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Developmental Biology 279 (2005) 336 – 344 www.elsevier.com/locate/ydbio Bone marrow contribution to skeletal muscle: A physiological response to stress Adam T. Palermob,1, Mark A. LaBargeb,1, Regis Doyonnasa, Jason Pomerantza, Helen M. Blaua,b,* a Baxter Laboratory in Genetic Pharmacology, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305-5175, USA b Department of Molecular Pharmacology, Stanford University School of Medicine Stanford, CA 94305, USA Received for publication 1 November 2004, revised 16 December 2004, accepted 20 December 2004 Available online 22 January 2005 Abstract Adult bone marrow-derived stem cells (BMDC) have been shown to contribute to numerous tissues after transplantation into a new host. However, whether the participation of these cells is part of the normal response to injury remains a matter of debate. Using parabiotically joined pairs of genetically labeled and wildtype mice, we show here that irradiation-induced damage of the target tissue, injection of bone marrow into the circulation, and immunological perturbation that are consequences of bone marrow transplantation are not necessary for bone marrow contribution to myofibers. Moreover, severe toxin-induced damage is not a prerequisite, as BMDC contribution to muscle is enhanced in response to increased muscle activity resulting from muscle overloading or forced exercise. Indeed, these two forms of muscle stress result in much more rapid contribution (within 1 month) than voluntary running (6 months). These results indicate that BMDC contribute to myofibers in response to physiologic stresses encountered by healthy organisms throughout life. D 2005 Elsevier Inc. All rights reserved. Keywords: Adult stem cells; Bone marrow-derived cells; Muscle; Transplantation; Irradiation; Regeneration; Damage; Exercise; Parabiosis Introduction Following transplantation with genetically marked bone marrow, donor-derived cells can be found integrated into diverse somatic tissues of the transplant recipient, such as skeletal muscle, brain, liver, kidney, lung, and gastrointestinal tract (Alvarez-Dolado et al., 2003; Brazelton et al., 2000, 2003; Camargo et al., 2004; Ferrari et al., 1998; Abbreviations: BMDC, bone marrow-derived cells; NTX, Notexin; BMT, bone marrow transplantation; HSC, hematopoietic stem cell; TA, tibialis anterior; EDL, extensor digitorum longus. * Corresponding author. Department of Molecular Pharmacology, Baxter Laboratory in Genetic Pharmacology, Stanford University School of Medicine, 269 W. Campus Drive CCSR 4215, Stanford, CA 943055175, USA. Fax: +1 650 736 0080. E-mail address: [email protected] (H.M. Blau). 1 These authors contributed equally to this work. 0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2004.12.024 Gussoni et al., 1999; Harris et al., 2004; Houghton et al., 2004; Kale et al., 2003; Krause et al., 2001; LaBarge and Blau, 2002; Lagasse et al., 2000; Mezey et al., 2000; Theise et al., 2002; Weimann et al., 2003a,b; for reviews, see Pomerantz and Blau, 2004; Wagers and Weissman, 2004). In one striking example, the contribution of BMDC to hepatocytes by fusion rescued mice with a lethal metabolic liver disease, indicating that BMDC can activate previously silent genes and function as nonhematopoietic cells (Camargo et al., 2004; Lagasse et al., 2000; Vassilopoulos et al., 2003; Wang et al., 2002). In other cases, mononucleate BMDC have been shown to assume different phenotypes in the absence of fusion and become epithelial cells (Harris et al., 2004) or muscle satellite cells (Dreyfus et al., 2004; Fukada et al., 2002; LaBarge and Blau, 2002; Sherwood et al., 2004). However, these experiments have all used bone marrow transplantation, which is highly invasive, and associated A.T. Palermo et al. / Developmental Biology 279 (2005) 336–344 with lethal irradiation as well as massive disregulation of cytokines. These confounding variables do not allow conclusions to be made regarding the potential of BMDC to contribute to tissues under normal physiological conditions. In the case of skeletal muscle, extensive research has convincingly shown that bone marrow-derived nuclei can become incorporated into mature myofibers of transplant recipients (reviewed in Grounds et al., 2002). Bone marrow contains a variety of cell types, such as hematopoietic cells, stromal cells, and endothelial progenitors, any of which could theoretically contribute to muscle in experiments in which whole bone marrow is transplanted. That at least some bone marrow-derived myonuclei are of hematopoietic origin has been clearly shown by transplantation of single hematopoietic stem