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Cellular and molecular bases of muscle regeneration: The critical role of insulin-like growth factor-1
International Congress Series 1302 (2007) 89 – 100
www.ics-elsevier.com
Cellular and molecular bases of muscle regeneration: The critical role of insulin-like growth factor-1 Antonio Musarò a,b,⁎, Cristina Giacinti a , Laura Pelosi a , Bianca M. Scicchitano a , Mario Molinaro a a
Department of Histology and Medical Embryology, CE-BEMM and IIM, University of Rome “La Sapienza”, Italy b Edith Cowan University, Perth WA, Australia
Abstract. One of the most exciting aspirations of current medical science is the regeneration of damaged body parts. The capacity of adult tissues to regenerate in response to injury stimuli represents an important homeostatic process that until recently was thought to be limited in mammals to tissues with high turnover such as blood and skin. However, this central dogma of cell biology has been revised on the basis of recent experimental evidence that even the adult brain is able to undergo repair. It is now generally accepted that each tissue type, even those such as nerves or muscle that are considered postmitotic, contains a reserve of undifferentiated progenitor cells, loosely termed stem cells, that participate in tissue regeneration and repair. Regeneration represents a coordinate process in which these stem cell populations are activated to maintain and preserve tissue structure and function upon injured stimuli. In this review we will discuss the molecular and cellular basis of muscle regeneration, the critical role of IGF-1 on muscle homeostasis, and its potential therapeutic approach to improve muscle regeneration and to attenuate atrophy and frailty associated with muscle diseases. © 2007 Elsevier B.V. All rights reserved. Keywords: Muscle regeneration; Insulin-like growth factor-1; Stem cells; Aging; Muscular dystrophy
1. Introduction The decline in functional performance and restriction of adaptability represents the hallmark of skeletal muscle pathologies. The characteristic loss in muscle mass, coupled with a decrease in strength and force output, has been associated with a selective activation of apoptotic pathways and a general reduction in survival mechanisms [1]. ⁎ Corresponding author. Via A. Scarpa, 14 Rome 00161 Italy. Tel.: +39 06 49766956; fax: +39 06 4462854. E-mail address: [email protected] (A. Musarò). 0531-5131/ © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2006.09.022
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The maintenance of a working skeletal musculature is conferred by its remarkable ability to regenerate after injury. Most muscle pathologies are characterized by the progressive loss of muscle tissue due to chronic degeneration combined with the inability of the regeneration machinery to replace damaged myofibers. Aging, cancer, AIDS, heart failure and genetic myopathies are all characterized by alterations in metabolic and physiological parameters and the inability to regenerate and repair the injured muscle represents a serious complication in such pathologies. Although the general mechanism underlies muscle regeneration has been identified, less is known about the limitation of pathological muscle to sustain an efficient process of repair. The reduction in the number satellite cells, the poor recruitment of circulating stem cells into damaged area, the persistence of the inflammatory response, and the altered expression of growth factor mediated-regeneration represent the potential limiting mechanisms. 2. Stem cells and muscle regeneration 2.1. The contribution of satellite cells to muscle regeneration Regeneration is a highly coordinated program in adult skeletal muscle that partially recapitulates the embryonic developmental program. The major role in the growth, remodeling and regeneration is played by satellite cells, a quiescent population of myogenic cells that reside between the basal lamina and plasmalemma [2] and that are rapidly activated in response to appropriate stimuli. RT-PCR analysis and gene targeting strategies [3,4] revealed that satellite cells present a plasmalemma profile of gene expression depending on the functional stage of the myogenic program. Once activated, satellite cells express factors involved in the specification of the myogenic program such as Pax-7, desmin, MNFα, Myf-5, and MyoD [4]. Activated satellite cells proliferate as indicated by the expression of factors involved in cell cycle progression such as PCNA and by incorporation of BrDU. Ultimately the committed satellite cells fuse together or to the existing fibers to form new muscle fibers during regeneration and muscle repair [4]. This aspect of muscle regeneration is hampered in several muscle diseases, including aging and muscular dystrophies. It has been suggested that the decline in the regenerative potential of senescent muscle is mainly due to a decline in satellite cell number [5]. However, other evidences suggested alternative explanations. Conboy et al. [6] reported that the dramatic age-related decline in myoblast generation in response to injury is due to an impairment of activation rather than a decline in number of satellite cells [6], demonstrating that Notch signaling plays a pivotal role in satellite cell activation and cell fate determination. Indeed, to examine the influence of systemic factors on aged progenitor muscle cells, this group recently established parabiotic pairings (that is, a shared circulatory system) between young and old mice (heterochronic parabioses), exposing old mice to factors present in young serum [7]. Notably, heterochronic parabiosis restored the activation of Notch signalling as well as the proliferation and regenerative capacity of aged satellite cells [7].
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The limitation of senescent skeletal muscle to sustain an efficient regenerative mechanism puts a question as to whether this is due to the intrinsic aging of stem cells or, rather, to the impairment of stem-cell function in the aged tissue environment. 2.2. The contribution of stem cells to muscle regeneration The discovery of stem cell lineages in many adult tissues has challenged the classic concept that stem cells in the adult are present in only a few locations, such as the skin or bone marrow, and are committed to differentiate into the tissue in which they reside. In addition, several evidences suggested that the migration of circulating stem cells into the injured area represents the mechanisms by which different tissues are repaired [8]. Indeed, accounts of the repopulation of adult organs by bone marrow-derived stem cells suggest that under the right conditions, they can contribute to virtually any part of the body. However this seems a rare event and presents limitations for an efficient tissue repair. It has been proposed that adult bone marrow-derived cells contribute to muscle tissue in a step-wise biological progression [9]. Following irradiation-induced damage, transplanted bone marrow-derived cells become satellite cell [9]; alternatively, they may fuse directly into regenerating muscle fibers [10]. However, in all animal studies to date, it has been necessary to replace host bone marrow with marked progenitor cells to prove their provenance. This experimental manipulation inevitably involves lethal irradiation of the host animal, a process that is emerging as necessary prerequisite for bone marrow engraftment into injured muscle [11]. In any case, the total number of bone marrow stem cells recruited to a muscle fate in these studies appears still insufficient to be of therapeutic benefit. In fact it has been reported that the poor recruitment of hematopoietic stem cells into the dystrophic muscle of the mdx mouse is the major obstacle for muscle regeneration and therefore for the rescue of the genetic disease [12]. A new class of vessel associated fetal stem cells, termed mesoangioblasts, has been isolated [13]. These cells show profiles of gene expression similar to that reported for hematopoietic, neural, and embryonic stem cells [13]. Mesoangioblasts can differentiate into most mesoderm (but not other germ layer) cell types when exposed to certain cytokines or to differentiating cells [13]. Intra-arterial mesoangioblast delivery was effective in restoring expression of α-sarcoglycan protein and of the other members of the dystrophin glycoprotein complex in treated α-sarcoglycan null mice [14]. Restoration of sarcoglycan expression was also associated with a marked reduction of the fibrosis and complete functional recovery of treated muscle. Although stem cells offer a new tool for regeneration in muscle disease, the signalling and molecular pathways involved in recruitment and myogenic commitment of progenitors cells is an important question that remains to be satisfactorily addressed. In addition, the environment in which these stem cells operate represents another important determinant for cell survival and differentiation, which may be compromised in the dystrophic milieu. The regenerative capacity of skeletal muscle is influenced by several factors [4], including growth factors and hormones, secreted in an autocrine/paracrine manner. Alterations in these parameters compromise the ability of skeletal muscle to sustain a regenerative
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process, leading to repeated episodes of incomplete muscle repair and therefore to muscle wasting. Among growth factors, IGF-1 plays a key role in growth, hypertrophy and muscle regeneration [15]. 3. The anabolic effect of the local isoform of IGF-1 (mIGF-1) on skeletal muscle 3.1. The molecular structure of the IGF-1 gene The IGF-1 gene is highly conserved in numerous species. The rodent IGF-1 gene contains six exons, separated by five introns (Fig. 1) [15]. Exons 1 and 2 encode distinct 5′UTRs, as well as different parts of the signal peptide, and are therefore termed leader exons. Exon 3 encodes 27 amino acids that are part of the signal peptide and common to all isoforms, as well as part of the mature IGF-1 peptide. Exon 4 encodes the rest of the mature peptide and 16 amino acids of the amino-terminal region of the E-peptide, which is also common to all IGF-1 mRNAs. Exons 5 and 6 encode two distinct carboxy-terminal E-peptides and the 3′UTR (Fig. 1). Although IGF-1 transcripts are not exclusively tissue-restricted, those that initiate at exon 2 predominate in the liver, are highly growth hormone responsive and their products are major endocrine effectors of GH [15]. By contrast, transcripts initiating at exon 1 are widely expressed in all tissues, and are less effected by circulating growth hormone levels, presumably performing autocrine or paracrine functions. The alternate splicing at the 5′ ends of these two IGF-1 transcripts generates different signal peptides, which purportedly affects the precise N-terminal pro-peptide cleavage site [15]. The function of the proteins encoded by these different transcripts is widely debated but a cohesive picture has yet to emerge. It has been reported that the circulating isoform of IGF-1 can also induce adverse side effect, including heart hypertrophy, hyperplasia, dermal abnormality and spontaneous tumor formation [15]. The neoplastic potential of at least certain IGF-1 isoforms is an obvious concern to be taken into account when designing IGF therapeutic strategies for human pathologies. In this context, restricting the action of supplemental IGF-1 to the tissue of origin by use of a local IGF-1 isoform (mIGF-1) will allow the assessment of its autocrine/paracrine role in skeletal muscle throughout the life-span of the animal, exclusive of possible endocrine effects on other tissues.
