Mesenchymal Stem Cells Augment the Adaptive Response to Eccentric Exercise KAI ZOU1,2, HEATHER D. HUNTSMAN1,2, M. CARMEN VALERO1,2, JOSEPH ADAMS1,2, JACK SKELTON1,2, MICHAEL DE LISIO1,2, TOR JENSEN3, and MARNI D. BOPPART1,2 1
Department of Kinesiology and Community Health, University of Illinois, Urbana, IL; 2Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana, IL; and 3Division of Biomedical Sciences, University of Illinois, Urbana, IL
ABSTRACT
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contraction). The need for the muscle to contract during lengthening is most evident during activities that require transfer of a load away from the body (weightlifting) or during downward locomotion to prevent imbalance and falling. While repeatedly shortening the muscle confers predominantly metabolic adaptations that provide resistance to fatigue, single or multiple bouts of lengthening can cause damage to muscle ultrastructure, initiating a long-term regenerative response that may positively affect growth and strength after exercise (14,31). Downhill running has been used as a model to investigate the natural processes of skeletal muscle injury and repair after eccentric exercise (1,23,24). Similar to injury created by other methods, such as cardiotoxin and crush injury, a single bout of downhill running can impair sarcolemmal integrity, evoke an immune response, and reduce maximal isometric force within 24 h after exercise (4,5). Within 2–4 d, satellite cell number is increased and newly formed fibers with centrally located nuclei (CLN) are apparent throughout the muscle (22). Although previous studies suggest that repeated bouts of downhill running do not exacerbate muscle damage or lengthen the recovery process (29), few studies, to our knowledge, have evaluated the long-term adaptive response to downhill running.
keletal muscle is a highly structured and intricately organized organ that provides the basis for human movement. Physical interaction between the essential myofibrillar proteins, actin and myosin, within each myofibril of a skeletal muscle cell can allow for the development of tension and the transfer of force to tendons and bones. Although concentric (shortening) contractions are most commonly used for movement of the peripheral limbs during physical activity, lengthening of the muscle can also occur during actin–myosin cross-bridge formation (eccentric
Address for correspondence: Marni D. Boppart, Sc.D., Beckman Institute for Advanced Science and Technology, 405 N. Mathews Avenue, MC-251 Urbana, IL 61801; E-mail: [email protected]. Submitted for publication February 2014. Accepted for publication May 2014. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.acsm-msse.org). 0195-9131/15/4702-0315/0 MEDICINE & SCIENCE IN SPORTS & EXERCISEÒ Copyright Ó 2014 by the American College of Sports Medicine DOI: 10.1249/MSS.0000000000000405
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ZOU, K., H. D. HUNTSMAN, M. CARMEN VALERO, J. ADAMS, J. SKELTON, M. DE LISIO, T. JENSEN, and M. D. BOPPART. Mesenchymal Stem Cells Augment the Adaptive Response to Eccentric Exercise. Med. Sci. Sports Exerc., Vol. 47, No. 2, pp. 315–325, 2015. Purpose: The >7A1 integrin is a transmembrane protein expressed in the skeletal muscle that can link the actin cytoskeleton to the surrounding basal lamina. We have previously demonstrated that transgenic mice overexpressing the >7B integrin in the skeletal muscle (MCK:>7B; >7Tg) mount an enhanced satellite cell and growth response to single or multiple bouts of eccentric exercise. In addition, interstitial stem cells characterized as mesenchymal stem cells (MSCs) accumulate in >7Tg muscle (mMSCs) in the sedentary state and after exercise. The results from these studies prompted us to determine the extent to which mMSC underlie the beneficial adaptive responses observed in >7Tg skeletal muscle after exercise. Methods: mMSCs (Sca-1+CD45j) were isolated from >7Tg mice, dyelabeled, and intramuscularly injected into adult wild type recipient mice. After injection of mMSCs or saline, mice remained sedentary (SED) or were subjected to eccentric exercise training (TR) (downhill running) on a treadmill (three times per week) for 2 or 4 wk. Gastrocnemius–soleus complexes were collected 24 h after the last bout of exercise. Results: mMSCs did not directly fuse with existing fibers; however, mMSCs injection enhanced Pax7+ satellite cell number and myonuclear content compared with all other groups at 2 wk after exercise. Mean CSA, percentage of larger caliber fibers (93000 Km2), and grip strength were increased in mMSCs/TR compared with saline/SED and mMSCs/SED at 4 wk. mMSC transplantation did not enhance repair or growth in the absence of exercise. Conclusions: The results from this study demonstrate that mMSCs contribute to beneficial changes in satellite cell expansion and growth in >7Tg muscle after eccentric exercise. Thus, MSCs that naturally accumulate in the muscle after eccentric contractions may enhance the adaptive response to exercise. Key Words: A7 INTEGRIN, SATELLITE CELLS, STEM CELL ANTIGEN-1, DOWNHILL RUNNING, REPAIR, GROWTH
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The >7A1 integrin is a critical transmembrane protein that links laminin in the basement membrane with the actin cytoskeleton across the sarcolemma (2,7). Our previous work has established that total >7B integrin protein expression is upregulated and transgenic expression of the >7BX2 integrin in the skeletal muscle (MCK:>7BX2; >7Tg) can reinforce sarcolemmal integrity as well as reduce macrophage content and preserve function after an acute bout of downhill running in young healthy mice (4,5). We subsequently demonstrated that >7Tg mice exhibit a robust regenerative response to single or multiple bouts (three times per week, 4 wk) of downhill running, including increased satellite cell quantity, new fiber synthesis, hypertrophic signaling, myofibrillar content, individual fiber and whole muscle cross-sectional area (CSA), and/or maximal isometric force (22,37). The lack of growth in wild type (WT) mice in response to repeated bouts of downhill running suggests that enhancement of >7BX2 integrin protein in muscle can confer a unique adaptive response to this type of exercise, yet the mechanisms that provide such an advantage have not been determined. Nonsatellite stem cells that can significantly enhance repair after injury have been identified in the skeletal muscle. Despite lack of consensus regarding nomenclature and classification, the majority of Pax7j mononuclear cells in murine skeletal muscle express stem cell antigen-1 (Sca-1) and a variety of mesenchymal stem cell (MSCs) markers, including CD90 and platelet-derived growth factor receptor > (PDGFR>) (3,10,21,27,33). Nonsatellite stem cells in the muscle are uniquely isolated and categorized as side population cells, pericytes, mesenchymal progenitors, fibroadipogenic progenitors, and PW1+ interstitial cells (3,12,21,27,28,33). Whereas some investigators have demonstrated that nonsatellite cells have some potential to become myogenic, the majority suggests an indirect and paracrine factor-mediated contribution to muscle healing (3,11). The extent to which nonsatellite stem cells contribute to postexercise repair has not been thoroughly evaluated. Our laboratory recently established that Sca-1+CD45j MSCs increase in muscle (mMSCs) in an >7 integrindependent manner and contribute to satellite cell accumulation and new fiber synthesis 1 wk after a single bout of eccentric exercise (34). Although some heterogeneity exists in the population of cells captured by positive selection for Sca-1 and negative selection for CD45 (hematopoietic cell marker), we were able to verify after short-term maintenance and expansion in culture that these cells do not express satellite cell markers yet highly express mesenchymal and pericyte markers and remain multipotent (34). Similar to the majority of studies examining a role for MSCs in muscle repair process after cardiotoxin injection (13,28,33), mMSCs did not directly contribute to new fiber formation or fuse with preexisting fibers but rather indirectly influenced satellite cell number and new fiber synthesis via a paracrine mechanism after an acute bout of exercise (34). The extent to which mMSCs account for acceleration of muscle repair and beneficial adaptations in fiber size and
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strength previously observed in >7Tg muscle in response to acute eccentric exercise and exercise training (22,37) has not been determined. Specific ablation of mMSCs is not possible, given the lack of consensus on whether distinct nonsatellite stem cell fractions exist in the muscle and/or whether these populations stably express one marker necessary for cell survival and/or function. In this study, we assess indices of postexercise repair, growth, and function in WT mice supplemented with mMSCs isolated from >7Tg muscle. We hypothesized that mMSCs could facilitate long-term repair and beneficial adaptations after repeated bouts of eccentric exercise.