cells (HSC) (Camargo et al., 2003; Corbel et al., 2003). Further investigation has revealed that one type of HSC derivative, the myelomonocytic cell, can contribute to both skeletal muscle and liver (Camargo et al., 2003, 2004; Doyonnas et al., 2004; Willenbring et al., 2004). To date, a major caveat of most reports of bone marrow contribution to myofibers has been that they utilized bone marrow transplantation (BMT), as this method enables bone marrow genetically marked with green fluorescent protein (GFP), beta-galactosidase, or sex chromosome differences to be tracked and studied in a background of unmarked tissues. The problem is that BMT is a severe procedure that introduces a number of complex variables. Specifically, transplantation involves the delivery of high doses of whole body radiation to ablate the endogenous bone marrow, and create a niche that is essential for donor marrow engraftment and reconstitution of the blood lineages. Such radiation doses are well documented to damage numerous tissues that contain proliferative cells in addition to the marrow. For example, irradiation of skeletal muscle impairs the local satellite cell population (muscle stem cells) and can lead to muscle atrophy, inflammation, and vascular leakiness (Baker and Krochak, 1989; Rosenblatt and Parry, 1992; Van der Meeren et al., 2001). Furthermore, the mechanical extraction of bone marrow from its native environment and injection directly into the circulation may allow cells that are normally confined to the bone marrow compartment to access tissues where they are not found under normal circumstances. Thus, published results to date do not effectively address whether bone marrow contribution to non-hematopoietic tissues occurs during the normal life of an organism. Consequently, it is critically important to determine whether the contribution of BMDC to muscle can also occur independently of BMT-associated variables. BMDC contribution to muscle is a response to tissue damage and was previously missed when studied in the absence of overt muscle injury (Wagers et al., 2002). By contrast, damage induced by delivery of toxins such as 337 notexin or cardiotoxin has resulted in an increased contribution to skeletal muscle (Abedi et al., 2004; Camargo et al., 2003; Corbel et al., 2003; Fukada et al., 2002). Although these stimuli have resulted in significant increases in the number of myofibers that integrate bone marrow-derived nuclei, these are severe forms of muscle damage in which widespread necrosis and inflammation are typical (Lefaucheur and Sebille, 1995). The question remains as to whether BMDC respond in this manner to stresses typical of normal life such as increased muscle use. In this study, we determine whether bone marrowderived cells contribute to muscle in response to physiological stimuli encountered throughout life. To determine whether damage due to muscle overuse could substitute for toxins, we exposed mice to treadmill running or surgically removed a muscle so that an otherwise underutilized muscle was overloaded. In both cases, a significant increase in the number of GFP+ muscle fibers was observed after 1 month. To eliminate transplant-associated variables, we used parabiotically joined mice to genetically label and track blood cells. The data reported here provide evidence that cells from the circulation, which derive from the bone marrow, can incorporate into myofibers independently of transplantation and in response to damageinduced signals typically encountered in the course of normal adult life. Materials and methods Mice and bone marrow transplantation C57/B6 (wildtype) mice were purchased from Jackson laboratory (Bar Harbor, ME) and C57/B6 GFP transgenic (GFP) mice (gift from Dr. Okabe, Osaka, Suita, Japan) were bred in-house and maintained in a pathogen-free environment. Bone marrow of GFP donor mice was flushed from the femurs and tibias to make single cell suspensions. Recipient wt mice that had received total body irradiation (9.6 Gy) were transplanted at 8 weeks of age with 106 cells of total bone marrow. Peripheral blood of transplanted recipients was analyzed at various time points after transplantation and only mice with more than 90% GFP+ cells in their peripheral blood were used. All protocols were approved by the Administrative Panel on Laboratory Animal Care at Stanford University School of Medicine. Parabiosis Parabiotic pairs were created by a modification of the procedure of Bunster and Meyer (1933). Mice were anesthetized with a cocktail of Ketamine (2.4 mg per mouse, Fort Dodge Animal Health, Overland Park, KS) and Xylazine (240 Ag per mouse, Phoenix Scientific, St. Joseph, 338 