Fig. 1. Schematic representation of rodent IGF-1 gene. Translated regions are shown in filled boxes. Exons 1 and 2 contain multiple transcription start sites (horizontal arrows). Translation initiation codons (AUG) are located at exons 1, 2 and 3 (vertical arrows). Exons 5 and 6 each encode distinct portions of the E-peptides.
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3.2. mIGF-1 and muscle aging IGF-1 declines during postnatal life, raising the prospect that this decline contributes to the progress of muscle atrophy in senescence, and limits the ability of skeletal muscle tissue to effect repair or to regenerate. To test this possibility we generated a transgenic mouse in which the local isoform of IGF-1 (mIGF-1) is driven by MLC promoter (MLC/mIGF-1) [16]. Transgenic animals exhibit marked skeletal muscle hypertrophy with dramatic reduction in body fat and no undesirable side effects such as tumor formation, as revealed in transgenic mice overexpressing the circulating IGF-1 isoform. The increased muscle mass in mIGF-1 transgenic mice was associated with augmented force generation compared to age-matched wild type littermates [16]. Examination of twoyear old animals revealed that whereas wild type mice underwent characteristic muscle atrophy, expression of the mIGF-1 transgene was protective against normal loss of muscle mass during senescence [16]. Thus, IGF-1 expression preserves both muscle integrity and the heterogeneity of myofibers, two fundamental parameters of muscle function. Overexpression of the mIGF-1 transgene also promoted and preserved the regenerative capacity of muscle tissues and the ability to repair damaged muscle during aging [16]. Thus mIGF-1 can overcome the normal inability of skeletal muscle to sustain regeneration and repair and as such represents a potentially effective gene therapeutic strategy to combat muscle wasting. In this context we demonstrated that the action of mIGF-1 is not dependent on life-long expression. Introduction of mIGF-1 somatically using an Adeno-Associated-Viral (AAV) vector was sufficient to rejuvenate the leg muscles of 27-month old mice, which exhibited the same mechanical force as legs of younger mice, and did not develop the pathological characteristics of senescent muscle [17]. 3.3. mIGF-1 and muscular dystrophy Because it is clear that mIGF-1 can prevent aging-related loss of muscle function [16,17], it is possible that mIGF-1 can prevent or diminish muscle loss associated with disease. To prove this hypothesis, we introduced mIGF-1 into the mdx dystrophic animals (mdx/ mIGF-1) [18]. By analyzing both muscle morphology and function in transgenic mdx/mIGF-1 we observed significant improvement in muscle mass and strength, a decrease in myonecrosis, and a reduction in fibrosis in aged diaphragms [18]. In particular, even though IGF-1 has been shown to stimulate fibroblasts, there was a net decrease in fibrosis in diaphragms of the mdx/mIGF-1 mice [18]. In fact, age-related fibrosis in the mdx diaphragm was effectively eliminated by mIGF-1 expression. It may be that the efficient and rapid repair of the mdx/mIGF-1 muscles prevents the establishment of an environment into which the fibroblasts migrate. This is of particular relevance to the human dystrophic condition where virtually all skeletal muscles succumb to fibrosis. Thus, the results found in the mouse diaphragm suggest that mIGF-1 might be effective not only in increasing muscle mass and strength, but also in reducing fibrosis associated with the disease.
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Finally, signaling pathways associated with muscle regeneration and protection against apoptosis were significantly elevated [18]. These results suggest that a combination of promoting muscle regenerative capacity and preventing muscle necrosis could be an effective treatment for the secondary symptoms caused by the primary loss of dystrophin. More recently, Abmayr et al. [19] demonstrated that co-injection of the rAAVmicrodystrophin and rAAV-mIGF-1 vectors resulted in increased muscle mass and strength, reduced myofiber degeneration, and increased protection against contraction-induced injury. These results suggest that a dual-gene, combinatorial strategy could enhance the efficacy of gene therapy of DMD and underscored the importance of rAAV vectors due to their relative lack of immunologic and toxic side effects combined with their potential for body-wide systemic gene delivery to muscle [19]. 3.4. mIGF-1 and muscle regeneration Muscle regeneration is a coordinate process in which several factors are sequentially activated to maintain and preserve muscle structure and function upon injured stimuli. Although adult skeletal muscle is composed of fully differentiated fibers, it retains the capacity to regenerate in response to injury, activating a quiescent population of myogenic cells, namely satellite cells, that reside between the basal lamina and plasmalemma [2]. The anabolic effects of IGF-1 may be due in part to stimulation of activation of satellite cells. It is not known whether in MLC/mIGF-1 transgenic animals, the satellite cells have an increased ability for self-renewal or whether there is an increased recruitment of nonsatellite cells. Our recent experimental evidences indicate that mIGF-1 promotes the two suggested pathways which can be considered two temporally separated events of the same biological process. We demonstrated that upon muscle injury, stem cells expressing c-Kit, Sca-1, and CD45 antigens increased locally and the percentage of the recruited cells were conspicuously enhanced by mIGF-1 expression [20]. FACS profiles of tissues from wild type and MLC/mIGF-1 transgenic mice, whose muscles were injured with cardiotoxin, revealed a consistent increase of Side Population (SP) cells in the bone marrow, compared to non-injured controls which percentage increase in MLC/mIGF-1 transgenic mice. In contrast, the number of SP cells remained unchanged in other tissues, such as spleen and liver of both wild type and MLC/mIGF-1 transgenic mice after muscle injury [20]. Thus, humoral signals emanating from the injured muscles were sufficient to induce stem cell proliferation in the bone marrow, but not in other tissues. Indeed, treatment of injured mice with 5-fluorouracil (5-FU), a cytotoxic agent which depletes cycling stem cells, was sufficient to block proliferation of bone marrow SP and expansion of the CD45+/Sca-1+ population in injured muscle. In addition, to definitely demonstrate the recruitment of bone marrow-stem cells into the site of muscle injury and the role of mIGF-1 in this process, we performed a bone marrow transplantation in both wild type and MLC/mIGF-1 transgenic mice using bone marrow-SP cells of c-kit/GFP transgenic mice [20]. FACS analysis revealed the presence of c-kit/GFP positive cells in the injured area of skeletal muscle and confirms the effect
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Fig. 2. Model of stem cell-mediated muscle regeneration (modified from Ref. [20]). Muscle injury involves the activation of satellite cells and the recruitment of circulating stem cells, which when penetrating the muscle compartment receive myogenic signals and may contribute to muscle regeneration and repair. This process is enhanced by mIGF-1 expression.