METHODS Animals. Protocols for animal use were approved by the Institutional Animal Care and Use Committee of the University of Illinois at Urbana-Champaign. Animal experiments in this study were conducted in accordance with the policy statement of the American College of Sports Medicine. Threemonth-old female C57/BL6 mice were purchased for injection of saline or mMSC transplantation (Charles River, Wilmington, MA). Five-week-old transgenic mice overexpressing the >7BX2 integrin in the skeletal muscle (SJ6/ C57BL6; MCK:>7BX2; >7Tg) were used for extraction and isolation of mMSCs. Transgenic mice were produced at the University of Illinois Transgenic Animal Facility as described (4,8). All mice were kept in a temperature-controlled animal room maintained on a 12:12 light–dark cycle at the University of Illinois. Mice were fed standard laboratory chow and water ad libitum. Isolation of Sca-1+CD45j cells from the skeletal muscle. Sca-1+CD45j cells were isolated via fluorescenceactivated cell sorting, as previously described (34). Briefly, gastrocnemius–soleus complexes were harvested from 5-wkold >7Tg mice 24 h after an acute bout of downhill running exercise, as to be described later. After enzymatic digestion of the muscle tissue, filtered samples were incubated on ice with antimouse CD16/CD32 (1 Kg per 106 cells) (eBioscience, San Diego, CA) for 10 min to block nonspecific Fc-mediated interactions. Cells were stained in a cocktail of monoclonal antimouse antibodies Sca-1-PE (600 ng per 106 cells) and CD45-APC (300 ng per 106 cells) (eBioscience, San Diego, CA) diluted in 2% fetal bovine serum in phosphate-buffered saline (PBS). Fluorescence-activated cell sorting was performed using an iCyt Reflection System located at Carle Hospital (Urbana, IL). Negative and single-stained controls were used to establish gates. Sca-1+CD45j cells were collected in medium for culture (high-glucose Dulbecco_s Modified Eagle_s Medium, 10% fetal bovine serum, gentamicin 5 KgImLj1) and seeded onto uncoated plastic tissue culture dishes at 2.5 104 cells per square centimeter. Cultures were incubated at 37-C and 5% CO2. Media were replaced every 3 d until the cells reached 80%–90% confluency. On average, cells were grown in culture for 7 d and were not passaged
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Briefly, slides were dried and fixed in acetone for 10 min followed by several washes in 1X PBS. Sections were then blocked with serum for 1 h and incubated with 70 KgImLj1 of goat antimouse monovalent Fab fragments (1:20, AffiniPure Fab Fragment Goat Anti-Mouse IgG (H+L); Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 30 min. After blocking, sections were incubated in the primary antibody for 1 h at room temperature (Pax7 and eMHC) or 30 min at 37-C (dystrophin). After several washes with 1% bovine serum albumin (BSA) in PBS, sections were then incubated in the appropriate secondary antibodies. IgG antibody was applied to tissue sections at 4-C overnight. Sections were washed with 1% BSA and stained with 4¶,6-diamidino-2-phenylindole (DAPI, 1:20000) (Sigma-Aldrich, St. Louis, MO) before mounting with Vectashield (Vector Laboratories). Immunohistochemistry analysis. Immunohistochemical images were obtained at 20 magnification for measurement of CLN and myofiber CSA and 40 for muscle damage and myonuclei-to-fiber ratio (N/F ratio) using a Leica DMRXA2 microscope. Images were acquired using a Zeiss AxioCam digital camera and Axiovision software (Zeiss, Thornwood, NY). For assessing satellite cell content, slides were imaged and captured on the NanoZoomer digital slide scanner (Hamamatsu Corp., Bridgewater, NJ) using the 40 objective setting. Investigators were blinded to sample information for all assessments. Immunohistochemistry quantification. To assess muscle damage, IgG+ fibers were counted in three sections per sample. The total number of IgG+ fibers for three sections was normalized to total area (mm2). Dystrophin and DAPI stains were performed to quantify the CLN, myofiber CSA, and N/F ratio (three sections per sample). To assess CLN, total number of fibers and the number of fibers with CLN were manually counted. At least 1000 fibers per sample were counted. The results for each sample were then averaged. To assess myofiber CSA, Adobe Photoshop was used to quantitate images acquired with a Zeiss AxioCam digital camera. Briefly, dystrophin–fluorescein isothiocyanate images acquired at 20 magnification from each sample were imported into Adobe Photoshop (CS5 Extended) where an average of 500 fibers per sample were manually circled using the magnetic lasso tool, which grabs the positively stained pixels and decreases subjectivity and interassessment error. CSA for each fiber was recorded in the measurement log. The results for each sample were then averaged. To determine the myonuclei content, the number of nuclei for each myofiber was counted. Briefly, myofibers were manually counted using Adobe Photoshop count tool. The total number of nuclei associated with each prenumbered myofiber was then manually counted. To make the distinction between myonuclei and mononuclear cells in the interstitial space, only nuclei that had a clear dystrophin line on only one side and those that were contained within the myofiber were counted. Two hundred fibers per sample were analyzed. The results for each sample were then averaged. Quiescent and activated satellite cells were detected with Pax7 antibody. The total number of Pax7+ cells per tissue section was manually counted. Only Pax7+DAPI+ cells immediately adjacent to
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during this time. Cells were then labeled with lipophilic dye (DiI) before injection. Eccentric exercise training. All mice were subjected to a single bout of downhill running exercise (j20-, 17 mIminj1, 30 min) 1 h before injection of either saline or mMSCs. Mice then remained sedentary or were subjected to multiple bouts of eccentric exercise. Exercise consisted of downhill running three times per week (Monday, Wednesday, and Friday) for 2 or 4 wk (j20-, 17 mIminj1, 30 min) on a treadmill (Exer-6M; Columbus Instruments). For each exercise bout, the speed was gradually increased from 10 to 17 mIminj1 over the first 2 min as a warm-up period. mMSC transplantation. One hour after downhill running, animals were anesthetized with 2% isoflurane administered by inhalation. For the 2-wk study, the right gastrocnemius muscle of each animal was transplanted with 4 104 cells suspended in 50-KL Hank’s balanced salt solution (mMSCs), whereas the left gastrocnemius muscle was transplanted with an equal volume of Hank’s balanced salt solution (saline). For mice exercising for 4 wk, both legs were transplanted with either cells or saline to measure grip strength. For both the 2and 4-wk studies, animals were randomly assigned to either a sedentary (SED) or exercise training (TR) group. Thus, the four groups compared were as follows: saline/SED, saline/TR, mMSCs/SED, and mMSCs/TR (n = 5–6 per group). Because of the limited number of mMSCs that can be isolated from the muscle, we were unable to inject all animals to finish both exercise training studies at the same time. Therefore, two studies (2 and 4 wk) were performed separately at different times. Tissue collection and preservation. Twenty-four hours after the final training session, all mice were euthanized via carbon dioxide asphyxiation. Gastrocnemius–soleus muscles were rapidly dissected and either frozen in liquid nitrogen before protein extraction or frozen in liquid nitrogencooled isopentane for immunohistochemistry studies. Immunohistochemistry. Muscle complexes were divided at the midline along the axial plane. The distal end was embedded in an optimum cutting temperature compound (TissueTek; Fischer Scientific). Three transverse cryosections per sample (8-Km nonserial sections, each separated by a minimum of 40 Km) were cut for each histological assessment using a CM3050S cryostat (Lecia, Wezlar, Germany). Sections were placed on microscope slides (Superfrost; Fischer Scientific, Hanover Park, IL) and stored at j80-C before staining. Sections were stained with antibodies against dystrophin (1:100, MANDRA-1; Sigma, St. Louis, MO), Pax7 (1:10; DSHB, Iowa City, IA) and embryonic myosin heavy chain (eMHC) (1:8, BF-45; DSHB, Iowa City, IA). All species-appropriate secondary antibodies used for immunofluorescence studies were applied at 1:200 (Pax7), 1:250 (dystrophin), or 1:400 (eMHC) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). For examination of damage, sections were incubated with fluorescein isothiocyanate–conjugated antimouse immunoglobulin G (IgG) antibodies (FI2000, 1:100) (Vector Laboratories, Burlingame, CA). Immunohistochemical methods were adapted from previously published methods from our laboratory (22,37).
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and outside the myofiber were counted. All data are presented as percentage of Pax7+ cells to total myonuclei. The average number was assessed for three sections per animal. Western blot analysis. All frozen gastrocnemius–soleus complexes collected after exercise training were manually ground with a mortar and pestle. Powdered tissue was homogenized in ice-cold buffer, as previously described (37). Protein concentration was determined with the Bradford protein assay using BSA for the standard curve. Equal amounts of protein (60 Kg) were reduced, separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis using 6%–8% acrylamide gels, and transferred to nitrocellulose membranes. Equal protein loading was verified by Ponceau S staining. Membranes were blocked in Tris-buffered saline (pH, 7.8) containing 5% BSA and then were incubated with the appropriate phosphospecific antibody (phospho–mammalian target of rapamycin (mTOR), phospho-p70s6k) overnight at 4-C (1:1000; Cell Signaling Technology, Danvers, MA). After multiple washes, horseradish peroxidase–conjugated antirabbit secondary antibodies (1:2000; Jackson ImmunoResearch, West Grove, PA) were applied to the blots for 1 h at room temperature. Bands were detected using: enhanced chemiluminescence western blotting substrate (SuperSignal West Dura; Thermo Fisher Scientific, Rockford, IL) and a Bio-Rad ChemiDoc XRS system (Bio-Rad, Hercules, CA). Bands were quantified using Quantity One software (Bio-Rad). After detection of phosphorylated proteins, blots were washed and stripped with Restore Western Blot Stripping Buffer (Thermo Fisher Scientific, Rockford, IL) for 20 min at room temperature. Membranes were washed and reprobed with total-specific antibodies (mTOR, p70S6K; 1:1000; Cell Signaling Technology, Danvers, MA) in the same manner as phosphospecific protein detection. Hind limb grip strength. Grip strength measurements for hind limbs were tested once a week using an electronic grip strength meter (Columbus Instruments, Columbus, OH). All mice were trained with the meter for 3 d before the initiation of the study. Each week, 24 h after the last exercise session, hind limb strength was measured. To assess strength, mice were passed over a metal mesh grid connected to a force transducer. Mice gripped the bar with their hind limbs and were then tugged gently away until the grip was released. The peak force in grams was recorded by the transducer. Three trials were measured on each mouse during each test. The highest of the three values for each was recorded for analysis. Total body weight was measured before each grip strength test. The individual strength was indicated as limb strength (g) divided by body weight (g). All measurements were performed by the same examiner. Statistical analysis. All averaged data are presented as means T SEM. To determine significance, comparisons between groups were performed by two-way ANOVA (treatment: SA or mMSCs and intervention: SED or TR) followed by Tukey post hoc test when significant interactions were detected. Repeated-measures ANOVA was used to determine between- and within-group differences in absolute and relative force as a result of treatment (mMSCs) and intervention
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(exercise). Data that were not normally distributed (as tested by the Kolmogorov–Smirnov test) were log-transformed. All calculations were performed with SPSS statistical software (20.0; SPSS, Inc., Chicago, IL). Differences were considered significant at P G 0.05.