A.T. Palermo et al. / Developmental Biology 279 (2005) 336–344 MO) delivered by intraperitoneal injection. Mice were shaved on either their right side or left side. The shaved skin of each mouse was sterilized and incisions were made from the olecranon to the knee joint, and the subcutaneous fascia was bluntly dissected to create free skin. A single 4-0 silk suture and tie was used to join the opposing olecranon and knee joints, and opposing dorsal and ventral skins were approximated by 9 mm wound clips (Becton-Dickinson, Franklin Lakes, NJ). Mice were given supportive care including buprenorphine, moistened food, and subcutaneous saline as needed, and each pair was housed separately. Cross-circulation and the degree of peripheral blood chimerism was evaluated by retro-orbital bleeding followed by analysis on a FACSCalibur flow cytometer (BectonDickinson, Franklin Lakes, NJ). Notexin injury Mice were anesthetized with inhaled methoxyflurane, the skin over the tibialis anterior (TA) was shaved, and a single 10 Al dose of 10 Ag/ml NTX (Sigma, St. Louis, MO) in PBS was injected. Forced exercise C57Bl/6 mice were transplanted with bone marrow from GFP transgenic mice 3 months prior to exposure to forced exercise. The mice ran on a motor-driven treadmill (Exercise 4 treadmill, Columbus Instrument, Columbus, OH) at a constant speed of 20 m/min on a 108 decline (downhill) for 60 min, 3 times per week with 1 day of rest between sessions. All mice were trained for 15 min before the first session. Training involved 5 min at 10 m/min followed by a progressive increase in speed from 10 m/min to 20 m/min at a rate of 1 m/min. To encourage animals to move forward and start running, a mild electric shock was applied from a grid behind the treadmill. Mice in the 4 week group were submitted to the protocol described above for 4 consecutive weeks. For both groups (1 or 4 weeks running), the TA, extensor digitorum longus (EDL), and soleus muscles were harvested for analysis 8 weeks after the onset of the exercise regimen. EDL overload Mice were anesthetized with Avertin (30 mg/kg, SigmaAldrich, St. Louis, MO) delivered by intraperitoneal injection. Their left and right legs were sterilized and incisions were made from below the ankle joint to above the knee, along the front of the leg. An incision was made in the epimysium, and the TA was bluntly dissected away from the rest of the leg. In sham-operated control mice, the skin was closed at this point, while in the contralateral experimental legs, the TA was then removed by first severing it below the myotendonous junction and gently separating it from the underlying structures before cutting just below the knee. The skin was then closed using a single 4-0 silk suture. Mice were given supportive care including buprenorphine, moistened food, and subcutaneous saline as above. Tissue collection and immunohistochemistry TA, EDL, or soleus muscles were dissected and immersed in PBS/0.5% EM grade paraformaldehyde for 2 h at room temperature followed by overnight immersion in PBS/20% sucrose at 48C. Fixed tissue was imbedded in OCT, snap-frozen in isobutane and liquid nitrogen, and then sectioned at 10 Am thickness. Sections were blocked in PBS/20% normal goat serum/0.3% Triton X-100 for 2 h, then incubated with primary antibodies against GFP (rabbit, 1:1000, Molecular Probes, Eugene, OR), and laminin-a2 (rat anti-mouse, 1:400, Upstate Biotech Associates, Waltham, MA), in blocking solution for 1 h. Following PBS/ 0.1% Triton X-100 washes, sections were incubated in secondary antibodies (goat anti-rabbit Alexa488, goat antirat Alexa546, Molecular Probes, Eugene, OR) in blocking solution for 1 h. Following short washes in PBS/0.1% Triton X-100, the sections were washed overnight in PBS, before mounting them with Fluoromount G (Southern Biotechnology Associates, Birmingham, AL). Some sections were counterstained with 5 Ag/ml Hoechst 33342 (Sigma, St. Louis, MO). Quantification of GFP+ muscle fibers To quantitate muscle fibers, at least three sections from each mouse that were z100 Am apart from each other were imaged on a Zeiss Axioplan epifluorescent microscope (Carl Zeiss Inc., Thornwood, NY) with a low magnification objective (5). Nuclear staining (Hoechst 33342) was visualized using a 345–385 nm BP excitation filter and a 435–485 nm BP emission filter. Laminin immunofluorescence images (Alexa546) were acquired using a 530–560 nm BP excitation filter and a 573–648 nm BP emission filter. GFP immunofluorescence images (Alexa488) were acquired using a 460–500 nm BP excitation filter and a 510–560 nm BP emission filter. All putative GFP+ fibers were visually confirmed using a dual-band red-green