of mIGF-1 in the enhancement of circulating stem cells into the site of muscle injury [20]. To follow the recruitment of these cells in regenerating muscle tissue, we performed histochemical analysis of wild type and MLC/mIGF-1 transgenic muscle after cardiotoxin injection using antibodies against Sca-1, as well as markers of myogenic commitment and differentiation. Regenerating muscles express Sca-1 and Pax-7 [20], a homeodomain protein implicated in the specification of satellite cells from multipotent stem cells and muscle regeneration [21,22]. Increased Sca-1 positive cells in the region of mononuclear infiltration and in blood vessels of MLC/mIGF-1 damaged muscle suggested a mobilization of the stem cell population in the injured area. Interestingly, a sub-population of cells adjoining a vessel expressed Pax-7, whereas vascular tissues did not express Pax-7, suggesting that vessel is the niche of circulating stem cells which, when penetrating the interstitium receive myogenic signals and are committed to the muscle phenotype, participating therefore to muscle regeneration and repair. In addition, when isolated from regenerating MLC/mIGF-1 muscles the recruited cells (Sca-1+/c-kit+) exhibit accelerated myogenic differentiation in culture, and readily induced muscle-specific markers in a subset of co-cultured bone marrow cells [20]. The results in this study are consistent with a model (Fig. 2) whereby injury stimulates the mobilization of uncommitted stem cells in the bone marrow, or in other sources, which migrate to sites of tissue damage and participate either directly or indirectly in the regeneration process. The homing of bone marrow cells to injured muscle dramatically increased in the MLC/mIGF-1 transgenic background. These results establish mIGF-1 as a potent enhancer of stem cell-mediated regeneration and provide a baseline to develop strategies to improve muscle regeneration in muscle diseases. Acknowledgements Work in the authors' laboratories has been supported by the Italian Telethon, MDA, AFM and ASI.
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References [1] A. Musarò, N. Rosenthal, Attenuating muscle wasting: cell and gene therapy approaches, Curr. Genomics 4 (7) (2003) 575–585. [2] A. Mauro, Satellite cell of skeletal muscle fibers, J. Biophys. Biochem. Cytol. 9 (1965) 493–495. [3] D.D. Cornelison, B.J. Wold, Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells, Dev. Biol. 191 (2) (1997) 270–283. [4] S.B. Charge, M.A. Rudnicki, Cellular and molecular regulation of muscle regeneration, Physiol. Rev. 84 (1) (2004) 209–238. [5] E. Schultz, B.H. Lipton, Skeletal muscle satellite cells: changes in proliferation potential as a function of age, Mech. Ageing Dev. 20 (4) (1982) 377–383. [6] I.M. Conboy, et al., Notch-mediated restoration of regenerative potential to aged muscle, Science 302 (5650) (2003) 1575–1577. [7] I.M. Conboy, et al., Rejuvenation of aged progenitor cells by exposure to a young systemic environment, Nature 433 (7027) (2005) 760–764. [8] H.M. Blau, T.R. Brazelton, J.M. Weimann, The evolving concept of a stem cell: entity or function? Cell 105 (7) (2001) 829–841. [9] M.A. LaBarge, H.M. Blau, Biological progression from adult bone marrow to mononucleate muscle stem cell to multinucleate muscle fiber in response to injury, Cell 111 (4) (2002) 589–601. [10] F.D. Camargo, et al., Single hematopoietic stem cells generate skeletal muscle through myeloid intermediates, Nat. Med. 9 (12) (2003) 1520–1527. [11] J.E. Morgan, et al., Myogenic cell proliferation and generation of a reversible tumorigenic phenotype are triggered by preirradiation of the recipient site, J. Cell Biol. 157 (4) (2002) 693–702. [12] G. Ferrari, A. Stornaiuolo, F. Mavilio, Failure to correct murine muscular dystrophy, Nature 411 (6841) (2001) 1014–1015. [13] G. Cossu, P. Bianco, Mesoangioblasts-vascular progenitors for extravascular mesodermal tissues, Curr. Opin. Genet. Dev. 13 (5) (2003) 537–542. [14] M. Sampaolesi, et al., Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts, Science 301 (5632) (2003) 487–492. [15] A. Musarò, N. Rosenthal, The critical role of insulin-like growth factor-1 isoforms in the physiopathology of skeletal muscle, Curr. Genomics 3 (7) (2006) 19–32. [16] A. Musaro, et al., Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle, Nat. Genet. 27 (2) (2001) 195–200. [17] E.R. Barton-Davis, et al., Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function, Proc. Natl. Acad. Sci. U. S. A. 95 (26) (1998) 15603–15607. [18] E.R. Barton, et al., Muscle-specific expression of insulin-like growth factor I counters muscle decline in mdx mice, J. Cell Biol. 157 (1) (2002) 137–148. [19] S. Abmayr, et al., Phenotypic improvement of dystrophic muscles by rAAV/microdystrophin vectors is augmented by Igf-1 codelivery, Molec. Ther. 12 (3) (2005) 441–450. [20] A. Musaro, et al., Stem cell-mediated muscle regeneration is enhanced by local isoform of insulin-like growth factor 1, Proc. Natl. Acad. Sci. U. S. A. 101 (5) (2004) 1206–1210. [21] F. Relaix, et al., Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells, J. Cell Biol. 172 (1) (2006) 91–102. [22] S. Kuang, et al., Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis, J. Cell Biol. 172 (1) (2006) 103–113.
Discussion Jeffrey Johnson In the case of muscle denervation crush, do you see somewhere kinds of responses in the muscle to that, because the muscle will go away very fast in that kind of a situation.