RESULTS Sca-1+CD45j mMSCs localize to regenerating myofibers after eccentric exercise training. The experimental design for examination of mMSCs contribution to muscle repair and growth is provided in Figure 1A. The majority of fluorescently labeled mMSCs were randomly present in the muscle interstitium at 2 wk after injection. Newly formed or regenerating fibers are identified in the muscle by the presence of a nucleus in the central location of the cell. Consistent with previously reported observations at 1 wk after injection (34), mMSCs migrated to fibers with CLN (Fig. 1B). Clustering of DiI-labeled cells in some areas of the muscle prevented the ability to quantitate cell number by immunofluorescence detection. However, at 2 wk, the presence of DiI-labeled cells in the muscle tissue between mMSCs/ SED and mMSCs/TR was not visibly different. mMSC transplantation allows for a small reduction in body weight during 4 wk of eccentric exercise training. Whereas body weight and muscle weight were not significantly altered in any group at 2 wk, body weight (g) and percent change in body weight were lower in mice that completed training for 4 wk compared with those in mice that remained sedentary (main effect of training, P = 0.003 for BW and P = 0.023 for percent change in body weight (BW%)) (Table 1). mMSCs/TR lost 1.1% of body weight compared with sedentary groups, both of which increased body weight by approximately 4% (Table 1). Despite the difference in percentage change over the duration of the study, the total difference in body weight between mMSCs/TR and saline/ SED at the end of the study was only 2.7 g. No significant group differences in absolute or relative muscle weight were observed after 4 wk of eccentric exercise training (Table 1). mMSC transplantation does not influence damage or macrophage content but reduces the appearance of CLN+ fibers in the muscle after 2 wk of eccentric exercise training. A trend toward an increase in muscle damage, as assessed by the number of IgG+ fibers per square millimeter, was noted after 2 wk of exercise training (trend for main effect of training, P = 0.067) (Fig. 2A). The trend in muscle damage coincided with F4/80+ macrophage content, which remained slightly elevated after exercise training (main effect of training, P = 0.028) (Fig. 2B). New fiber formation, as identified by eMHC expression, was not observed in any sample at 2 wk (data not shown) likely because of conversion to the adult isoform of MHC at this late time point. Although no differences were detected in damage or macrophage content after exercise with mMSC transplantation, the percentage of CLN+ fibers was elevated almost twofold in saline/TR compared with that in both saline/SED and mMSCs/SED but
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BASIC SCIENCES FIGURE 1—Sca-1+CD45- mMSCs localize to regenerating myofibers after eccentric exercise training. A. Experimental design for injection of saline or DiI-labeled mMSCs into the gastrocnemius–soleus muscle followed by 2 or 4 wk of exercise training. B. Representative cross-section of the gastrocnemius–soleus muscle transplanted with mMSCs at 2 wk after exercise. Arrows indicate DiI-labeled mMSCs at high resolution. Unmerged and merged images of DiI-labeled mMSCs (red), dystrophin (green), and nuclei (blue) at 20 (scale bar, 20 Km) and 40 (scale bar, 40 Km) magnifications are provided.
not in mMSCs/TR at 2 wk (treatment–intervention interaction, P = 0.048) (Fig. 2C). mMSC transplantation significantly enhances satellite cell number after 2 wk of eccentric exercise training. We previously reported that satellite cell number was increased 1 wk after mMSC transplantation in the muscle, and this effect is enhanced if recipient mice exercised before injection (34). In the current study, Pax7+ cell number was approximately twofold higher in mMSCs/TR compared with that in all other groups at 2 wk (treatment–intervention interaction, P = 0.009) (Fig. 3A). Satellite cells were quantified using Pax7/Dystrophin costaining to ensure that all Pax7+ cells
were properly localized within the basal lamina surrounding each fiber. No changes in Pax7+ cell number were observed in any group at the end of the 4-wk training program (data not shown). Myonuclear content is increased with mMSC transplantation after eccentric exercise training. Myonuclear accretion as a result of satellite cell fusion with existing myofibers can potentially contribute to muscle growth after exercise training (6,16). Consistent with our previously reported results, we did not observe direct fusion of DiI-labeled mMSCs with existing fibers (34). To assess overall myonuclear content, we counted the number of myonuclei per
TABLE 1. Body and muscle weight. After 2 wk of Training Groups Saline/SED mMSCs/SED Saline/TR mMSCs/TR
BW (g)
BW%
22.8 T 0.7
1.1 T 2.0
21.3 T 0.3
j0.4 T 0.5
After 4 wk of Training
MW (mg) 208.2 210.1 211.3 211.5
T T T T
9.2 21.4 16.2 11.4
rMW 9.2 9.9 9.9 9.7
T T T T
0.5 0.7 1.0 0.5
BW (g) 23.8 22.8 21.1 21.1
T T T T
0.8 0.8 0.7* 0.4*
BW% 4.4 3.9 2.1 j1.1
T T T T
2.7 0.4 2.0* 1.0*
MW (mg) 207.5 202.8 183.9 220.0
T T T T
8.7 17.0 16.6 14.3
rMW 8.8 9.0 8.7 10.6
T T T T
0.4 1.0 0.9 0.9
Values are presented as mean T SEM. *P G 0.05 main effect of training at 4 wk. n = 6 per group. BW, body weight; MW, muscle weight; rMW, relative muscle weight (normalized to body weight).
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BASIC SCIENCES FIGURE 2—mMSCs transplantation does not influence damage or macrophage content but reduces the appearance of CLN+ fibers in muscle after 2 wk of eccentric exercise training. A. Quantitation and visualization of damaged myofibers after saline or mMSCs injection at 2 wk; SED or TR. 20, scale bar = 50 Km. B. Quantitation and visualization of F4/80 positive cells after saline or mMSCs injection at 2 wk. 20, scale bar = 20 Km. C. Quantitation and visualization of myofibers with CLN after saline or mMSCs injection at 2 wk. 20, scale bar = 20 Km. *P G 0.05 compared with saline/SED. §P G 0.05 compared with mMSCs/SED. n = 5–6 per group. Values are presented as mean T SEM.
fiber (N/F ratio). The N/F ratio was significantly higher in mMSCs/TR (2.08 T 0.13) compared with that in all other groups after 2 wk of exercise training (treatment–intervention interaction, P = 0.026) (Fig. 3B). The N/F ratio was further elevated in mice receiving mMSC transplantation at 4 wk (main effect of mMSC injection, P = 0.009) (Fig. 3C).
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mMSC transplantation increases myofiber hypertrophy and hind limb strength after eccentric exercise training. A trend for an increase in mean fiber CSA was noted in mMSCs/TR after 2 wk of downhill running exercise (trend for treatment–intervention interaction, P = 0.065; trend for main effect of mMSCs injection, P = 0.061)
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BASIC SCIENCES FIGURE 3—mMSC transplantation significantly enhances satellite cell number and myonuclear content after eccentric exercise training. A. Quantitation and visualization of Pax7+ satellite cells after saline or mMSCs injection at 2 wk. 40, scale bar = 10 Km. Quantitation of the number of myonuclei per fiber after saline or mMSCs injection at 2 (B) and 4 wk (C). *P G 0.05 compared with saline/SED. #P G 0.05 compared with saline/TR. §P G 0.05 compared with mMSCs/SED. n = 5–6 per group. Values are presented as mean T SEM.
(Fig. 4A). Average myofiber CSA was significantly increased in mMSCs/TR compared with that in saline/SED (23%) and mMSCs/SED (17%) after 4 wk of downhill running exercise (treatment–intervention interaction, P = 0.045) (Fig. 4B). A rightward shift in fiber size distribution was observed in mMSCs/TR at 4 wk, reflecting an increase in the percentage of large caliber fibers (Fig. 4C). The percentage of fibers ranging 93000 Km2 was significantly increased in mMSCs/TR compared with that in all the other groups (treatment– intervention interaction, P = 0.017) (Fig. 4C). The gastrocnemius muscle has been shown to contribute to hind limb grip strength (25). Therefore, we used grip strength as a noninvasive measure of hind limb function during the course of the 4-wk study. This measurement allowed us to repeatedly and noninvasively assess force development as training progressed. Statistically significant changes in absolute or relative hind limb grip strength were not detected (Fig. 5A
MESENCHYMAL STEM CELLS AND SKELETAL MUSCLE
and B). However, the percent change in grip strength after 4 wk of exercise training compared with baseline was significantly higher in mMSCs/TR compared with that in both sedentary groups (treatment–intervention interaction, P = 0.049) (Fig. 5C). Hypertrophic signaling is upregulated with mMSC transplantation after 4 wk of eccentric exercise training. mTOR-p70S6K signaling can initiate protein synthesis after mechanical stimuli (17–19). Consistent with small and insignificant increases in CSA and grip strength in saline/ TR at 4 wk, mTOR and p70S6K phosphorylation (normalized to total protein) remained unchanged (see Figure, Supplemental Digital Content 1, http://links.lww.com/MSS/A410, Hypertrophic signaling is upregulated with mMSC transplantation after 4 wk of eccentric exercise training). However, both mTOR and p70S6K phosphorylation were increased almost twofold in mMSCs/TR at 4 wk compared with those in all other groups
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BASIC SCIENCES FIGURE 4—mMSC transplantation increases myofiber hypertrophy after eccentric exercise training. Mean CSA of muscle fibers in the gastrocnemius– soleus muscle after saline or mMSCs injection at 2 (A) or 4 wk (B). C. Muscle fiber CSA distribution at 4 wk. *P G 0.05 compared with saline/SED. #P G 0.05 compared with saline/TR. §P G 0.05 compared with mMSCs/SED. n = 5–6 per group. Values are presented as mean T SEM.