filter set exciting at 485–505 nm/560– 585 nm with emissions at 515–550 nm/590–660 nm. This filter set is useful for readily discriminating between Alexa488 fluorescence and autofluorescence. GFP immunofluorescence images were then overlaid on laminin immunofluorescence images and GFP+ muscle fibers were only counted if they were completely surrounded by an intact basal laminal membrane. Counting only fibers with intact basal laminal membranes ensures that dying fibers or large conglomerations of blood cells are excluded from the analysis. The confocal images (1 Am optical sections) were acquired on a Zeiss laser-scanning microscope (LSM 510). A.T. Palermo et al. / Developmental Biology 279 (2005) 336–344 339 Results Tissue irradiation is not a prerequisite for contribution to muscle from the circulation To determine whether irradiation of the muscle and associated variables of bone marrow transplantation are necessary for incorporation of circulating cells into myofibers, we generated surgically joined parabiotic pairs of mice. In such pairs, one partner was wildtype (C57/B6) and the other partner was a syngenic mouse in which the green fluorescent protein was under the control of the beta actin promoter and therefore expressed in all tissues (Fig. 1A). In parabiotic animals, anastamosis of the circulatory systems occurs and the peripheral blood can freely exchange between the two animals as early as 2 weeks after joining (Bunster and Meyer, 1933). Parabiosis thus enables GFP-labeled circulatory cells to gain access to the tissues of wildtype mice. Three weeks after parabiotic joining, the peripheral blood was evaluated by flow cytometry to confirm cross-circulation between the animals and the extent of blood chimerism. Mononuclear cells from peripheral blood were doublestained with either Mac1/Gr1 or B220/CD3 to identify their myeloid or lymphoid origin, respectively. FACS analysis indicated that peripheral blood of the wildtype partners contained 37–51% GFP+ cells (Figs. 1B, E), and normal proportions of the lymphoid and myeloid subsets. The wildtype partner was then injured by injection of NTX, a myotoxin commonly used to damage muscle, into the TA contralateral to the surgical site. Mice were sacrificed 2 or 4 weeks later and both injured and non-injured TA muscles of the wildtype mice were harvested. The TA adjacent to the surgical site served as a negative control for NTX damage. Circulating cell contribution to myofibers in the TAs was evaluated by immunofluorescence staining with antibodies against GFP and laminin, and Hoechst dye was used to visualize the nuclei. To control for the possibility that NTX injection had not been administered to the muscle in question, central nucleation of myofibers was used to assess whether the TA had been damaged and regeneration had been induced. This approach is based on the well-established finding that the myonuclei in healthy muscle are on the perimeter of myofibers, whereas in muscle regenerating either because of severe dystrophy (Coulton et al., 1988) or because of myotoxin injection (Martin and Ontell, 1988), myonuclei are in the center of fibers. Only mice in which the contralateral TAs contained N80% centrally nucleated fibers, indicative of muscle regeneration, are reported here. Our analysis indicated that the undamaged TAs contained very few GFP+ myofibers (Figs. 1C, E), whereas the regenerating contralateral TAs contained substantially more GFP+ myofibers (Figs. 1D, E). In this study, the average number of myofibers in a single transverse section of the TA approximated 3000. Thus, the average undamaged TA had less than 0.05% GFP+ myofibers per cross-section as Fig. 1. Contribution of circulating cells to skeletal muscle in parabiotic mice in the absence of irradiation. (A) Wildtype C57/Bl6 mice were parabiotically conjoined with GFP transgenic mice. Three weeks after joining, the wt partner was injured by notexin (NTX) injection into the tibialis anterior muscle (TA) contralateral to the surgical site. TA muscles were harvested 2 or 4 weeks later. (B) Peripheral blood mononuclear cells were evaluated by flow cytometry (FACS) for expression of GFP as well as staining with either a cocktail of lymphoid markers (B220/CD3) or a cocktail of myeloid markers (Mac1/GR1). FACS plots are shown for a GFP transgenic mouse (GFP+ ctrl), a C57/Bl6 control mouse (wt ctrl), and for either partner of a parabiotic pair. (C) Representative transverse section from a TA of a wildtype parabiotic partner not treated with NTX, showing intact, nonGFP+ muscle fibers. (D) GFP+ muscle fibers (green) surrounded by intact basal laminal membranes (red) in a representative TA transverse