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Antonio Musarò We analyzed the effect of IGF-1 not just in the innervated muscle, but in a model which is correlated to muscle denervation, which is the amiotrophic lateral sclerosis transgenic mouse in which there is a degeneration of motor neurons. What basically we found is that the IGF-1 not only preserves muscle mass in this pathological model, but was also in some way able to protect motor neurons from degeneration. So what we found is that if you maintain the muscle in good condition, in good shape, the muscle is also able to signal to motor neurons trying to activate antiapoptic pathways. And the idea is that the skeletal muscle is a source of signals that help not only the muscle, but also help to save motor neurons from degeneration. Jeffrey Johnson Was that in the G-93A mice? Antonio Musarò Yes, it’s the SODG-93 mouse crossed with our transgenic mouse. Jeffrey Johnson Why type-1 versus type-2 differential sensitivity to degeneration? We have studied the GA-93 mice a lot. And when you look at the muscles, it’s clear the type-2 fibers are the ones that are going, very early in the pathology. And the type-1s are there right to the end, when they become paralyzed… so why? Antonio Musarò This is a good question that probably no-one has a good answer to. I can guess the reason is that the slow fibers are more resistant to degeneration compared to the fast fibers and, thinking about the evolutionary aspect, probably the idea is that it’s better to lose fast fibers rather than slow fibers, because the slow fibers are involved in the maintenance of the muscle, of the body part, the fast fibers are involved in muscle strength. Pura Muñoz It’s reported that calcineurin is expressed in slow fibers, and in the case of the dystrophic mice calcineurin stimulates utrophin expression, thereby compensating for the lack of dystrophin. So the slow fibers are endowed with an additional mechanism, at least in dystrophic conditions. Reinhard Fässler You showed that locally produced IGF is producing all these good effects on muscle, but you didn’t mention how it works. Is it working on satellite cells or on the myofibers or is it working through IGF receptors? Where are the IGF receptors expressed? Which IGF receptors are they, because IGF receptors are very, very low-expressed in muscle? Antonio Musarò The signal transduction pathway goes through the IGF receptor, and the specific action of IGF-1 is to activate the satellite cells. And the hypertrophic phenotype actually involves not only increasing the protein synthesis, but also IGF-1 inducing the activation of satellite cells: these satellite cells are fused with the pre-existing fibers and increase the muscle phenotype. I don’t know if there is any correlation between, for example, IGF-1 and the other growth factors, but the interesting thing about IGF-1 is that it’s one of the growth factors that can stimulate the two events that are mutually exclusive, proliferation and differentiation, in contrast with, for example, FGF which stimulates proliferation but blocks differentiation. So, in vitro, we demonstrated that IGF-1 activates
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two different signal transduction pathways. We induce proliferation activating MAP kinase, and then induce calcineurin and the EKT pathway to induce differentiation in the hypertrophy. Reinhard Fässler Are the IGF-1 receptors expressed on myofibers? Antonio Musarò Yes, they’re expressed there. Reinhard Fässler At high levels or very low levels, or only after regeneration? Are they expressed on resting muscle? Antonio Musarò They are also expressed in resting muscle. I don’t have evidence for an increase in its expression during muscle regeneration. Martin Schwab I have a question about neuronal activity which regulates, of course, muscle development and also trophic status of muscle mass in a very crucial way. Is IGF-1 a direct downstream gene regulated by neuronal activity? Did you look for that or did anyone? Antonio Musarò No, we don’t have evidence for that, but IGF-1 is one of the growth factors that is expressed early on during muscle development. And after birth IGF-1 expression decreases progressively, and in humans over 30 years old the levels of IGF-1 have really dropped. So I don’t know if there is any connection between nerve activity and IGF-1 production. But, as I said before, when we maintain IGF-1 over-expression in the SODG-93A mouse model, where there is a degeneration of a motor neuron, we are able to demonstrate that, in this situation, we also preserve not only motor neuron activity, but also the numbers of motor neurons. Martin Schwab But this could even be a direct effect of IGF-1 on the motor neurons. It has been used as you know in clinical trials Antonio Musarò Exactly. Jeffrey Davidson In your adoptive transfer experiment with the AP-marked cells, what fraction of marrowderived circulating cells are participators in the repair process? Antonio Musarò We actually analyzed several compartments of the bone marrow derived stem cells and we found that the SP population plays the major role or, let’s say, has the best effect. Because when we transplant, for example, stem cells which lack c-kit, we couldn’t get the same results. So we analyzed a sort of different population that expressed all the markers, for example, ESK1, CD45, CD11b and C-kit, plus others that express, for example, CD45 and CD11b. Comparing these two populations, using the first one we got the best results, but when we transplanted CD-45 positive, CD-11b positive, but CKit negative and ESK1 negative, we didn’t obtain good results in terms of positive fibers.
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Jeffrey Davidson But following that, what fraction of the regenerative capacity in the repairing muscle comes from the circulating cells versus the endogenous precursors? Antonio Musarò Probably, under our conditions, less than 10%. Less than 10% of these transplanted stems cell can participate in regeneration. I mean the major role is played by satellite cells, definitely by satellite cells. Jeffrey Davidson In experiments where you’ve shown that IGF is accelerating the decline in inflammatory signals, what is the cellular source of those cytokines? Is it myocytes or inflammatory cells? Antonio Musarò We used by cytofluorimetric analysis, and the IGF-1 modulates, for example, the recruitment of macrophages and of neutrophils. So these are the two main populations that are modulated by IGF-1 expression and that produce these pro-inflammatory cytokines. Norbert Fusenig I have two questions concerning your "bodybuilder mouse". First, are all muscles involved in the hypertrophy and hyperplasia, or only those in physical activity? Antonio Musarò The MLC promoter is activated specifically in the fast fibers. For example, the soleus is composed mainly by slow fibers, the EDL by fast fibers and if you compare the muscle mass between the soleus and the EDL, there is a significant difference. So the EDL is more of a hypertrophic muscle than the soleus, and this phenotype is induced just by overexpression, so the mice don’t need it to exercise to show this hypertrophy… Norbert Fusenig So what happens to the heart? Antonio Musarò The heart is basically normal. I mean, we analyze the histological level and also the functional levels, and this local expression of IGF-1 doesn’t induce hypertrophic phenotype in the heart. Norbert Fusenig And a second question. Is it an ongoing process or is it limited to some time. Do the mice grow and grow until the skin is exploding? Antonio Musarò No, actually. This phenotype starts at six weeks after birth and you can maintain it for the entire life, so there is no continuous increase in the muscle mass. Definitely there is some fat or other mechanism that balances the effect. Norbert Fusenig Do you see any therapeutic application? Has something been done in that direction? Antonio Musarò Not yet. Most of the clinical trials were done using the other isoform of IGF-1, and the problem is that this circulating form that induces adverse side effects. Actually there is a link between increased level of IGF-1 and tumor formation. So the idea is to use this local form and since this local form doesn’t circulate you have to inject it directly into the muscle. An obvious approach was to place this IGF-1 in a vector and inject this vector in a senescent muscle. Using this gene therapeutic approach we demonstrated that we can rescue the
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senescent skeletal muscle, improving muscle mass and also muscle strength in senescent muscle. Snorri Thorgeirsson I have an obligatory stem cell question. So is this a fusion or a transdifferentiation? And if it is a fusion, what is the cell it fuses with? Antonio Musarò The answer is that I don’t know. And there is, let’s say, a big fight between two groups: there are some who believe that it’s transdifferentiation, others that it’s fusion. I mean in skeletal muscle it doesn’t matter too much whether it’s fusion of transdifferentiation, because in the end what is important is to get functional recovery. But it’s a good point. So far nobody has found a way to discriminate between transdifferentiation and fusion in skeletal muscle. Snorri Thorgeirsson But you would think though that if you fuse satellite cells, that would be different than if you fused with a more committed cell. Antonio Musarò Yes, sure, if your bone marrow stem cells are fused with pre-existing fibers or with the satellite cells and become muscle, the muscle is helping as well. Snorri Thorgeirsson You’ve shown us the benefit of IGF-1, but is it necessary? What happens if you knock it out selectively in the muscle? Antonio Musarò It’s great point, but nobody has knocked out specifically this isoform of IGF-1. All the evidence reported is on knocking out the circulating form.