(treatment–intervention interaction, P = 0.047 for mTOR and P = 0.038 for p70S6K).
DISCUSSION Our previous work has established that the >7A1 integrin is an important regulator of beneficial adaptations to eccentric exercise, including protection from damage, accelerated regeneration, and myofiber hypertrophy (4,5,22,37). Recently, we reported that multipotent MSCs increase in >7Tg skeletal muscle within 24 h after an acute bout of eccentric exercise (34) and extraction and transplantation of these cells into muscle of recipient mice can stimulate satellite cell proliferation, new fiber synthesis, and vascular growth after exercise (20,34). In the current study, we provide the first demonstration that MSCs isolated from >7Tg muscle can effectively enhance the adaptive response to eccentric exercise training in WT mice over a period of 2 or 4 wk, including positive changes in satellite cell quantity, myofiber growth, and function. Therefore, mMSCs likely contribute to improvements in satellite cell number and growth previously observed in >7Tg mice after exercise. Transplantation of MSCs can effectively repair muscle and restore skeletal muscle function in response to a variety of different types of injury, including resection, crush, or
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chemical-based injuries (9,15,26,35,36). In this study, we examined the extent to which MSCs isolated from >7Tg muscle could facilitate the process of repair after prolonged eccentric exercise training. CLN+ fibers can reflect either newly synthesized fibers or established myofibers undergoing repair. In this study, CLN+ fibers were significantly elevated in the saline-injected muscle yet not present in the mMSCs-injected muscle 2 wk after training (Fig. 2C). The lack of CLN+ fibers in the mMSCs group would suggest that either 1) repair (either new fiber synthesis or myoblast fusion with existing fibers) did not occur or 2) repair occurred at an earlier time point during training and evidence of this event was no longer present at 2 wk. mMSCs secrete anti-inflammatory cytokines, such as interleukins 10 and 1rn, that have the potential to suppress exercise-induced damage, an effect that would reduce the need for repair. The fact that macrophage content was elevated to a small, yet similar, extent at 2 wk of training (main effect for exercise) suggests that mMSCs may not have suppressed inflammation or damage but rather enhanced the rate of repair. The rapid formation of new fibers in >7Tg mice 2 d after an acute bout of eccentric exercise, followed by downregulation at 7 d, also supports this hypothesis (22). Whether mMSCs directly allow for rapid and transient fiber repair or induce a change in macrophage phenotype that would indirectly facilitate this event was not discerned in the
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current study. However, studies conducted in our laboratory suggest that mMSCs-conditioned media can increase myoblast proliferation and myotube formation in vitro and presence of the macrophage is not necessary for these events. Although it will be important to delineate the effects of mMSCs on macrophage function in the muscle after exercise, current evaluation is limited by lack of single and unique stable cell surface markers necessary for identification of the M1 and M2 macrophages (30). Therefore, further short-term studies are necessary to determine the extent to which exogenous mMSCs can influence early (24 and 48 h) damage and repair responses as well as macrophage migration and macrophage polarization/ function immediately after an acute bout of eccentric exercise. Previous studies demonstrate that satellite cell number is elevated in >7Tg mice after exercise and mMSC transplantation
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FIGURE 5—mMSC transplantation increases hind limb strength after 4 wk of eccentric exercise training. Hind limb grip strength was assessed every week. A. Absolute hind limb grip strength (peak tension, g). B. Relative hind limb grip strength (peak tension/body weight, gIgj1). C. Percentage changes in relative hind limb grip strength after 4 wk of training compared to sedentary state. *P G 0.05 compared with saline/ SED. §P G 0.05 compared with mMSCs/SED. n = 5–6 per group. Values are presented as mean T SEM.
can significantly increase Pax7+ cell content in the skeletal muscle 1 wk after exercise (22,34). In the current study, we similarly demonstrate enhancement of satellite cell number (1.5%–2.5%) with mMSCs injection at 2 wk (Fig. 3A). The sustained rise in satellite cell number would not be expected if regeneration was complete. Therefore, mMSCs enrichment may robustly increase satellite cell proliferation and survival, perhaps maintaining an elevated quantity of satellite cells in the muscle even when repair is no longer necessary. It is important to note that although we made every attempt to carefully count only Pax7+DAPI+ cells in contact with a muscle fiber, background staining inherent to the application of mouse hybridoma supernatant may have influenced our results and further studies are necessary to validate our observations. In addition, information regarding satellite cell activation (myogenesis) should be ascertained. Our attempts to costain satellite cells for myogenic differentiation antigen or myogenin were not successful, but such information would be helpful in determining whether these cells were quiescent or activated at 2 wk. Finally, the fact that mMSC transplantation by itself did not elicit increase in satellite cell number suggests that additional factors associated with eccentric exercise (mechanical strain, pro-inflammatory cytokines) are necessary for mMSCs contribution to satellite cell expansion and/or survival. mMSC transplantation increased myofiber growth 4 wk after initiation of exercise training. The enhancement in fiber CSA observed in the present study (Fig. 4A–C) was consistent with physiological growth observed in >7Tg muscle after repeated bouts of eccentric exercise (37). The facts that the N/F ratio was elevated as early as 2 wk after exercise in mMSCs-treated muscle and that the ratio was further increased at 4 wk (Fig. 3B and C) suggest that addition of myonuclei may have contributed to growth. As previously stated, we did not observe DiI+ fibers at any time point in the current study. Thus, satellite cell expansion and subsequent myoblast fusion likely accounted for the myofiber growth observed. The twofold enhancement of mTOR and p70S6K phosphorylation by mMSC transplantation is noteworthy and suggests some capacity for these cells to influence protein synthesis. Regardless of the precise mechanism responsible for fiber growth, these changes likely contributed to the significant improvement in strength observed in MSCs-injected mice at 4 wk after exercise compared with that in baseline (Fig. 5 C). We previously evaluated Sca-1+CD45j mMSCs for satellite cell markers and a variety of nonsatellite cell markers via flow cytometry and polymerase chain reaction (34). While sorting nonsatellite cells on the basis of two markers, Sca-1 (positive selection) and CD45 (negative selection), increases the potential for retrieving a heterogeneous mononuclear cell population, we previously determined that mMSCs do not express Pax7 yet are positive for several MSCs (CD90, CD105, CD29, and CD73) and pericyte markers (NG2, PDGFRA, and CD146). Although not reported, mMSCs are also positive for PDGFR>. The extent to which these cells represent type 1
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pericytes (Sca-1+NG2+PDGFR>+) (3), fibroadipogenic progenitors (Sca-1+PDGFR>+linj) (21), or PW1+ interstitial cells (Sca-1+PW1+PDGFR>+) (27) previously reported in the literature is not known, yet the potential is high given their limited capacity for myogenesis and their ability to provide stromal contribution to repair (34). MSCs, true to their mesodermal origin, have the capacity to differentiate into adipocytes and fibroblasts. Because mechanical strain can inhibit MSC adipogenesis (32), it will be important to determine the extent to which tension created in the muscle during eccentric exercise has the capacity to inhibit adipogenesis/ fibrogenesis and promote the release of factors that can modulate the environment in a manner that supports tissue health. In conclusion, the results from this study suggest that mMSCs contribute to satellite cell expansion and accelerated growth in >7Tg mice after exercise. In addition, it provides the first evidence of a role for mMSCs in the adaptive response to
eccentric exercise. Further studies are necessary to determine the origin of MSCs that accumulate in the muscle after exercise, the factors in the microenvironment that regulate mMSC migration and function, and the precise mechanisms by which mMSCs confer the beneficial effects of eccentric exercise. We believe that this information will guide us toward the development of methods to enhance muscle repair and interventions that can effectively preserve muscle mass and function with age, immobilization, and disease. The authors would like to thank Anthony Zhang and Zak Kammer for their assistance in data analysis. This work was supported by a grant from the Ellison Medical Foundation (AG-NS-0547-09 to M. D. B.). H. D. H. was supported by a National Science Foundation Integrative Graduate Education and Research Traineeship (IGERT) in Cellular and Molecular Mechanics and BioNanotechnology. The authors declare no conflicts of interest. The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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15. Ferrari G, Cusella-De Angelis G, Coletta M, et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science. 1998;279(5356):1528–30. 16. Guerci A, Lahoute C, Hebrard S, et al. Srf-dependent paracrine signals produced by myofibers control satellite cell-mediated skeletal muscle hypertrophy. Cell Metab. 2012;15(1):25–37. 17. Higbie EJ, Cureton KJ, Warren GL 3rd, Prior BM. Effects of concentric and eccentric training on muscle strength, cross-sectional area, and neural activation. J Appl Physiol (1985). 1996;81(5): 2173–81. 18. Hornberger TA, Chien S. Mechanical stimuli and nutrients regulate rapamycin-sensitive signaling through distinct mechanisms in skeletal muscle. J Cell Biochem. 2006;97(6):1207–16. 19. Hornberger TA, Esser KA. Mechanotransduction and the regulation of protein synthesis in skeletal muscle. Proc Nutr Soc. 2004; 63(2):331–5. 20. Huntsman HD, Zachwieja N, Zou K, et al. Mesenchymal stem cells contribute to vascular growth in skeletal muscle in response to eccentric exercise. Am J Physiol Heart Circ Physiol. 2013;304(1): H72–81. 21. Joe AW, Yi L, Natarajan A, et al. Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis. Nat Cell Biol. 2010;12(2):153–63. 22. Lueders TN, Zou K, Huntsman HD, et al. The >7A1-integrin accelerates fiber hypertrophy and myogenesis following a single bout of eccentric exercise. Am J Physiol Cell Physiol. 2011;301(4):C938–46. 23. Malm C, Sjodin TL, Sjoberg B, et al. Leukocytes, cytokines, growth factors and hormones in human skeletal muscle and blood after uphill or downhill running. J Physiol. 2004;556(Pt 3):983–1000. 24. Maughan RJ, Donnelly AE, Gleeson M, Whiting PH, Walker KA, Clough PJ. Delayed-onset muscle damage and lipid peroxidation in man after a downhill run. Muscle Nerve. 1989;12(4):332–6. 25. Maurissen JP, Marable BR, Andrus AK, Stebbins KE. Factors affecting grip strength testing. Neurotoxicol Teratol. 2003;25(5): 543–53. 26. Merritt EK, Cannon MV, Hammers DW, et al. Repair of traumatic skeletal muscle injury with bone-marrow-derived mesenchymal stem cells seeded on extracellular matrix. Tissue Eng Part A. 2010;16(9): 2871–81. 27. Mitchell KJ, Pannerec A, Cadot B, et al. Identification and characterization of a non-satellite cell muscle resident progenitor during postnatal development. Nat Cell Biol. 2010;12(3):257–66. 28. Motohashi N, Uezumi A, Yada E, et al. Muscle CD31(-) CD45(-) side population cells promote muscle regeneration by stimulating
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Department of Kinesiology and Community Health, University of Illinois, Urbana, IL; 2Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana, IL; and 3Division of Biomedical Sciences, University of Illinois, Urbana, IL
ABSTRACT
S
contraction). The need for the muscle to contract during lengthening is most evident during activities that require transfer of a load away from the body (weightlifting) or during downward locomotion to prevent imbalance and falling. While repeatedly shortening the muscle confers predominantly metabolic adaptations that provide resistance to fatigue, single or multiple bouts of lengthening can cause damage to muscle ultrastructure, initiating a long-term regenerative response that may positively affect growth and strength after exercise (14,31). Downhill running has been used as a model to investigate the natural processes of skeletal muscle injury and repair after eccentric exercise (1,23,24). Similar to injury created by other methods, such as cardiotoxin and crush injury, a single bout of downhill running can impair sarcolemmal integrity, evoke an immune response, and reduce maximal isometric force within 24 h after exercise (4,5). Within 2–4 d, satellite cell number is increased and newly formed fibers with centrally located nuclei (CLN) are apparent throughout the muscle (22). Although previous studies suggest that repeated bouts of downhill running do not exacerbate muscle damage or lengthen the recovery process (29), few studies, to our knowledge, have evaluated the long-term adaptive response to downhill running.