section from a NTX-injured parabiotic mouse (nuclei in blue, Hoechst 33342). (E) Number of GFP+ fibers observed in mice at either 2 or 4 weeks post injury, along with the percentage of GFP+ cells (chimerism) in the peripheral blood (% chim), and whether central nuclei were observed. Number of fibers is reported as mean F SD for 3 sections separated by at least 100 Am. 340 A.T. Palermo et al. / Developmental Biology 279 (2005) 336–344 compared to approximately 0.4% GFP+ myofibers in NTXinjured TAs, an increase of almost one order of magnitude. Thus, using parabiosis, we could demonstrate that cells present in the circulation become incorporated into regenerating muscle even when the muscle has not been damaged by irradiation and the local muscle-specific stem cells known as satellite cells are intact. Note that by contrast, the level of irradiation required for a bone marrow transplant ablates two thirds of the satellite cell population (LaBarge and Blau, 2002). While this experiment demonstrates the existence of circulating cells that can contribute to muscle, it does not identify the source of these cells, as all tissues of the GFP transgenic partner expressed GFP. Thus, GFP+ cells from tissues other than bone marrow could have been present in the circulation and become incorporated into myofibers of the wildtype partner. Circulating cells contributing to muscle regeneration are of bone marrow origin To test the hypothesis that the circulating cells with the ability to incorporate into myofibers can arise from the bone marrow, wildtype mice were joined to wildtype mice that had received bone marrow transplants from GFP transgenic mice 6 months earlier (Fig. 2A). In these experiments, bone marrow-derived cells, not cells from other tissues, are labeled with GFP in the parabiotic animals. Three weeks after joining, the peripheral blood of the non-transplanted partner was found to be 22–42% GFP+ by flow cytometry (Fig. 2D). Contralateral TAs of the non-transplanted mice were injured with NTX and evaluated 2 or 4 weeks later. In this experiment, as above, localized NTX-induced injury caused a substantial increase in the number of GFP+ myofibers compared to non-injured TA muscles (Figs. 2B, C, D). The fraction of GFP+ myofibers increased from less than 0.04% in undamaged muscle to approximately 0.4% in NTX-injured muscle, a magnitude similar to that observed when GFP transgenic rather than transplanted partners were used. We therefore conclude that the population of circulating cells able to incorporate into muscle in the previous experiment can be functionally reconstituted by transplanted bone marrow alone and does not require other cells. Taken together, these data demonstrate that BMDC incorporation into myofibers following transplantation is similar to that observed in GFP–wildtype parabiotic pairs, i.e., independent of irradiation or other bone marrow transplantation-associated variables. Forced downhill running elicits BMDC contribution to muscle fibers Most previous studies of BMDC contribution to muscle have employed injury models unrelated to the physiological stresses experienced by muscle in the course of normal life, or have required half a year to see effects. BMDC incorporation Fig. 2. Contribution of circulating bone marrow-derived cells to skeletal muscle in parabiotic mice. (A) Wildtype C57/Bl6 mice were parabiotically conjoined with wildtype mice that had received bone marrow transplants from GFP transgenic mice 6 months earlier. Three weeks after joining, the wt partner was injured by notexin (NTX) injection into the tibialis anterior muscle (TA) contralateral to the surgical site at which the mice were conjoined. TA muscles were harvested 2 or 4 weeks later. (B) Representative transverse section from non-notexin-injured TA of wildtype parabiotic partner, showing a single rare, GFP+ muscle fiber. (C) GFP+ muscle fibers (green) surrounded by intact basal laminal membranes (red) in a representative TA transverse section from a NTX-injured parabiotic mouse (nuclei in blue, Hoechst 33342). (D) Number of GFP+ fibers observed in mice at either 2 or 4 weeks post injury, along with the percentage of GFP+ cells (chimerism) in the peripheral blood (% chim), and whether central nuclei were observed. Number of GFP+ fibers is reported as mean F SD for 3 sections separated by at least 100 Am. has been shown to be elevated after acute myotoxin injury which results in chronic inflammation, necrosis, and regeneration (Abedi et al., 2004; Bossolasco et al., 2004; Camargo et al., 2003; Corbel et al., 2003; Fukada et al., 2002). In the course of normal life, however, the majority of ongoing myogenesis is the result of