IGF-I, limiting its access to the cell. Experiments with B104 cells may provide useful information about the role of IGFs and their binding proteins as potential regulators of growth and differentiation of the primitive neuroblast.     

A synthetic mechano-growth factor E peptide promotes rat tenocyte migration by lessening cell stiffness and increasing F-actin formation via the FAK-ERK1/2 signaling pathway

Tendon injuries are common in sports and are frequent reasons for orthopedic consultations. The management of damaged tendons is one of the most challenging problems in orthopedics. Mechano-growth factor (MGF), a recently discovered growth repair factor, plays positive roles in tissue repair through the improvement of cell proliferation and migration and the protection of cells against injury-induced apoptosis. However, it remains unclear whether MGF has the potential to accelerate tendon repair. We used a scratch wound assay in this study to demonstrate that MGF-C25E (a synthetic mechano-growth factor E peptide) promotes the migration of rat tenocytes and that this promotion is accompanied by an elevation in the expression of the following signaling molecules: focal adhesion kinase (FAK) and extracellular signal regulated kinase1/2 (ERK1/2). Inhibitors of the FAK and ERK1/2 pathways inhibited the MGF-C25E-induced tenocyte migration, indicating that MGF-C25E promotes tenocyte migration through the FAK-ERK1/2 signaling pathway. The analysis of the mechanical properties showed that the Young's modulus of tenocytes was decreased through treatment of MGF-C25E, and an obvious formation of pseudopodia and F-actin was observed in MGF-C25E-treated tenocytes. The inhibition of the FAK or ERK1/2 signals restored the decrease in Young's modulus and inhibited the formation of pseudopodia and F-actin. Overall, our study demonstrated that MGF-C25E promotes rat tenocyte migration by lessening cell stiffness and increasing pseudopodia formation via the FAK-ERK1/2 signaling pathway.     

Fibroblast growth factor inhibits insulin-like growth factor-II (IGF-II) gene expression and increases IGF-I receptor abundance in BC3H-1 muscle cells.

Muscle is an important target tissue for insulin-like growth factor (IGF) action. We have previously reported that muscle cell differentiation is associated with down-regulation of the IGF-I receptor at the level of gene expression that is concomitant with an increase in the expression and secretion of IGF-II. Furthermore, treatment of myoblasts with IGF-II resulted in a similar decrease in IGF-I receptor mRNA abundance, suggesting an autocrine role of IGF-II in IGF-I receptor regulation. To explore further the role of IGF-II in IGF-I receptor regulation, BC3H-1 mouse muscle cells were exposed to differentiation medium in the presence of basic fibroblast growth factor (FGF), a known inhibitor of myogenic differentiation. FGF treatment of cells resulted in a 50% inhibition of IGF-II gene expression compared to that in control myoblasts and markedly inhibited IGF-II secretion. Concomitantly, FGF resulted in a 60-70% increase in IGF-I binding compared to that in control myoblasts. Scatchard analyses and studies of gene expression demonstrated that the increased IGF-I binding induced by FGF reflected parallel increases in IGF-I receptor content and mRNA abundance. These studies indicate that FGF may up-regulate IGF-I receptor expression in muscle cells through inhibition of IGF-II peptide expression and further support the concept of an autocrine role of IGF-II in IGF-I receptor regulation. In addition, these studies suggest that one mechanism by which FGF inhibits muscle cell differentiation is through inhibition of IGF-II expression.