keletal muscle is a highly structured and intricately organized organ that provides the basis for human movement. Physical interaction between the essential myofibrillar proteins, actin and myosin, within each myofibril of a skeletal muscle cell can allow for the development of tension and the transfer of force to tendons and bones. Although concentric (shortening) contractions are most commonly used for movement of the peripheral limbs during physical activity, lengthening of the muscle can also occur during actin–myosin cross-bridge formation (eccentric
Address for correspondence: Marni D. Boppart, Sc.D., Beckman Institute for Advanced Science and Technology, 405 N. Mathews Avenue, MC-251 Urbana, IL 61801; E-mail: [email protected]. Submitted for publication February 2014. Accepted for publication May 2014. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.acsm-msse.org). 0195-9131/15/4702-0315/0 MEDICINE & SCIENCE IN SPORTS & EXERCISEÒ Copyright Ó 2014 by the American College of Sports Medicine DOI: 10.1249/MSS.0000000000000405
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ZOU, K., H. D. HUNTSMAN, M. CARMEN VALERO, J. ADAMS, J. SKELTON, M. DE LISIO, T. JENSEN, and M. D. BOPPART. Mesenchymal Stem Cells Augment the Adaptive Response to Eccentric Exercise. Med. Sci. Sports Exerc., Vol. 47, No. 2, pp. 315–325, 2015. Purpose: The >7A1 integrin is a transmembrane protein expressed in the skeletal muscle that can link the actin cytoskeleton to the surrounding basal lamina. We have previously demonstrated that transgenic mice overexpressing the >7B integrin in the skeletal muscle (MCK:>7B; >7Tg) mount an enhanced satellite cell and growth response to single or multiple bouts of eccentric exercise. In addition, interstitial stem cells characterized as mesenchymal stem cells (MSCs) accumulate in >7Tg muscle (mMSCs) in the sedentary state and after exercise. The results from these studies prompted us to determine the extent to which mMSC underlie the beneficial adaptive responses observed in >7Tg skeletal muscle after exercise. Methods: mMSCs (Sca-1+CD45j) were isolated from >7Tg mice, dyelabeled, and intramuscularly injected into adult wild type recipient mice. After injection of mMSCs or saline, mice remained sedentary (SED) or were subjected to eccentric exercise training (TR) (downhill running) on a treadmill (three times per week) for 2 or 4 wk. Gastrocnemius–soleus complexes were collected 24 h after the last bout of exercise. Results: mMSCs did not directly fuse with existing fibers; however, mMSCs injection enhanced Pax7+ satellite cell number and myonuclear content compared with all other groups at 2 wk after exercise. Mean CSA, percentage of larger caliber fibers (93000 Km2), and grip strength were increased in mMSCs/TR compared with saline/SED and mMSCs/SED at 4 wk. mMSC transplantation did not enhance repair or growth in the absence of exercise. Conclusions: The results from this study demonstrate that mMSCs contribute to beneficial changes in satellite cell expansion and growth in >7Tg muscle after eccentric exercise. Thus, MSCs that naturally accumulate in the muscle after eccentric contractions may enhance the adaptive response to exercise. Key Words: A7 INTEGRIN, SATELLITE CELLS, STEM CELL ANTIGEN-1, DOWNHILL RUNNING, REPAIR, GROWTH
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The >7A1 integrin is a critical transmembrane protein that links laminin in the basement membrane with the actin cytoskeleton across the sarcolemma (2,7). Our previous work has established that total >7B integrin protein expression is upregulated and transgenic expression of the >7BX2 integrin in the skeletal muscle (MCK:>7BX2; >7Tg) can reinforce sarcolemmal integrity as well as reduce macrophage content and preserve function after an acute bout of downhill running in young healthy mice (4,5). We subsequently demonstrated that >7Tg mice exhibit a robust regenerative response to single or multiple bouts (three times per week, 4 wk) of downhill running, including increased satellite cell quantity, new fiber synthesis, hypertrophic signaling, myofibrillar content, individual fiber and whole muscle cross-sectional area (CSA), and/or maximal isometric force (22,37). The lack of growth in wild type (WT) mice in response to repeated bouts of downhill running suggests that enhancement of >7BX2 integrin protein in muscle can confer a unique adaptive response to this type of exercise, yet the mechanisms that provide such an advantage have not been determined. Nonsatellite stem cells that can significantly enhance repair after injury have been identified in the skeletal muscle. Despite lack of consensus regarding nomenclature and classification, the majority of Pax7j mononuclear cells in murine skeletal muscle express stem cell antigen-1 (Sca-1) and a variety of mesenchymal stem cell (MSCs) markers, including CD90 and platelet-derived growth factor receptor > (PDGFR>) (3,10,21,27,33). Nonsatellite stem cells in the muscle are uniquely isolated and categorized as side population cells, pericytes, mesenchymal progenitors, fibroadipogenic progenitors, and PW1+ interstitial cells (3,12,21,27,28,33). Whereas some investigators have demonstrated that nonsatellite cells have some potential to become myogenic, the majority suggests an indirect and paracrine factor-mediated contribution to muscle healing (3,11). The extent to which nonsatellite stem cells contribute to postexercise repair has not been thoroughly evaluated. Our laboratory recently established that Sca-1+CD45j MSCs increase in muscle (mMSCs) in an >7 integrindependent manner and contribute to satellite cell accumulation and new fiber synthesis 1 wk after a single bout of eccentric exercise (34). Although some heterogeneity exists in the population of cells captured by positive selection for Sca-1 and negative selection for CD45 (hematopoietic cell marker), we were able to verify after short-term maintenance and expansion in culture that these cells do not express satellite cell markers yet highly express mesenchymal and pericyte markers and remain multipotent (34). Similar to the majority of studies examining a role for MSCs in muscle repair process after cardiotoxin injection (13,28,33), mMSCs did not directly contribute to new fiber formation or fuse with preexisting fibers but rather indirectly influenced satellite cell number and new fiber synthesis via a paracrine mechanism after an acute bout of exercise (34). The extent to which mMSCs account for acceleration of muscle repair and beneficial adaptations in fiber size and
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strength previously observed in >7Tg muscle in response to acute eccentric exercise and exercise training (22,37) has not been determined. Specific ablation of mMSCs is not possible, given the lack of consensus on whether distinct nonsatellite stem cell fractions exist in the muscle and/or whether these populations stably express one marker necessary for cell survival and/or function. In this study, we assess indices of postexercise repair, growth, and function in WT mice supplemented with mMSCs isolated from >7Tg muscle. We hypothesized that mMSCs could facilitate long-term repair and beneficial adaptations after repeated bouts of eccentric exercise.