exercise-induced damage. Our previous work suggested that exercise-induced damage could elicit BMDC contribution to myofibers, but this occurred slowly over the course of 6 months (LaBarge and Blau, 2002). To determine whether a more acute stress could elicit the same effect, we placed wildtype mice 3 months post-GFP+ BMT on inclined treadmills and forced the mice to run for 1 h, 3 times per week for 1 or 4 weeks (Fig. 3A) (Lerman et al., 2002). Forced downhill running A.T. Palermo et al. / Developmental Biology 279 (2005) 336–344 Fig. 3. Bone marrow contribution to myofibers increases in frequency in response to physiological cues. (A) Wildtype C57/Bl6 mice that had received bone marrow transplants from GFP transgenic mice 3 months earlier were placed on a treadmill on a 108 decline at a speed of 20 m/min (downhill) for 60 min, 3 times per week with 1 day rest between sessions. TA, EDL, and soleus muscles were harvested after 1 or 4 weeks of exercise followed by 7 or 4 weeks of recovery. (B) Wildtype C57/Bl6 mice that had received bone marrow transplants from GFP transgenic mice 4 months earlier underwent surgery to remove their TA muscles. This procedure results in compensatory hypertrophy of the underlying EDL muscle. EDL muscles were harvested after 4 weeks. (C) Number of mice per group and number of GFP+ fibers per muscle for mice after forced exercise or EDL overload. GFP+ fibers are reported as mean F SD per section analyzed in each experimental group. involves acute eccentric contraction of the TA, EDL, and soleus muscles (Smith et al., 1997), which is distinct from voluntary running on a wheel (LaBarge and Blau, 2002). Eccentric muscle contraction can lead to muscle damage and delayed-onset muscle soreness in humans and mice (Kuipers, 1994). On the eighth week after the initiation of exercise, lower leg muscles of the exercised mice were evaluated and compared to control legs from unexercised, transplanted animals by immunostaining for GFP and laminin. Significant increases in GFP+ myofibers were observed in these animals after both 1 and 4 weeks of forced exercise (Fig. 3C), a result never seen with voluntary exercise. The fraction of GFP+ fibers increased from 0.02% in unexercised mice to 0.2% and 0.1% in mice exercised for 1 and 4 weeks, respectively. These results indicate that sustained exercise, in which fibers are thought to acquire membrane damage due to shear stress, is sufficient to elicit BMDC incorporation into muscle. Furthermore, these experiments demonstrate that BMDC can respond extremely rapidly to muscle damage, incorporating into myofibers in a single week. Overloading causes GFP+ myofibers to appear in the EDL BMDC incorporation into myofibers was also tested in another model of acute physiological muscle stress, in 341 which hypertrophy of the EDL muscle is induced by surgically resecting the TA (Fig. 3B). The TA and the EDL normally function synergistically during ankle flexion, and resecting the TA increases demand on the EDL, which undergoes a compensatory hypertrophy. This treatment has been shown to induce satellite cell contribution to myofibers as the fibers compensate, thus enabling the EDL to function alone (Rosenblatt and Parry, 1992). To test the hypothesis that EDL hypertrophy also results in increased BMDC contribution to myofibers, we resected the TA muscles of wildtype mice that had received GFP+ BMT 4 months earlier. In the contralateral leg, a sham operation was performed in which the TA was bluntly dissected away from the underlying EDL, but left intact. Mice were sacrificed 4 weeks after surgery. The EDL muscles of experimental legs were grossly hypertrophic as compared to the sham-operated legs. EDL sections from both legs were immunostained for GFP and laminin expression. Very low numbers of GFP+ myofibers were observed in the control muscles, whereas the hypertrophic EDLs all exhibited significant GFP incorporation (Fig. 3C). On average, 0.05% of fibers were GFP+ in non-overloaded muscle, whereas 1.6% of fibers were GFP+ in overloaded muscle, a 30-fold increase. From these two experiments, in which muscle usage was forcibly and acutely increased, we conclude that BMDCs can respond relatively rapidly to physiological stresses as well as to more extreme damage by incorporating into myofibers. Discussion This report demonstrates that previous findings by us and others that bone marrow-derived cells can contribute to muscle fibers are not merely a result of the harsh experimental paradigms used, but also occur under physiological conditions. To date, most studies, including our own, have used bone marrow transplantation to follow the fate of genetically marked cells