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Cellular and molecular bases of muscle regeneration: The critical role of insulin-like growth factor-1 Antonio Musarò a,b,⁎, Cristina Giacinti a , Laura Pelosi a , Bianca M. Scicchitano a , Mario Molinaro a a
Department of Histology and Medical Embryology, CE-BEMM and IIM, University of Rome “La Sapienza”, Italy b Edith Cowan University, Perth WA, Australia
Abstract. One of the most exciting aspirations of current medical science is the regeneration of damaged body parts. The capacity of adult tissues to regenerate in response to injury stimuli represents an important homeostatic process that until recently was thought to be limited in mammals to tissues with high turnover such as blood and skin. However, this central dogma of cell biology has been revised on the basis of recent experimental evidence that even the adult brain is able to undergo repair. It is now generally accepted that each tissue type, even those such as nerves or muscle that are considered postmitotic, contains a reserve of undifferentiated progenitor cells, loosely termed stem cells, that participate in tissue regeneration and repair. Regeneration represents a coordinate process in which these stem cell populations are activated to maintain and preserve tissue structure and function upon injured stimuli. In this review we will discuss the molecular and cellular basis of muscle regeneration, the critical role of IGF-1 on muscle homeostasis, and its potential therapeutic approach to improve muscle regeneration and to attenuate atrophy and frailty associated with muscle diseases. © 2007 Elsevier B.V. All rights reserved. Keywords: Muscle regeneration; Insulin-like growth factor-1; Stem cells; Aging; Muscular dystrophy
1. Introduction The decline in functional performance and restriction of adaptability represents the hallmark of skeletal muscle pathologies. The characteristic loss in muscle mass, coupled with a decrease in strength and force output, has been associated with a selective activation of apoptotic pathways and a general reduction in survival mechanisms [1]. ⁎ Corresponding author. Via A. Scarpa, 14 Rome 00161 Italy. Tel.: +39 06 49766956; fax: +39 06 4462854. E-mail address: [email protected] (A. Musarò). 0531-5131/ © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2006.09.022
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The maintenance of a working skeletal musculature is conferred by its remarkable ability to regenerate after injury. Most muscle pathologies are characterized by the progressive loss of muscle tissue due to chronic degeneration combined with the inability of the regeneration machinery to replace damaged myofibers. Aging, cancer, AIDS, heart failure and genetic myopathies are all characterized by alterations in metabolic and physiological parameters and the inability to regenerate and repair the injured muscle represents a serious complication in such pathologies. Although the general mechanism underlies muscle regeneration has been identified, less is known about the limitation of pathological muscle to sustain an efficient process of repair. The reduction in the number satellite cells, the poor recruitment of circulating stem cells into damaged area, the persistence of the inflammatory response, and the altered expression of growth factor mediated-regeneration represent the potential limiting mechanisms. 2. Stem cells and muscle regeneration 2.1. The contribution of satellite cells to muscle regeneration Regeneration is a highly coordinated program in adult skeletal muscle that partially recapitulates the embryonic developmental program. The major role in the growth, remodeling and regeneration is played by satellite cells, a quiescent population of myogenic cells that reside between the basal lamina and plasmalemma [2] and that are rapidly activated in response to appropriate stimuli. RT-PCR analysis and gene targeting strategies [3,4] revealed that satellite cells present a plasmalemma profile of gene expression depending on the functional stage of the myogenic program. Once activated, satellite cells express factors involved in the specification of the myogenic program such as Pax-7, desmin, MNFα, Myf-5, and MyoD [4]. Activated satellite cells proliferate as indicated by the expression of factors involved in cell cycle progression such as PCNA and by incorporation of BrDU. Ultimately the committed satellite cells fuse together or to the existing fibers to form new muscle fibers during regeneration and muscle repair [4]. This aspect of muscle regeneration is hampered in several muscle diseases, including aging and muscular dystrophies. It has been suggested that the decline in the regenerative potential of senescent muscle is mainly due to a decline in satellite cell number [5]. However, other evidences suggested alternative explanations. Conboy et al. [6] reported that the dramatic age-related decline in myoblast generation in response to injury is due to an impairment of activation rather than a decline in number of satellite cells [6], demonstrating that Notch signaling plays a pivotal role in satellite cell activation and cell fate determination. Indeed, to examine the influence of systemic factors on aged progenitor muscle cells, this group recently established parabiotic pairings (that is, a shared circulatory system) between young and old mice (heterochronic parabioses), exposing old mice to factors present in young serum [7]. Notably, heterochronic parabiosis restored the activation of Notch signalling as well as the proliferation and regenerative capacity of aged satellite cells [7].
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The limitation of senescent skeletal muscle to sustain an efficient regenerative mechanism puts a question as to whether this is due to the intrinsic aging of stem cells or, rather, to the impairment of stem-cell function in the aged tissue environment. 2.2. The contribution of stem cells to muscle regeneration The discovery of stem cell lineages in many adult tissues has challenged the classic concept that stem cells in the adult are present in only a few locations, such as the skin or bone marrow, and are committed to differentiate into the tissue in which they reside. In addition, several evidences suggested that the migration of circulating stem cells into the injured area represents the mechanisms by which different tissues are repaired [8]. Indeed, accounts of the repopulation of adult organs by bone marrow-derived stem cells suggest that under the right conditions, they can contribute to virtually any part of the body. However this seems a rare event and presents limitations for an efficient tissue repair. It has been proposed that adult bone marrow-derived cells contribute to muscle tissue in a step-wise biological progression [9]. Following irradiation-induced damage, transplanted bone marrow-derived cells become satellite cell [9]; alternatively, they may fuse directly into regenerating muscle fibers [10]. However, in all animal studies to date, it has been necessary to replace host bone marrow with marked progenitor cells to prove their provenance. This experimental manipulation inevitably involves lethal irradiation of the host animal, a process that is emerging as necessary prerequisite for bone marrow engraftment into injured muscle [11]. In any case, the total number of bone marrow stem cells recruited to a muscle fate in these studies appears still insufficient to be of therapeutic benefit. In fact it has been reported that the poor recruitment of hematopoietic stem cells into the dystrophic muscle of the mdx mouse is the major obstacle for muscle regeneration and therefore for the rescue of the genetic disease [12]. A new class of vessel associated fetal stem cells, termed mesoangioblasts, has been isolated [13]. These cells show profiles of gene expression similar to that reported for hematopoietic, neural, and embryonic stem cells [13]. Mesoangioblasts can differentiate into most mesoderm (but not other germ layer) cell types when exposed to certain cytokines or to differentiating cells [13]. Intra-arterial mesoangioblast delivery was effective in restoring expression of α-sarcoglycan protein and of the other members of the dystrophin glycoprotein complex in treated α-sarcoglycan null mice [14]. Restoration of sarcoglycan expression was also associated with a marked reduction of the fibrosis and complete functional recovery of treated muscle. Although stem cells offer a new tool for regeneration in muscle disease, the signalling and molecular pathways involved in recruitment and myogenic commitment of progenitors cells is an important question that remains to be satisfactorily addressed. In addition, the environment in which these stem cells operate represents another important determinant for cell survival and differentiation, which may be compromised in the dystrophic milieu. The regenerative capacity of skeletal muscle is influenced by several factors [4], including growth factors and hormones, secreted in an autocrine/paracrine manner. Alterations in these parameters compromise the ability of skeletal muscle to sustain a regenerative
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process, leading to repeated episodes of incomplete muscle repair and therefore to muscle wasting. Among growth factors, IGF-1 plays a key role in growth, hypertrophy and muscle regeneration [15]. 3. The anabolic effect of the local isoform of IGF-1 (mIGF-1) on skeletal muscle 3.1. The molecular structure of the IGF-1 gene The IGF-1 gene is highly conserved in numerous species. The rodent IGF-1 gene contains six exons, separated by five introns (Fig. 1) [15]. Exons 1 and 2 encode distinct 5′UTRs, as well as different parts of the signal peptide, and are therefore termed leader exons. Exon 3 encodes 27 amino acids that are part of the signal peptide and common to all isoforms, as well as part of the mature IGF-1 peptide. Exon 4 encodes the rest of the mature peptide and 16 amino acids of the amino-terminal region of the E-peptide, which is also common to all IGF-1 mRNAs. Exons 5 and 6 encode two distinct carboxy-terminal E-peptides and the 3′UTR (Fig. 1). Although IGF-1 transcripts are not exclusively tissue-restricted, those that initiate at exon 2 predominate in the liver, are highly growth hormone responsive and their products are major endocrine effectors of GH [15]. By contrast, transcripts initiating at exon 1 are widely expressed in all tissues, and are less effected by circulating growth hormone levels, presumably performing autocrine or paracrine functions. The alternate splicing at the 5′ ends of these two IGF-1 transcripts generates different signal peptides, which purportedly affects the precise N-terminal pro-peptide cleavage site [15]. The function of the proteins encoded by these different transcripts is widely debated but a cohesive picture has yet to emerge. It has been reported that the circulating isoform of IGF-1 can also induce adverse side effect, including heart hypertrophy, hyperplasia, dermal abnormality and spontaneous tumor formation [15]. The neoplastic potential of at least certain IGF-1 isoforms is an obvious concern to be taken into account when designing IGF therapeutic strategies for human pathologies. In this context, restricting the action of supplemental IGF-1 to the tissue of origin by use of a local IGF-1 isoform (mIGF-1) will allow the assessment of its autocrine/paracrine role in skeletal muscle throughout the life-span of the animal, exclusive of possible endocrine effects on other tissues.