METHODS Animals. Protocols for animal use were approved by the Institutional Animal Care and Use Committee of the University of Illinois at Urbana-Champaign. Animal experiments in this study were conducted in accordance with the policy statement of the American College of Sports Medicine. Threemonth-old female C57/BL6 mice were purchased for injection of saline or mMSC transplantation (Charles River, Wilmington, MA). Five-week-old transgenic mice overexpressing the >7BX2 integrin in the skeletal muscle (SJ6/ C57BL6; MCK:>7BX2; >7Tg) were used for extraction and isolation of mMSCs. Transgenic mice were produced at the University of Illinois Transgenic Animal Facility as described (4,8). All mice were kept in a temperature-controlled animal room maintained on a 12:12 light–dark cycle at the University of Illinois. Mice were fed standard laboratory chow and water ad libitum. Isolation of Sca-1+CD45j cells from the skeletal muscle. Sca-1+CD45j cells were isolated via fluorescenceactivated cell sorting, as previously described (34). Briefly, gastrocnemius–soleus complexes were harvested from 5-wkold >7Tg mice 24 h after an acute bout of downhill running exercise, as to be described later. After enzymatic digestion of the muscle tissue, filtered samples were incubated on ice with antimouse CD16/CD32 (1 Kg per 106 cells) (eBioscience, San Diego, CA) for 10 min to block nonspecific Fc-mediated interactions. Cells were stained in a cocktail of monoclonal antimouse antibodies Sca-1-PE (600 ng per 106 cells) and CD45-APC (300 ng per 106 cells) (eBioscience, San Diego, CA) diluted in 2% fetal bovine serum in phosphate-buffered saline (PBS). Fluorescence-activated cell sorting was performed using an iCyt Reflection System located at Carle Hospital (Urbana, IL). Negative and single-stained controls were used to establish gates. Sca-1+CD45j cells were collected in medium for culture (high-glucose Dulbecco_s Modified Eagle_s Medium, 10% fetal bovine serum, gentamicin 5 KgImLj1) and seeded onto uncoated plastic tissue culture dishes at 2.5 104 cells per square centimeter. Cultures were incubated at 37-C and 5% CO2. Media were replaced every 3 d until the cells reached 80%–90% confluency. On average, cells were grown in culture for 7 d and were not passaged
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Briefly, slides were dried and fixed in acetone for 10 min followed by several washes in 1X PBS. Sections were then blocked with serum for 1 h and incubated with 70 KgImLj1 of goat antimouse monovalent Fab fragments (1:20, AffiniPure Fab Fragment Goat Anti-Mouse IgG (H+L); Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 30 min. After blocking, sections were incubated in the primary antibody for 1 h at room temperature (Pax7 and eMHC) or 30 min at 37-C (dystrophin). After several washes with 1% bovine serum albumin (BSA) in PBS, sections were then incubated in the appropriate secondary antibodies. IgG antibody was applied to tissue sections at 4-C overnight. Sections were washed with 1% BSA and stained with 4¶,6-diamidino-2-phenylindole (DAPI, 1:20000) (Sigma-Aldrich, St. Louis, MO) before mounting with Vectashield (Vector Laboratories). Immunohistochemistry analysis. Immunohistochemical images were obtained at 20 magnification for measurement of CLN and myofiber CSA and 40 for muscle damage and myonuclei-to-fiber ratio (N/F ratio) using a Leica DMRXA2 microscope. Images were acquired using a Zeiss AxioCam digital camera and Axiovision software (Zeiss, Thornwood, NY). For assessing satellite cell content, slides were imaged and captured on the NanoZoomer digital slide scanner (Hamamatsu Corp., Bridgewater, NJ) using the 40 objective setting. Investigators were blinded to sample information for all assessments. Immunohistochemistry quantification. To assess muscle damage, IgG+ fibers were counted in three sections per sample. The total number of IgG+ fibers for three sections was normalized to total area (mm2). Dystrophin and DAPI stains were performed to quantify the CLN, myofiber CSA, and N/F ratio (three sections per sample). To assess CLN, total number of fibers and the number of fibers with CLN were manually counted. At least 1000 fibers per sample were counted. The results for each sample were then averaged. To assess myofiber CSA, Adobe Photoshop was used to quantitate images acquired with a Zeiss AxioCam digital camera. Briefly, dystrophin–fluorescein isothiocyanate images acquired at 20 magnification from each sample were imported into Adobe Photoshop (CS5 Extended) where an average of 500 fibers per sample were manually circled using the magnetic lasso tool, which grabs the positively stained pixels and decreases subjectivity and interassessment error. CSA for each fiber was recorded in the measurement log. The results for each sample were then averaged. To determine the myonuclei content, the number of nuclei for each myofiber was counted. Briefly, myofibers were manually counted using Adobe Photoshop count tool. The total number of nuclei associated with each prenumbered myofiber was then manually counted. To make the distinction between myonuclei and mononuclear cells in the interstitial space, only nuclei that had a clear dystrophin line on only one side and those that were contained within the myofiber were counted. Two hundred fibers per sample were analyzed. The results for each sample were then averaged. Quiescent and activated satellite cells were detected with Pax7 antibody. The total number of Pax7+ cells per tissue section was manually counted. Only Pax7+DAPI+ cells immediately adjacent to
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during this time. Cells were then labeled with lipophilic dye (DiI) before injection. Eccentric exercise training. All mice were subjected to a single bout of downhill running exercise (j20-, 17 mIminj1, 30 min) 1 h before injection of either saline or mMSCs. Mice then remained sedentary or were subjected to multiple bouts of eccentric exercise. Exercise consisted of downhill running three times per week (Monday, Wednesday, and Friday) for 2 or 4 wk (j20-, 17 mIminj1, 30 min) on a treadmill (Exer-6M; Columbus Instruments). For each exercise bout, the speed was gradually increased from 10 to 17 mIminj1 over the first 2 min as a warm-up period. mMSC transplantation. One hour after downhill running, animals were anesthetized with 2% isoflurane administered by inhalation. For the 2-wk study, the right gastrocnemius muscle of each animal was transplanted with 4 104 cells suspended in 50-KL Hank’s balanced salt solution (mMSCs), whereas the left gastrocnemius muscle was transplanted with an equal volume of Hank’s balanced salt solution (saline). For mice exercising for 4 wk, both legs were transplanted with either cells or saline to measure grip strength. For both the 2and 4-wk studies, animals were randomly assigned to either a sedentary (SED) or exercise training (TR) group. Thus, the four groups compared were as follows: saline/SED, saline/TR, mMSCs/SED, and mMSCs/TR (n = 5–6 per group). Because of the limited number of mMSCs that can be isolated from the muscle, we were unable to inject all animals to finish both exercise training studies at the same time. Therefore, two studies (2 and 4 wk) were performed separately at different times. Tissue collection and preservation. Twenty-four hours after the final training session, all mice were euthanized via carbon dioxide asphyxiation. Gastrocnemius–soleus muscles were rapidly dissected and either frozen in liquid nitrogen before protein extraction or frozen in liquid nitrogencooled isopentane for immunohistochemistry studies. Immunohistochemistry. Muscle complexes were divided at the midline along the axial plane. The distal end was embedded in an optimum cutting temperature compound (TissueTek; Fischer Scientific). Three transverse cryosections per sample (8-Km nonserial sections, each separated by a minimum of 40 Km) were cut for each histological assessment using a CM3050S cryostat (Lecia, Wezlar, Germany). Sections were placed on microscope slides (Superfrost; Fischer Scientific, Hanover Park, IL) and stored at j80-C before staining. Sections were stained with antibodies against dystrophin (1:100, MANDRA-1; Sigma, St. Louis, MO), Pax7 (1:10; DSHB, Iowa City, IA) and embryonic myosin heavy chain (eMHC) (1:8, BF-45; DSHB, Iowa City, IA). All species-appropriate secondary antibodies used for immunofluorescence studies were applied at 1:200 (Pax7), 1:250 (dystrophin), or 1:400 (eMHC) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). For examination of damage, sections were incubated with fluorescein isothiocyanate–conjugated antimouse immunoglobulin G (IgG) antibodies (FI2000, 1:100) (Vector Laboratories, Burlingame, CA). Immunohistochemical methods were adapted from previously published methods from our laboratory (22,37).
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and outside the myofiber were counted. All data are presented as percentage of Pax7+ cells to total myonuclei. The average number was assessed for three sections per animal. Western blot analysis. All frozen gastrocnemius–soleus complexes collected after exercise training were manually ground with a mortar and pestle. Powdered tissue was homogenized in ice-cold buffer, as previously described (37). Protein concentration was determined with the Bradford protein assay using BSA for the standard curve. Equal amounts of protein (60 Kg) were reduced, separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis using 6%–8% acrylamide gels, and transferred to nitrocellulose membranes. Equal protein loading was verified by Ponceau S staining. Membranes were blocked in Tris-buffered saline (pH, 7.8) containing 5% BSA and then were incubated with the appropriate phosphospecific antibody (phospho–mammalian target of rapamycin (mTOR), phospho-p70s6k) overnight at 4-C (1:1000; Cell Signaling Technology, Danvers, MA). After multiple washes, horseradish peroxidase–conjugated antirabbit secondary antibodies (1:2000; Jackson ImmunoResearch, West Grove, PA) were applied to the blots for 1 h at room temperature. Bands were detected using: enhanced chemiluminescence western blotting substrate (SuperSignal West Dura; Thermo Fisher Scientific, Rockford, IL) and a Bio-Rad ChemiDoc XRS system (Bio-Rad, Hercules, CA). Bands were quantified using Quantity One software (Bio-Rad). After detection of phosphorylated proteins, blots were washed and stripped with Restore Western Blot Stripping Buffer (Thermo Fisher Scientific, Rockford, IL) for 20 min at room temperature. Membranes were washed and reprobed with total-specific antibodies (mTOR, p70S6K; 1:1000; Cell Signaling Technology, Danvers, MA) in the same manner as phosphospecific protein detection. Hind limb grip strength. Grip strength measurements for hind limbs were tested once a week using an electronic grip strength meter (Columbus Instruments, Columbus, OH). All mice were trained with the meter for 3 d before the initiation of the study. Each week, 24 h after the last exercise session, hind limb strength was measured. To assess strength, mice were passed over a metal mesh grid connected to a force transducer. Mice gripped the bar with their hind limbs and were then tugged gently away until the grip was released. The peak force in grams was recorded by the transducer. Three trials were measured on each mouse during each test. The highest of the three values for each was recorded for analysis. Total body weight was measured before each grip strength test. The individual strength was indicated as limb strength (g) divided by body weight (g). All measurements were performed by the same examiner. Statistical analysis. All averaged data are presented as means T SEM. To determine significance, comparisons between groups were performed by two-way ANOVA (treatment: SA or mMSCs and intervention: SED or TR) followed by Tukey post hoc test when significant interactions were detected. Repeated-measures ANOVA was used to determine between- and within-group differences in absolute and relative force as a result of treatment (mMSCs) and intervention
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(exercise). Data that were not normally distributed (as tested by the Kolmogorov–Smirnov test) were log-transformed. All calculations were performed with SPSS statistical software (20.0; SPSS, Inc., Chicago, IL). Differences were considered significant at P G 0.05.