and have monitored the contribution of bone marrow derivatives to tissues extensively damaged due to destructive toxins. Here, using skeletal muscle as a model, we provide evidence that cells from the bone marrow can contribute to other tissues in response to types of acute stress that occur during the normal life of a animal. First, in parabiotically joined animals, blood chimerism allowed tracking of circulating BMDC originating in the bone marrow of one mouse and detection of those cells in the damaged muscle of its parabiotic partner in the absence of BMT and associated variables. Second, during hypertrophy, a physiologic response to two forms of forced increased muscle use, BMDC incorporated into muscle as efficiently and rapidly as after severe toxin-induced damage. Together, these experiments rule out a number of confounding variables, and suggest that bone marrow contribution to tissues occurs relatively rapidly in response to injury associated with strenuous physical activity. 342 A.T. Palermo et al. / Developmental Biology 279 (2005) 336–344 Bone marrow transplantation (BMT) involves wholebody irradiation, mechanical extraction, and injection of bone marrow cells into the bloodstream. In the course of BMT, marked systemic alterations occur, such as changes in the balance of cytokines (Holler et al., 1997). If these transplant-associated variables were prerequisites for the contribution of BMDC to muscle fibers, BMDC nuclei would not be expected to be found in skeletal muscle fibers in the course of normal life, a finding that would significantly limit the therapeutic potential of BMDC. The parabiosis studies presented here show that when GFP transgenic mice were surgically joined to wildtype mice, GFP+ muscle fibers were detected in the wildtype partner, and their frequency was significantly increased by NTXinduced muscle damage. These experiments show that a cell with the ability to transit through the circulation can contribute to non-irradiated regenerating muscle in the absence of bone marrow transplantation. Although enhanced by NTX-mediated muscle damage, this participation of BMDC also occurred in low numbers in the absence of NTX, in the leg adjacent to the conjoining surgical site. When GFP+ bone marrow transplant recipients were used as the parabiotic donors, similar results were found. This finding suggests that the population of cells that contributed to muscle was not derived from cells which entered the bloodstream from other tissues, but rather from the bone marrow via the circulation, in a manner similar to that previously described using bone marrow transplantation. Although parabiotic joining avoids most variables associated with bone marrow transplantation and irradiation, transient inflammation is associated with the surgical procedure, similar to that which occurs after wounding. We found evidence of systemic inflammation in a separate cohort of parabiotic mice, but it occurred only in a subset of animals and the extent of inflammation was not outside the range of the response observed during wound repair, which is a common physiologic occurrence (data not shown). Additionally, serum creatine phosphokinase (CPK) levels, indicative of muscle damage, were measured in parabiotic animals. The results were within the normal range of the assay (158 F 87 IU/L and 244 F 125 IU/L at 2 and 14 days after surgery) and were similar to CPK levels measured in uninjured mice. In contrast, work by others has shown that in the dystrophic mdx mouse, CPK levels approximate 5000 IU/L (Hoyte et al., 2004). Therefore, by this measure, parabiotic joining does not induce significant muscle damage. Other groups have also used parabiotic joining to emulate physiologic conditions, and have observed that parabiosis does not alter the mobilization of bone marrow progenitor cells in comparison with non-parabiotic mice (Wright et al., 2001). Moreover, it should be noted that the same group, using parabiotic animals in which no muscle damage was induced, observed very few GFP+ muscle fibers (Wagers et al., 2002), in good agreement with our findings. Thus, the inflammation associated with the surgery required for joining mice does not by itself account for bone marrow contribution to myofibers. Nonetheless, the possibility that some systemic inflammation is necessary for BMDC to contribute to muscle cannot be ruled out by this or other published reports to date. The nature of such inflammatory signals, their identity, and their potential role in the recruitment and activation of BMDC is a subject of major interest. In a second set of experiments, we explored the nature of regenerative stimuli that are able to elicit