Fig. 1. Schematic representation of rodent IGF-1 gene. Translated regions are shown in filled boxes. Exons 1 and 2 contain multiple transcription start sites (horizontal arrows). Translation initiation codons (AUG) are located at exons 1, 2 and 3 (vertical arrows). Exons 5 and 6 each encode distinct portions of the E-peptides.
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3.2. mIGF-1 and muscle aging IGF-1 declines during postnatal life, raising the prospect that this decline contributes to the progress of muscle atrophy in senescence, and limits the ability of skeletal muscle tissue to effect repair or to regenerate. To test this possibility we generated a transgenic mouse in which the local isoform of IGF-1 (mIGF-1) is driven by MLC promoter (MLC/mIGF-1) [16]. Transgenic animals exhibit marked skeletal muscle hypertrophy with dramatic reduction in body fat and no undesirable side effects such as tumor formation, as revealed in transgenic mice overexpressing the circulating IGF-1 isoform. The increased muscle mass in mIGF-1 transgenic mice was associated with augmented force generation compared to age-matched wild type littermates [16]. Examination of twoyear old animals revealed that whereas wild type mice underwent characteristic muscle atrophy, expression of the mIGF-1 transgene was protective against normal loss of muscle mass during senescence [16]. Thus, IGF-1 expression preserves both muscle integrity and the heterogeneity of myofibers, two fundamental parameters of muscle function. Overexpression of the mIGF-1 transgene also promoted and preserved the regenerative capacity of muscle tissues and the ability to repair damaged muscle during aging [16]. Thus mIGF-1 can overcome the normal inability of skeletal muscle to sustain regeneration and repair and as such represents a potentially effective gene therapeutic strategy to combat muscle wasting. In this context we demonstrated that the action of mIGF-1 is not dependent on life-long expression. Introduction of mIGF-1 somatically using an Adeno-Associated-Viral (AAV) vector was sufficient to rejuvenate the leg muscles of 27-month old mice, which exhibited the same mechanical force as legs of younger mice, and did not develop the pathological characteristics of senescent muscle [17]. 3.3. mIGF-1 and muscular dystrophy Because it is clear that mIGF-1 can prevent aging-related loss of muscle function [16,17], it is possible that mIGF-1 can prevent or diminish muscle loss associated with disease. To prove this hypothesis, we introduced mIGF-1 into the mdx dystrophic animals (mdx/ mIGF-1) [18]. By analyzing both muscle morphology and function in transgenic mdx/mIGF-1 we observed significant improvement in muscle mass and strength, a decrease in myonecrosis, and a reduction in fibrosis in aged diaphragms [18]. In particular, even though IGF-1 has been shown to stimulate fibroblasts, there was a net decrease in fibrosis in diaphragms of the mdx/mIGF-1 mice [18]. In fact, age-related fibrosis in the mdx diaphragm was effectively eliminated by mIGF-1 expression. It may be that the efficient and rapid repair of the mdx/mIGF-1 muscles prevents the establishment of an environment into which the fibroblasts migrate. This is of particular relevance to the human dystrophic condition where virtually all skeletal muscles succumb to fibrosis. Thus, the results found in the mouse diaphragm suggest that mIGF-1 might be effective not only in increasing muscle mass and strength, but also in reducing fibrosis associated with the disease.
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Finally, signaling pathways associated with muscle regeneration and protection against apoptosis were significantly elevated [18]. These results suggest that a combination of promoting muscle regenerative capacity and preventing muscle necrosis could be an effective treatment for the secondary symptoms caused by the primary loss of dystrophin. More recently, Abmayr et al. [19] demonstrated that co-injection of the rAAVmicrodystrophin and rAAV-mIGF-1 vectors resulted in increased muscle mass and strength, reduced myofiber degeneration, and increased protection against contraction-induced injury. These results suggest that a dual-gene, combinatorial strategy could enhance the efficacy of gene therapy of DMD and underscored the importance of rAAV vectors due to their relative lack of immunologic and toxic side effects combined with their potential for body-wide systemic gene delivery to muscle [19]. 3.4. mIGF-1 and muscle regeneration Muscle regeneration is a coordinate process in which several factors are sequentially activated to maintain and preserve muscle structure and function upon injured stimuli. Although adult skeletal muscle is composed of fully differentiated fibers, it retains the capacity to regenerate in response to injury, activating a quiescent population of myogenic cells, namely satellite cells, that reside between the basal lamina and plasmalemma [2]. The anabolic effects of IGF-1 may be due in part to stimulation of activation of satellite cells. It is not known whether in MLC/mIGF-1 transgenic animals, the satellite cells have an increased ability for self-renewal or whether there is an increased recruitment of nonsatellite cells. Our recent experimental evidences indicate that mIGF-1 promotes the two suggested pathways which can be considered two temporally separated events of the same biological process. We demonstrated that upon muscle injury, stem cells expressing c-Kit, Sca-1, and CD45 antigens increased locally and the percentage of the recruited cells were conspicuously enhanced by mIGF-1 expression [20]. FACS profiles of tissues from wild type and MLC/mIGF-1 transgenic mice, whose muscles were injured with cardiotoxin, revealed a consistent increase of Side Population (SP) cells in the bone marrow, compared to non-injured controls which percentage increase in MLC/mIGF-1 transgenic mice. In contrast, the number of SP cells remained unchanged in other tissues, such as spleen and liver of both wild type and MLC/mIGF-1 transgenic mice after muscle injury [20]. Thus, humoral signals emanating from the injured muscles were sufficient to induce stem cell proliferation in the bone marrow, but not in other tissues. Indeed, treatment of injured mice with 5-fluorouracil (5-FU), a cytotoxic agent which depletes cycling stem cells, was sufficient to block proliferation of bone marrow SP and expansion of the CD45+/Sca-1+ population in injured muscle. In addition, to definitely demonstrate the recruitment of bone marrow-stem cells into the site of muscle injury and the role of mIGF-1 in this process, we performed a bone marrow transplantation in both wild type and MLC/mIGF-1 transgenic mice using bone marrow-SP cells of c-kit/GFP transgenic mice [20]. FACS analysis revealed the presence of c-kit/GFP positive cells in the injured area of skeletal muscle and confirms the effect
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Fig. 2. Model of stem cell-mediated muscle regeneration (modified from Ref. [20]). Muscle injury involves the activation of satellite cells and the recruitment of circulating stem cells, which when penetrating the muscle compartment receive myogenic signals and may contribute to muscle regeneration and repair. This process is enhanced by mIGF-1 expression.