RESULTS Sca-1+CD45j mMSCs localize to regenerating myofibers after eccentric exercise training. The experimental design for examination of mMSCs contribution to muscle repair and growth is provided in Figure 1A. The majority of fluorescently labeled mMSCs were randomly present in the muscle interstitium at 2 wk after injection. Newly formed or regenerating fibers are identified in the muscle by the presence of a nucleus in the central location of the cell. Consistent with previously reported observations at 1 wk after injection (34), mMSCs migrated to fibers with CLN (Fig. 1B). Clustering of DiI-labeled cells in some areas of the muscle prevented the ability to quantitate cell number by immunofluorescence detection. However, at 2 wk, the presence of DiI-labeled cells in the muscle tissue between mMSCs/ SED and mMSCs/TR was not visibly different. mMSC transplantation allows for a small reduction in body weight during 4 wk of eccentric exercise training. Whereas body weight and muscle weight were not significantly altered in any group at 2 wk, body weight (g) and percent change in body weight were lower in mice that completed training for 4 wk compared with those in mice that remained sedentary (main effect of training, P = 0.003 for BW and P = 0.023 for percent change in body weight (BW%)) (Table 1). mMSCs/TR lost 1.1% of body weight compared with sedentary groups, both of which increased body weight by approximately 4% (Table 1). Despite the difference in percentage change over the duration of the study, the total difference in body weight between mMSCs/TR and saline/ SED at the end of the study was only 2.7 g. No significant group differences in absolute or relative muscle weight were observed after 4 wk of eccentric exercise training (Table 1). mMSC transplantation does not influence damage or macrophage content but reduces the appearance of CLN+ fibers in the muscle after 2 wk of eccentric exercise training. A trend toward an increase in muscle damage, as assessed by the number of IgG+ fibers per square millimeter, was noted after 2 wk of exercise training (trend for main effect of training, P = 0.067) (Fig. 2A). The trend in muscle damage coincided with F4/80+ macrophage content, which remained slightly elevated after exercise training (main effect of training, P = 0.028) (Fig. 2B). New fiber formation, as identified by eMHC expression, was not observed in any sample at 2 wk (data not shown) likely because of conversion to the adult isoform of MHC at this late time point. Although no differences were detected in damage or macrophage content after exercise with mMSC transplantation, the percentage of CLN+ fibers was elevated almost twofold in saline/TR compared with that in both saline/SED and mMSCs/SED but
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BASIC SCIENCES FIGURE 1—Sca-1+CD45- mMSCs localize to regenerating myofibers after eccentric exercise training. A. Experimental design for injection of saline or DiI-labeled mMSCs into the gastrocnemius–soleus muscle followed by 2 or 4 wk of exercise training. B. Representative cross-section of the gastrocnemius–soleus muscle transplanted with mMSCs at 2 wk after exercise. Arrows indicate DiI-labeled mMSCs at high resolution. Unmerged and merged images of DiI-labeled mMSCs (red), dystrophin (green), and nuclei (blue) at 20 (scale bar, 20 Km) and 40 (scale bar, 40 Km) magnifications are provided.
not in mMSCs/TR at 2 wk (treatment–intervention interaction, P = 0.048) (Fig. 2C). mMSC transplantation significantly enhances satellite cell number after 2 wk of eccentric exercise training. We previously reported that satellite cell number was increased 1 wk after mMSC transplantation in the muscle, and this effect is enhanced if recipient mice exercised before injection (34). In the current study, Pax7+ cell number was approximately twofold higher in mMSCs/TR compared with that in all other groups at 2 wk (treatment–intervention interaction, P = 0.009) (Fig. 3A). Satellite cells were quantified using Pax7/Dystrophin costaining to ensure that all Pax7+ cells
were properly localized within the basal lamina surrounding each fiber. No changes in Pax7+ cell number were observed in any group at the end of the 4-wk training program (data not shown). Myonuclear content is increased with mMSC transplantation after eccentric exercise training. Myonuclear accretion as a result of satellite cell fusion with existing myofibers can potentially contribute to muscle growth after exercise training (6,16). Consistent with our previously reported results, we did not observe direct fusion of DiI-labeled mMSCs with existing fibers (34). To assess overall myonuclear content, we counted the number of myonuclei per
TABLE 1. Body and muscle weight. After 2 wk of Training Groups Saline/SED mMSCs/SED Saline/TR mMSCs/TR
BW (g)
BW%
22.8 T 0.7
1.1 T 2.0
21.3 T 0.3
j0.4 T 0.5
After 4 wk of Training
MW (mg) 208.2 210.1 211.3 211.5
T T T T
9.2 21.4 16.2 11.4
rMW 9.2 9.9 9.9 9.7
T T T T
0.5 0.7 1.0 0.5
BW (g) 23.8 22.8 21.1 21.1
T T T T
0.8 0.8 0.7* 0.4*
BW% 4.4 3.9 2.1 j1.1
T T T T
2.7 0.4 2.0* 1.0*
MW (mg) 207.5 202.8 183.9 220.0
T T T T
8.7 17.0 16.6 14.3
rMW 8.8 9.0 8.7 10.6
T T T T
0.4 1.0 0.9 0.9
Values are presented as mean T SEM. *P G 0.05 main effect of training at 4 wk. n = 6 per group. BW, body weight; MW, muscle weight; rMW, relative muscle weight (normalized to body weight).
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BASIC SCIENCES FIGURE 2—mMSCs transplantation does not influence damage or macrophage content but reduces the appearance of CLN+ fibers in muscle after 2 wk of eccentric exercise training. A. Quantitation and visualization of damaged myofibers after saline or mMSCs injection at 2 wk; SED or TR. 20, scale bar = 50 Km. B. Quantitation and visualization of F4/80 positive cells after saline or mMSCs injection at 2 wk. 20, scale bar = 20 Km. C. Quantitation and visualization of myofibers with CLN after saline or mMSCs injection at 2 wk. 20, scale bar = 20 Km. *P G 0.05 compared with saline/SED. §P G 0.05 compared with mMSCs/SED. n = 5–6 per group. Values are presented as mean T SEM.
fiber (N/F ratio). The N/F ratio was significantly higher in mMSCs/TR (2.08 T 0.13) compared with that in all other groups after 2 wk of exercise training (treatment–intervention interaction, P = 0.026) (Fig. 3B). The N/F ratio was further elevated in mice receiving mMSC transplantation at 4 wk (main effect of mMSC injection, P = 0.009) (Fig. 3C).
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mMSC transplantation increases myofiber hypertrophy and hind limb strength after eccentric exercise training. A trend for an increase in mean fiber CSA was noted in mMSCs/TR after 2 wk of downhill running exercise (trend for treatment–intervention interaction, P = 0.065; trend for main effect of mMSCs injection, P = 0.061)
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BASIC SCIENCES FIGURE 3—mMSC transplantation significantly enhances satellite cell number and myonuclear content after eccentric exercise training. A. Quantitation and visualization of Pax7+ satellite cells after saline or mMSCs injection at 2 wk. 40, scale bar = 10 Km. Quantitation of the number of myonuclei per fiber after saline or mMSCs injection at 2 (B) and 4 wk (C). *P G 0.05 compared with saline/SED. #P G 0.05 compared with saline/TR. §P G 0.05 compared with mMSCs/SED. n = 5–6 per group. Values are presented as mean T SEM.