BMDC contribution to muscle and whether physiologic stimuli encountered in daily life could suffice. Most muscle regeneration occurs in response to use-related damage. BMDC contribution to muscle was found to be inducible in two different models: forced downhill running on a treadmill and compensatory hypertrophy of the EDL after resection of the TA. Both of these stresses resulted in significant increases in GFP+ myofibers within 4 weeks after forced exercise or surgery. From these studies, we conclude that toxin-induced muscle damage that is typically associated with widespread necrosis and inflammation is not required for BMDC contribution to muscle. Indeed, we found that contribution of BMDC to muscle during the acquisition of new healthy muscle tissue is on a par with that seen after toxin damage. The number of bone marrow-derived nuclei in regenerating muscle is difficult to assess. In this, and other studies (Brazelton et al., 2003; LaBarge and Blau, 2002), we have observed muscles containing a range of 0.5% to 5% GFP+ myofibers. However, because a myofiber is a multinucleate syncytium, it is unclear what proportion of total nuclei in myofibers are derived from the bone marrow. Since GFP is not restricted to the vicinity of the nucleus that encodes it, but is a diffusible cytoplasmic protein, the fraction of myonuclei derived from bone marrow is likely to be lower than the fraction of GFP+ fibers. In addition, it is not known whether the amount of GFP expressed by a single nucleus, when distributed into the large volume of a myofiber, is detectable by our assay. It is also possible that BMDC nuclei fuse preferentially with certain fibers and not others. These complicating factors make it difficult to estimate from this type of study what fraction of myonuclei is of bone marrow origin. However, the possibility that this fraction may be very small does not affect the conclusion that it does occur, and responds to physiological cues. We hypothesize that BMDC contribution to muscle is mediated by a specific set of molecular cues associated with physiologic muscle injury, inflammation, and the resulting mobilization of BMDC. The identity of these cues is an important matter for future investigation. Factors that activate myelomonocytic progenitors, shown to be involved in bone marrow contribution to muscle (Camargo et al., 2003; Doyonnas et al., 2004), are a reasonable starting point. For example, G-CSF is known to mobilize myelomonocytic progenitors from the bone marrow of mice (Lapidot and Petit, 2002). Moreover, recent work in humans has shown that endurance exercise results not only in acutely higher blood levels of G-CSF and other cytokines, A.T. Palermo et al. / Developmental Biology 279 (2005) 336–344 but also in a chronic elevation of the number of circulating bone marrow-derived progenitor cells (Bonsignore et al., 2002). Another class of factors worthy of investigation includes molecules involved in the proliferation and differentiation of muscle stem cells (satellite cells), such as IGF-1 (Florini et al., 1996). It has already been shown that transgenic overexpression throughout life of the muscle isoform of IGF-1 can enhance BMDC recruitment to regenerating muscle in vivo (Musaro et al., 2004). Local in vivo delivery of such factors in wildtype mice would indicate whether these or other factors are involved in signaling BMDC to contribute to myofibers. In conclusion, the results reported here eliminate a number of variables inherent in previous reports and demonstrate that BMDC contribution to muscle occurs under circumstances compatible with daily activity and is a characteristic of normal physiology. These findings provide impetus for understanding the underlying mechanism and the role of inflammation and injury-related signals. From a therapeutic perspective, the frequency of contribution may currently be inadequate for improving muscle function. However, the prospect of recruiting circulating cells to regenerating muscle would circumvent the problems of cell distribution associated with intramuscular injection of myoblasts for therapy. We have shown here that this is a natural phenomenon that is a response to damage. Elucidation of the relevant damage-associated factors could potentially lead to a robust response sufficient for therapy. Acknowledgments We thank Frances L. Johnson for her teaching and assistance with the parabiosis protocols, Andrew Patterson for assistance with the treadmill exercise protocols, Peggy Kraft and Kassie Koleckar for their excellent technical support, and James Weimann and Alessandra Sacco for useful comments. 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