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【摘要】
Administration of recombinant human insulin-like growth factor-I (rhIGF-I) has beneficial effects in animal models of muscle injury and muscular dystrophy. However, the results of these studies may have been confounded by interactions of rhIGF-I with endogenous IGF-binding proteins (IGFBPs). To date, no study has examined whether inhibiting IGFBP interactions with endogenous IGF-I can improve muscle fiber regeneration or muscular pathologies. We tested the hypothesis that reducing IGFBP interactions with endogenous IGF-I would enhance muscle regeneration after myotoxic injury and improve the dystrophic pathology in mdx mice. We administered an IGF-I aptamer (NBI-31772; 6 mg/kg per day, continuous infusion) to C57BL/10 mice undergoing regeneration after myotoxic injury or to mdx dystrophic mice. NBI-31772 binds all six IGFBPs with high affinity and releases "free" endogenous IGF-I. NBI-31772 treatment increased the rate of functional repair in fast-twitch tibialis anterior muscles after notexin-induced injury as evidenced by an increase in maximum force producing capacity (Po) at 10 days after injury. In contrast, NBI-31772 administration for 28 days did not alter Po of extensor digitorum longus (EDL) and soleus muscles or normalized force of diaphragm muscle strips from mdx mice. Although IGFBP inhibition reduced the susceptibility of the fast-twitch EDL and the diaphragm muscle to contraction-mediated damage, it increased muscle fatigability during repeated maximal contractions. Although the results in the myotoxic injury model suggest IGF-I signaling is important in this model, the results in the mdx model are mixed.
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Skeletal muscle injury and degeneration can result from trauma caused by mechanical (laceration, crush, strain), chemical (myotoxin), or metabolic (temperature) factors and is a major contributor to the etiology of myopathies, such as the muscular dystrophies. Although muscle fibers have an inherent capacity to regenerate when damaged, the regeneration process is often slow, inefficient, and incomplete with respect to functional restoration.1 Treatment strategies to enhance the regenerative capacity of muscle are significant for patients suffering from muscle injuries and other muscle wasting conditions.
The muscular dystrophies define a group of genetic disorders characterized by progressive skeletal muscle wasting and weakness. The most severe and rapidly progressing of these conditions is Duchenne muscular dystrophy, affecting 1 in 3500 males born worldwide.2 The absence of a cytoskeletal protein, dystrophin, renders dystrophic muscles extremely fragile and easily damaged by everyday activities, especially by contractions where muscles are activated and then stretched forcibly.3,4 This increased susceptibility to contraction-mediated injury results in ongoing cycles of muscle fiber degeneration and less than adequate regeneration and contributes to an etiology of progressive muscle wasting and weakness.5
Although animal models can reproduce some (or most) of the pathologies associated with muscular dystrophies, models of muscle injury such as crush or strain injuries can be problematic especially for generating reproducible responses in animal models. On the other hand, injecting muscles with a myotoxic agent such as notexin or cardiotoxin results in complete and reproducible muscle fiber degeneration6 and the ability to monitor the events of and mechanisms controlling muscle fiber regeneration.
Regardless of the initial injury, effective muscle regeneration is dependent on the timed induction of myogenic regulatory factors and growth factors, including insulin-like growth factor-I (IGF-I).1,7 IGF-I activates both myoblast proliferation and subsequently differentiation, two processes that are crucial to muscle repair and regeneration.8,9 The importance of IGF-I in skeletal muscle regeneration has been demonstrated in transgenic mice, where overexpression of IGF-I localized to skeletal muscle maintained regenerative capacity in aged mice10 and reduced the dystrophic pathology in mdx mice, an animal model of Duchenne muscular dystrophy.11,12 In addition, we have shown previously that exogenous administration of recombinant human IGF-I (rhIGF-I) increased the rate of functional recovery after myotoxic injury13 and improved the dystrophic pathology in mdx mice.14-16 Although rhIGF-I administration and transgenic IGF-I overexpression have beneficial effects on skeletal muscle, their mechanism of action seems to be vastly different. For example in mice, transgenic IGF-I overexpression resulted in marked muscle hypertrophy,11 an effect not observed following rhIGF-I administration.14-16 This discrepancy is likely due to different effects of IGF-binding proteins (IGFBPs) on IGF-I in the bloodstream (systemic delivery) compared with muscle-specific transgenic overexpression of IGF-I.