of mIGF-1 in the enhancement of circulating stem cells into the site of muscle injury [20]. To follow the recruitment of these cells in regenerating muscle tissue, we performed histochemical analysis of wild type and MLC/mIGF-1 transgenic muscle after cardiotoxin injection using antibodies against Sca-1, as well as markers of myogenic commitment and differentiation. Regenerating muscles express Sca-1 and Pax-7 [20], a homeodomain protein implicated in the specification of satellite cells from multipotent stem cells and muscle regeneration [21,22]. Increased Sca-1 positive cells in the region of mononuclear infiltration and in blood vessels of MLC/mIGF-1 damaged muscle suggested a mobilization of the stem cell population in the injured area. Interestingly, a sub-population of cells adjoining a vessel expressed Pax-7, whereas vascular tissues did not express Pax-7, suggesting that vessel is the niche of circulating stem cells which, when penetrating the interstitium receive myogenic signals and are committed to the muscle phenotype, participating therefore to muscle regeneration and repair. In addition, when isolated from regenerating MLC/mIGF-1 muscles the recruited cells (Sca-1+/c-kit+) exhibit accelerated myogenic differentiation in culture, and readily induced muscle-specific markers in a subset of co-cultured bone marrow cells [20]. The results in this study are consistent with a model (Fig. 2) whereby injury stimulates the mobilization of uncommitted stem cells in the bone marrow, or in other sources, which migrate to sites of tissue damage and participate either directly or indirectly in the regeneration process. The homing of bone marrow cells to injured muscle dramatically increased in the MLC/mIGF-1 transgenic background. These results establish mIGF-1 as a potent enhancer of stem cell-mediated regeneration and provide a baseline to develop strategies to improve muscle regeneration in muscle diseases. Acknowledgements Work in the authors' laboratories has been supported by the Italian Telethon, MDA, AFM and ASI.
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References [1] A. Musarò, N. Rosenthal, Attenuating muscle wasting: cell and gene therapy approaches, Curr. Genomics 4 (7) (2003) 575–585. [2] A. Mauro, Satellite cell of skeletal muscle fibers, J. Biophys. Biochem. Cytol. 9 (1965) 493–495. [3] D.D. Cornelison, B.J. Wold, Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells, Dev. Biol. 191 (2) (1997) 270–283. [4] S.B. Charge, M.A. Rudnicki, Cellular and molecular regulation of muscle regeneration, Physiol. Rev. 84 (1) (2004) 209–238. [5] E. Schultz, B.H. Lipton, Skeletal muscle satellite cells: changes in proliferation potential as a function of age, Mech. Ageing Dev. 20 (4) (1982) 377–383. [6] I.M. Conboy, et al., Notch-mediated restoration of regenerative potential to aged muscle, Science 302 (5650) (2003) 1575–1577. [7] I.M. Conboy, et al., Rejuvenation of aged progenitor cells by exposure to a young systemic environment, Nature 433 (7027) (2005) 760–764. [8] H.M. Blau, T.R. Brazelton, J.M. Weimann, The evolving concept of a stem cell: entity or function? Cell 105 (7) (2001) 829–841. [9] M.A. LaBarge, H.M. Blau, Biological progression from adult bone marrow to mononucleate muscle stem cell to multinucleate muscle fiber in response to injury, Cell 111 (4) (2002) 589–601. [10] F.D. Camargo, et al., Single hematopoietic stem cells generate skeletal muscle through myeloid intermediates, Nat. Med. 9 (12) (2003) 1520–1527. [11] J.E. Morgan, et al., Myogenic cell proliferation and generation of a reversible tumorigenic phenotype are triggered by preirradiation of the recipient site, J. Cell Biol. 157 (4) (2002) 693–702. [12] G. Ferrari, A. Stornaiuolo, F. Mavilio, Failure to correct murine muscular dystrophy, Nature 411 (6841) (2001) 1014–1015. [13] G. Cossu, P. Bianco, Mesoangioblasts-vascular progenitors for extravascular mesodermal tissues, Curr. Opin. Genet. Dev. 13 (5) (2003) 537–542. [14] M. Sampaolesi, et al., Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts, Science 301 (5632) (2003) 487–492. [15] A. Musarò, N. Rosenthal, The critical role of insulin-like growth factor-1 isoforms in the physiopathology of skeletal muscle, Curr. Genomics 3 (7) (2006) 19–32. [16] A. Musaro, et al., Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle, Nat. Genet. 27 (2) (2001) 195–200. [17] E.R. Barton-Davis, et al., Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function, Proc. Natl. Acad. Sci. U. S. A. 95 (26) (1998) 15603–15607. [18] E.R. Barton, et al., Muscle-specific expression of insulin-like growth factor I counters muscle decline in mdx mice, J. Cell Biol. 157 (1) (2002) 137–148. [19] S. Abmayr, et al., Phenotypic improvement of dystrophic muscles by rAAV/microdystrophin vectors is augmented by Igf-1 codelivery, Molec. Ther. 12 (3) (2005) 441–450. [20] A. Musaro, et al., Stem cell-mediated muscle regeneration is enhanced by local isoform of insulin-like growth factor 1, Proc. Natl. Acad. Sci. U. S. A. 101 (5) (2004) 1206–1210. [21] F. Relaix, et al., Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells, J. Cell Biol. 172 (1) (2006) 91–102. [22] S. Kuang, et al., Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis, J. Cell Biol. 172 (1) (2006) 103–113.
Discussion Jeffrey Johnson In the case of muscle denervation crush, do you see somewhere kinds of responses in the muscle to that, because the muscle will go away very fast in that kind of a situation.