(Fig. 4A). Average myofiber CSA was significantly increased in mMSCs/TR compared with that in saline/SED (23%) and mMSCs/SED (17%) after 4 wk of downhill running exercise (treatment–intervention interaction, P = 0.045) (Fig. 4B). A rightward shift in fiber size distribution was observed in mMSCs/TR at 4 wk, reflecting an increase in the percentage of large caliber fibers (Fig. 4C). The percentage of fibers ranging 93000 Km2 was significantly increased in mMSCs/TR compared with that in all the other groups (treatment– intervention interaction, P = 0.017) (Fig. 4C). The gastrocnemius muscle has been shown to contribute to hind limb grip strength (25). Therefore, we used grip strength as a noninvasive measure of hind limb function during the course of the 4-wk study. This measurement allowed us to repeatedly and noninvasively assess force development as training progressed. Statistically significant changes in absolute or relative hind limb grip strength were not detected (Fig. 5A
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and B). However, the percent change in grip strength after 4 wk of exercise training compared with baseline was significantly higher in mMSCs/TR compared with that in both sedentary groups (treatment–intervention interaction, P = 0.049) (Fig. 5C). Hypertrophic signaling is upregulated with mMSC transplantation after 4 wk of eccentric exercise training. mTOR-p70S6K signaling can initiate protein synthesis after mechanical stimuli (17–19). Consistent with small and insignificant increases in CSA and grip strength in saline/ TR at 4 wk, mTOR and p70S6K phosphorylation (normalized to total protein) remained unchanged (see Figure, Supplemental Digital Content 1, http://links.lww.com/MSS/A410, Hypertrophic signaling is upregulated with mMSC transplantation after 4 wk of eccentric exercise training). However, both mTOR and p70S6K phosphorylation were increased almost twofold in mMSCs/TR at 4 wk compared with those in all other groups
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BASIC SCIENCES FIGURE 4—mMSC transplantation increases myofiber hypertrophy after eccentric exercise training. Mean CSA of muscle fibers in the gastrocnemius– soleus muscle after saline or mMSCs injection at 2 (A) or 4 wk (B). C. Muscle fiber CSA distribution at 4 wk. *P G 0.05 compared with saline/SED. #P G 0.05 compared with saline/TR. §P G 0.05 compared with mMSCs/SED. n = 5–6 per group. Values are presented as mean T SEM.
(treatment–intervention interaction, P = 0.047 for mTOR and P = 0.038 for p70S6K).
DISCUSSION Our previous work has established that the >7A1 integrin is an important regulator of beneficial adaptations to eccentric exercise, including protection from damage, accelerated regeneration, and myofiber hypertrophy (4,5,22,37). Recently, we reported that multipotent MSCs increase in >7Tg skeletal muscle within 24 h after an acute bout of eccentric exercise (34) and extraction and transplantation of these cells into muscle of recipient mice can stimulate satellite cell proliferation, new fiber synthesis, and vascular growth after exercise (20,34). In the current study, we provide the first demonstration that MSCs isolated from >7Tg muscle can effectively enhance the adaptive response to eccentric exercise training in WT mice over a period of 2 or 4 wk, including positive changes in satellite cell quantity, myofiber growth, and function. Therefore, mMSCs likely contribute to improvements in satellite cell number and growth previously observed in >7Tg mice after exercise. Transplantation of MSCs can effectively repair muscle and restore skeletal muscle function in response to a variety of different types of injury, including resection, crush, or
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chemical-based injuries (9,15,26,35,36). In this study, we examined the extent to which MSCs isolated from >7Tg muscle could facilitate the process of repair after prolonged eccentric exercise training. CLN+ fibers can reflect either newly synthesized fibers or established myofibers undergoing repair. In this study, CLN+ fibers were significantly elevated in the saline-injected muscle yet not present in the mMSCs-injected muscle 2 wk after training (Fig. 2C). The lack of CLN+ fibers in the mMSCs group would suggest that either 1) repair (either new fiber synthesis or myoblast fusion with existing fibers) did not occur or 2) repair occurred at an earlier time point during training and evidence of this event was no longer present at 2 wk. mMSCs secrete anti-inflammatory cytokines, such as interleukins 10 and 1rn, that have the potential to suppress exercise-induced damage, an effect that would reduce the need for repair. The fact that macrophage content was elevated to a small, yet similar, extent at 2 wk of training (main effect for exercise) suggests that mMSCs may not have suppressed inflammation or damage but rather enhanced the rate of repair. The rapid formation of new fibers in >7Tg mice 2 d after an acute bout of eccentric exercise, followed by downregulation at 7 d, also supports this hypothesis (22). Whether mMSCs directly allow for rapid and transient fiber repair or induce a change in macrophage phenotype that would indirectly facilitate this event was not discerned in the
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current study. However, studies conducted in our laboratory suggest that mMSCs-conditioned media can increase myoblast proliferation and myotube formation in vitro and presence of the macrophage is not necessary for these events. Although it will be important to delineate the effects of mMSCs on macrophage function in the muscle after exercise, current evaluation is limited by lack of single and unique stable cell surface markers necessary for identification of the M1 and M2 macrophages (30). Therefore, further short-term studies are necessary to determine the extent to which exogenous mMSCs can influence early (24 and 48 h) damage and repair responses as well as macrophage migration and macrophage polarization/ function immediately after an acute bout of eccentric exercise. Previous studies demonstrate that satellite cell number is elevated in >7Tg mice after exercise and mMSC transplantation
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FIGURE 5—mMSC transplantation increases hind limb strength after 4 wk of eccentric exercise training. Hind limb grip strength was assessed every week. A. Absolute hind limb grip strength (peak tension, g). B. Relative hind limb grip strength (peak tension/body weight, gIgj1). C. Percentage changes in relative hind limb grip strength after 4 wk of training compared to sedentary state. *P G 0.05 compared with saline/ SED. §P G 0.05 compared with mMSCs/SED. n = 5–6 per group. Values are presented as mean T SEM.
can significantly increase Pax7+ cell content in the skeletal muscle 1 wk after exercise (22,34). In the current study, we similarly demonstrate enhancement of satellite cell number (1.5%–2.5%) with mMSCs injection at 2 wk (Fig. 3A). The sustained rise in satellite cell number would not be expected if regeneration was complete. Therefore, mMSCs enrichment may robustly increase satellite cell proliferation and survival, perhaps maintaining an elevated quantity of satellite cells in the muscle even when repair is no longer necessary. It is important to note that although we made every attempt to carefully count only Pax7+DAPI+ cells in contact with a muscle fiber, background staining inherent to the application of mouse hybridoma supernatant may have influenced our results and further studies are necessary to validate our observations. In addition, information regarding satellite cell activation (myogenesis) should be ascertained. Our attempts to costain satellite cells for myogenic differentiation antigen or myogenin were not successful, but such information would be helpful in determining whether these cells were quiescent or activated at 2 wk. Finally, the fact that mMSC transplantation by itself did not elicit increase in satellite cell number suggests that additional factors associated with eccentric exercise (mechanical strain, pro-inflammatory cytokines) are necessary for mMSCs contribution to satellite cell expansion and/or survival. mMSC transplantation increased myofiber growth 4 wk after initiation of exercise training. The enhancement in fiber CSA observed in the present study (Fig. 4A–C) was consistent with physiological growth observed in >7Tg muscle after repeated bouts of eccentric exercise (37). The facts that the N/F ratio was elevated as early as 2 wk after exercise in mMSCs-treated muscle and that the ratio was further increased at 4 wk (Fig. 3B and C) suggest that addition of myonuclei may have contributed to growth. As previously stated, we did not observe DiI+ fibers at any time point in the current study. Thus, satellite cell expansion and subsequent myoblast fusion likely accounted for the myofiber growth observed. The twofold enhancement of mTOR and p70S6K phosphorylation by mMSC transplantation is noteworthy and suggests some capacity for these cells to influence protein synthesis. Regardless of the precise mechanism responsible for fiber growth, these changes likely contributed to the significant improvement in strength observed in MSCs-injected mice at 4 wk after exercise compared with that in baseline (Fig. 5 C). We previously evaluated Sca-1+CD45j mMSCs for satellite cell markers and a variety of nonsatellite cell markers via flow cytometry and polymerase chain reaction (34). While sorting nonsatellite cells on the basis of two markers, Sca-1 (positive selection) and CD45 (negative selection), increases the potential for retrieving a heterogeneous mononuclear cell population, we previously determined that mMSCs do not express Pax7 yet are positive for several MSCs (CD90, CD105, CD29, and CD73) and pericyte markers (NG2, PDGFRA, and CD146). Although not reported, mMSCs are also positive for PDGFR>. The extent to which these cells represent type 1
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pericytes (Sca-1+NG2+PDGFR>+) (3), fibroadipogenic progenitors (Sca-1+PDGFR>+linj) (21), or PW1+ interstitial cells (Sca-1+PW1+PDGFR>+) (27) previously reported in the literature is not known, yet the potential is high given their limited capacity for myogenesis and their ability to provide stromal contribution to repair (34). MSCs, true to their mesodermal origin, have the capacity to differentiate into adipocytes and fibroblasts. Because mechanical strain can inhibit MSC adipogenesis (32), it will be important to determine the extent to which tension created in the muscle during eccentric exercise has the capacity to inhibit adipogenesis/ fibrogenesis and promote the release of factors that can modulate the environment in a manner that supports tissue health. In conclusion, the results from this study suggest that mMSCs contribute to satellite cell expansion and accelerated growth in >7Tg mice after exercise. In addition, it provides the first evidence of a role for mMSCs in the adaptive response to
eccentric exercise. Further studies are necessary to determine the origin of MSCs that accumulate in the muscle after exercise, the factors in the microenvironment that regulate mMSC migration and function, and the precise mechanisms by which mMSCs confer the beneficial effects of eccentric exercise. We believe that this information will guide us toward the development of methods to enhance muscle repair and interventions that can effectively preserve muscle mass and function with age, immobilization, and disease. The authors would like to thank Anthony Zhang and Zak Kammer for their assistance in data analysis. This work was supported by a grant from the Ellison Medical Foundation (AG-NS-0547-09 to M. D. B.). H. D. H. was supported by a National Science Foundation Integrative Graduate Education and Research Traineeship (IGERT) in Cellular and Molecular Mechanics and BioNanotechnology. The authors declare no conflicts of interest. The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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BASIC SCIENCES
MESENCHYMAL STEM CELLS AND SKELETAL MUSCLE
Medicine & Science in Sports & Exercised
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