The biological actions of IGF-I in vivo are strongly mediated by IGFBPs, which bind 99% of IGF-I in the circulation.17 This large reservoir of bound, biologically inactive IGF-I has a prolonged half-life and is protected from degradation.17-19 IGFBPs are believed to primarily transport IGF-I to target tissues, modulate IGF-I action, and prevent hypoglycemia.17-19 In general, IGFBPs are thought to inhibit IGF-I actions in vitro and in vivo by preventing IGF-I binding to cell surface receptors.20-22
Although the effects of rhIGF-I administration and IGF-I overexpression on skeletal muscle regeneration have been well characterized,10-12,14-16 the role of IGFBPs in skeletal muscle regeneration remains poorly understood. Thus, the aim of the current study was to examine the effects of IGFBP inhibition on muscle regeneration after myotoxic injury and also in mdx dystrophic mice. We tested the hypothesis that inhibition of endogenous IGFBPs would enhance skeletal muscle regeneration and improve the dystrophic pathology in a manner similar to exogenous rhIGF-I administration.
【关键词】 modulation insulin-like igf-binding interactions enhances skeletal regeneration ameliorates dystrophic pathology
Materials and Methods
Experimental Animals
All procedures were approved by the Animal Experimentation Ethics Committee of The University of Melbourne and conformed to the Guidelines for the Care and Use of Experimental Animals as described by the National Health and Medical Research Council of Australia (Canberra, ACT, Australia). To examine the effect of IGFBP inhibition on muscle regeneration, male C57BL/10ScSn (BL/10) mice (12 to 14 weeks old, n > 5 per group) were used. To determine the role of IGFBPs in dystrophic skeletal muscles, male C57BL/10ScSn-mdx/J dystrophic (mdx) mice (8 to 10 weeks old, n = 8 per group) were used. All mice were obtained from the Animal Resource Centre (Canning Vale, WA, Australia) and housed in the Biological Research Facility at The University of Melbourne under a 12-hour light/dark cycle, with drinking water and standard chow provided ad libitum. For both experiments treated mice received continuous subcutaneous (s.c.) administration of the IGF-I aptamer NBI-31772 (6 mg/kg per day in 50% dimethyl sulfoxide and 50% polyethylene glycol), whereas control mice received vehicle alone (50% dimethyl sulfoxide and 50% polyethylene glycol). Continuous systemic delivery of NBI-31772 (or vehicle alone) was achieved by subcutaneous implantation of a micro-osmotic pump (Alzet model 1002; Alzet, Cupertino, CA) as described previously.14 NBI-31772 is a nonpeptide ligand that has high-affinity binding for all six IGF binding proteins,23 and in vitro binding assays have revealed that NBI-31772 displaces IGF-I from its interaction with binding proteins, thus releasing "free" biologically active IGF-I.24 BL/10 mice received NBI-31772 infusion for a period of 10, 14, or 21 days, whereas mdx mice received NBI-31772 for 28 days.
Myotoxic Injury of Tibialis Anterior Muscles
In BL/10 mice, tibialis anterior (TA) muscles were injured using the myotoxic agent notexin, as described previously.13 In brief, mice were anesthetized deeply with sodium pentobarbitone (60 mg/kg i.p., Nembutal; Rhone Merieux, Pinkenba, QLD, Australia), and an adequate depth of anesthesia was maintained, such that the mice were unresponsive to tactile stimulation. The right hindlimb was shaved, and a small portion of the anterior aspect of the TA muscle was surgically exposed. The muscle was injected with 40 µl of notexin (10 µg/ml in isotonic saline; Latoxan, Valence, France) with a 29-gauge fixed needle. Care was taken to prevent leakage of notexin from the muscle. An injection volume of 40 µl was the maximum holding capacity of a TA muscle and reliably produced complete degeneration of all myofibers.6,13 After the intramuscular injection, the skin incision was closed with surgical (Michel) clips (Aesculap, Tuttlingen, Germany), and the mouse was allowed to recover on a heat pad.
Contractile Properties Measured In Situ
The methods for in situ contractile analysis of TA muscles from mice have been described in detail elsewhere.16 In brief, TA muscles from injured and control BL/10 mice were stimulated by supramaximal (10 V) 0.2-ms square wave pulses of 300 ms in duration delivered via two wire electrodes adjacent to the femoral nerve. Optimum muscle length (Lo) was determined from maximum isometric twitch force (Pt), and maximum isometric tetanic force (Po) was recorded from the plateau of the frequency-force relationship.