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Antonio Musarò We analyzed the effect of IGF-1 not just in the innervated muscle, but in a model which is correlated to muscle denervation, which is the amiotrophic lateral sclerosis transgenic mouse in which there is a degeneration of motor neurons. What basically we found is that the IGF-1 not only preserves muscle mass in this pathological model, but was also in some way able to protect motor neurons from degeneration. So what we found is that if you maintain the muscle in good condition, in good shape, the muscle is also able to signal to motor neurons trying to activate antiapoptic pathways. And the idea is that the skeletal muscle is a source of signals that help not only the muscle, but also help to save motor neurons from degeneration. Jeffrey Johnson Was that in the G-93A mice? Antonio Musarò Yes, it’s the SODG-93 mouse crossed with our transgenic mouse. Jeffrey Johnson Why type-1 versus type-2 differential sensitivity to degeneration? We have studied the GA-93 mice a lot. And when you look at the muscles, it’s clear the type-2 fibers are the ones that are going, very early in the pathology. And the type-1s are there right to the end, when they become paralyzed… so why? Antonio Musarò This is a good question that probably no-one has a good answer to. I can guess the reason is that the slow fibers are more resistant to degeneration compared to the fast fibers and, thinking about the evolutionary aspect, probably the idea is that it’s better to lose fast fibers rather than slow fibers, because the slow fibers are involved in the maintenance of the muscle, of the body part, the fast fibers are involved in muscle strength. Pura Muñoz It’s reported that calcineurin is expressed in slow fibers, and in the case of the dystrophic mice calcineurin stimulates utrophin expression, thereby compensating for the lack of dystrophin. So the slow fibers are endowed with an additional mechanism, at least in dystrophic conditions. Reinhard Fässler You showed that locally produced IGF is producing all these good effects on muscle, but you didn’t mention how it works. Is it working on satellite cells or on the myofibers or is it working through IGF receptors? Where are the IGF receptors expressed? Which IGF receptors are they, because IGF receptors are very, very low-expressed in muscle? Antonio Musarò The signal transduction pathway goes through the IGF receptor, and the specific action of IGF-1 is to activate the satellite cells. And the hypertrophic phenotype actually involves not only increasing the protein synthesis, but also IGF-1 inducing the activation of satellite cells: these satellite cells are fused with the pre-existing fibers and increase the muscle phenotype. I don’t know if there is any correlation between, for example, IGF-1 and the other growth factors, but the interesting thing about IGF-1 is that it’s one of the growth factors that can stimulate the two events that are mutually exclusive, proliferation and differentiation, in contrast with, for example, FGF which stimulates proliferation but blocks differentiation. So, in vitro, we demonstrated that IGF-1 activates
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two different signal transduction pathways. We induce proliferation activating MAP kinase, and then induce calcineurin and the EKT pathway to induce differentiation in the hypertrophy. Reinhard Fässler Are the IGF-1 receptors expressed on myofibers? Antonio Musarò Yes, they’re expressed there. Reinhard Fässler At high levels or very low levels, or only after regeneration? Are they expressed on resting muscle? Antonio Musarò They are also expressed in resting muscle. I don’t have evidence for an increase in its expression during muscle regeneration. Martin Schwab I have a question about neuronal activity which regulates, of course, muscle development and also trophic status of muscle mass in a very crucial way. Is IGF-1 a direct downstream gene regulated by neuronal activity? Did you look for that or did anyone? Antonio Musarò No, we don’t have evidence for that, but IGF-1 is one of the growth factors that is expressed early on during muscle development. And after birth IGF-1 expression decreases progressively, and in humans over 30 years old the levels of IGF-1 have really dropped. So I don’t know if there is any connection between nerve activity and IGF-1 production. But, as I said before, when we maintain IGF-1 over-expression in the SODG-93A mouse model, where there is a degeneration of a motor neuron, we are able to demonstrate that, in this situation, we also preserve not only motor neuron activity, but also the numbers of motor neurons. Martin Schwab But this could even be a direct effect of IGF-1 on the motor neurons. It has been used as you know in clinical trials Antonio Musarò Exactly. Jeffrey Davidson In your adoptive transfer experiment with the AP-marked cells, what fraction of marrowderived circulating cells are participators in the repair process? Antonio Musarò We actually analyzed several compartments of the bone marrow derived stem cells and we found that the SP population plays the major role or, let’s say, has the best effect. Because when we transplant, for example, stem cells which lack c-kit, we couldn’t get the same results. So we analyzed a sort of different population that expressed all the markers, for example, ESK1, CD45, CD11b and C-kit, plus others that express, for example, CD45 and CD11b. Comparing these two populations, using the first one we got the best results, but when we transplanted CD-45 positive, CD-11b positive, but CKit negative and ESK1 negative, we didn’t obtain good results in terms of positive fibers.
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Jeffrey Davidson But following that, what fraction of the regenerative capacity in the repairing muscle comes from the circulating cells versus the endogenous precursors? Antonio Musarò Probably, under our conditions, less than 10%. Less than 10% of these transplanted stems cell can participate in regeneration. I mean the major role is played by satellite cells, definitely by satellite cells. Jeffrey Davidson In experiments where you’ve shown that IGF is accelerating the decline in inflammatory signals, what is the cellular source of those cytokines? Is it myocytes or inflammatory cells? Antonio Musarò We used by cytofluorimetric analysis, and the IGF-1 modulates, for example, the recruitment of macrophages and of neutrophils. So these are the two main populations that are modulated by IGF-1 expression and that produce these pro-inflammatory cytokines. Norbert Fusenig I have two questions concerning your "bodybuilder mouse". First, are all muscles involved in the hypertrophy and hyperplasia, or only those in physical activity? Antonio Musarò The MLC promoter is activated specifically in the fast fibers. For example, the soleus is composed mainly by slow fibers, the EDL by fast fibers and if you compare the muscle mass between the soleus and the EDL, there is a significant difference. So the EDL is more of a hypertrophic muscle than the soleus, and this phenotype is induced just by overexpression, so the mice don’t need it to exercise to show this hypertrophy… Norbert Fusenig So what happens to the heart? Antonio Musarò The heart is basically normal. I mean, we analyze the histological level and also the functional levels, and this local expression of IGF-1 doesn’t induce hypertrophic phenotype in the heart. Norbert Fusenig And a second question. Is it an ongoing process or is it limited to some time. Do the mice grow and grow until the skin is exploding? Antonio Musarò No, actually. This phenotype starts at six weeks after birth and you can maintain it for the entire life, so there is no continuous increase in the muscle mass. Definitely there is some fat or other mechanism that balances the effect. Norbert Fusenig Do you see any therapeutic application? Has something been done in that direction? Antonio Musarò Not yet. Most of the clinical trials were done using the other isoform of IGF-1, and the problem is that this circulating form that induces adverse side effects. Actually there is a link between increased level of IGF-1 and tumor formation. So the idea is to use this local form and since this local form doesn’t circulate you have to inject it directly into the muscle. An obvious approach was to place this IGF-1 in a vector and inject this vector in a senescent muscle. Using this gene therapeutic approach we demonstrated that we can rescue the
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senescent skeletal muscle, improving muscle mass and also muscle strength in senescent muscle. Snorri Thorgeirsson I have an obligatory stem cell question. So is this a fusion or a transdifferentiation? And if it is a fusion, what is the cell it fuses with? Antonio Musarò The answer is that I don’t know. And there is, let’s say, a big fight between two groups: there are some who believe that it’s transdifferentiation, others that it’s fusion. I mean in skeletal muscle it doesn’t matter too much whether it’s fusion of transdifferentiation, because in the end what is important is to get functional recovery. But it’s a good point. So far nobody has found a way to discriminate between transdifferentiation and fusion in skeletal muscle. Snorri Thorgeirsson But you would think though that if you fuse satellite cells, that would be different than if you fused with a more committed cell. Antonio Musarò Yes, sure, if your bone marrow stem cells are fused with pre-existing fibers or with the satellite cells and become muscle, the muscle is helping as well. Snorri Thorgeirsson You’ve shown us the benefit of IGF-1, but is it necessary? What happens if you knock it out selectively in the muscle? Antonio Musarò It’s great point, but nobody has knocked out specifically this isoform of IGF-1. All the evidence reported is on knocking out the circulating form.