Contractile Properties Measured In Vitro
The contractile properties of extensor digitorum longus (EDL) and soleus muscles and of diaphragm muscle strips from mdx mice were analyzed in vitro, as described previously.25 In brief, EDL, soleus, and diaphragm muscle preparations were stimulated by supramaximal (40 V) 0.2-ms square wave pulses of 350-, 1200-, and 450-ms duration, respectively, delivered via platinum plate electrodes that flanked both sides of the muscle. Lo was determined at Pt and Po was recorded from the plateau of the frequency-force curve. The EDL and soleus muscles from the right hindlimb, as well as a diaphragm muscle strip, were assessed for their susceptibility to contraction-induced injury, whereas the EDL and soleus muscles from the left hindlimb, as well as another diaphragm muscle strip, were assessed for their resistance to a fatiguing stimulation protocol.
The contraction-induced injury protocol used in this study was similar to others described previously.26,27 Muscles were lengthened at a velocity of two Lf/s at progressively increasing magnitudes of stretch beyond Lf (5, 10, 20, 30, 40, and 50%), with maximum isometric force being determined after each lengthening contraction. The "force deficit" after contraction-induced damage was determined by calculating the difference between the Po measured 2 minutes after the lengthening contractions and Po determined before any of the lengthening contractions and is expressed as a percentage of the maximum Po determined before the protocol of lengthening contractions.16,26
Muscle fatigue was assessed using a standard fatigue stimulation protocol we have described previously.25 Muscles were stimulated maximally once every 4 seconds for 4 minutes, with maximum force recorded every minute. During recovery, maximum force was determined at 5, 10, and 15 minutes after completion of the fatigue protocol.
Skeletal Muscle Morphology
At the completion of all of the contractile function analyses, the muscles were mounted in embedding medium, frozen in thawing isopentane, and stored at C80??C. A portion of each muscle was cryosectioned transversely (8 µm) through the midbelly region. Muscle sections were stained with hematoxylin and eosin (H&E) to determine general muscle architecture and to determine the cross-sectional area (CSA) of individual muscle fibers. Median values for CSA were calculated from at least 200 individual muscle fibers per cross section, as described previously.16
Myosin Heavy Chain Analysis
Myosin heavy chain (MyHC) isoform composition within TA muscles of BL/10 mice was determined via sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Muscle samples were homogenized on ice in phosphate-buffered saline and centrifuged at 300 x g for 2 minutes at 4??C. The pellet was resuspended in 100 µl of Guba-Straub buffer (300 mmol/L NaCl, 100 mmol/L NaH2PO4H2O, 50 mmol/L NaH2PO4, 1 mmol/L MgCl2?
【参考文献】
Huard J, Li Y, Fu FH: Muscle injuries and repair: current trends in research. J Bone Joint Surg Am 2002, 84-A:822-832
Emery AE: The muscular dystrophies. Lancet 2002, 359:687-695
Lynch GS: Role of contraction-induced injury in the mechanisms of muscle damage in muscular dystrophy. Clin Exp Pharmacol Physiol 2004, 31:557-561
Petrof BJ, Shrager JB, Stedman HH, Kelly AM, Sweeney HL: Dystrophin protects the sarcolemma from stresses developed during muscle contraction. Proc Natl Acad Sci USA 1993, 90:3710-3714
Blake DJ, Weir A, Newey SE, Davies KE: Function and genetics of dystrophin and dystrophin-related proteins in muscle. Physiol Rev 2002, 82:291-329
Plant DR, Colarossi FE, Lynch GS: Notexin causes greater myotoxic damage and slower functional repair in mouse skeletal muscles than bupivacaine. Muscle Nerve 2006, 34:577-585
Charg? SB, Rudnicki MA: Cellular and molecular regulation of muscle regeneration. Physiol Rev 2004, 84:209-238
Engert JC, Berglund EB, Rosenthal N: Proliferation precedes differentiation in IGF-I-stimulated myogenesis. J Cell Biol 1996, 135:431-440
Rosenthal SM, Cheng ZQ: Opposing early and late effects of insulin-like growth factor I on differentiation and the cell cycle regulatory retinoblastoma protein in skeletal myoblasts. Proc Natl Acad Sci USA 1995, 92:10307-10311
Musar
作者单位：From the Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, Melbourne, Victoria, Australia