Skeletal muscle pericytes increase in quantity following eccentric exercise (ECC) and contribute to myofiber repair and adaptation in mice. The purpose of the present investigation was to examine pericyte quantity in response to muscle-damaging ECC and protein supplementation in human skeletal muscle. Male subjects were divided into protein supplement (WHY; n = 12) or isocaloric placebo (CHO; n = 12) groups and completed ECC using an isokinetic dynamometer. Supplements were consumed 3 times/day throughout the experimental time course. Biopsies were collected prior to (PRE) and 3, 24, 48, and 168 h following ECC. Reflective of the damaging protocol, integrin subunits, including α7, β1A, and β1D, increased (3.8-fold, 3.6-fold and 3.9-fold, respectively, P < 0.01) 24 h post-ECC with no difference between supplements. Pericyte quantity did not change post-ECC. WHY resulted in a small, but significant, decrease in ALP + pericytes when expressed as a percentage of myonuclei (CHO 6.8 ± 0.3% vs. WHY 5.8 ± 0.3%, P < 0.05) or per myofiber (CHO 0.119 ± 0.01 vs. WHY 0.098 ± 0.01, P < 0.05). The quantity of myonuclei expressing serum response factor and the number of pericytes expressing serum response factor, did not differ as a function of time post-ECC or supplement. These data demonstrate that acute muscle-damaging ECC increases α7β1 integrin content in human muscle, yet pericyte quantity is largely unaltered. Future studies should focus on the capacity for ECC to influence pericyte function, specifically paracrine factor release as a mechanism toward pericyte contribution to repair and adaptation postexercise.

skeletal muscle has a remarkable capacity to repair and regenerate in response to injury, including damage caused by an acute bout of eccentric exercise (ECC). In response to ECC, myogenic stem cells, or satellite cells (27), become activated, proliferate and differentiate to repair damaged myofibers (17). Studies have confirmed that satellite cells are essential for repair postinjury (23, 28), yet factors in the stem cell microenvironment, or niche, that regulate satellite cell activation and proliferation following exercise-induced damage are not well-described. Recently, muscle-resident stromal cells have been identified in mouse (18, 42) and human skeletal muscle (11, 40) that secrete growth factors and cytokines that support muscle regeneration. These cells have been defined as neural/glial antigen 2 (NG2)+ pericytes (2, 3, 11, 12, 20), alkaline phosphatase (ALP)+ pericytes (12, 20), platelet-derived growth factor receptor alpha (PDGFRα)+ progenitor cells (49, 50), Sca-1+CD45− muscle-derived mesenchymal stem cells (mMSCs) (24, 42, 43), Sca-1+/− fibro-adipogenic progenitors (21), PW1+ interstitial cells (PICs) (31), side population (SP) cells (13, 32), and muscle-derived stem cells (35). Despite limited potential for myogenesis (11, 12, 40), in vitro and in vivo studies suggest that muscle-resident stromal cells synthesize and secrete a variety of factors, such as IL-6, HGF, and TGF-β, that have been implicated in satellite cell proliferation and fiber repair (13, 24, 32, 43). Currently, the extent to which non-satellite cells appear in human skeletal muscle following exercise-induced injury and secrete paracrine factor that can contribute to repair is not known.

We have previously demonstrated that a single bout of damaging eccentric exercise can increase transcription and protein expression of the α7β1 integrin transmembrane adhesion protein in mouse skeletal muscle (4, 6). Transgenic expression of this important cytoskeleton-ECM linkage protein in muscle (MCK:α7BX2) can protect against ECC-induced damage, as well as increase satellite cell content and promote myofiber growth (4, 25, 25). Additionally, mice overexpressing the α7 integrin subunit accumulate Sca-1+CD45− mMSCs, the majority of which express pericyte markers, following eccentric exercise (42), and mMSCs isolated from α7 integrin transgenic mice release factors that substantially promote satellite cell proliferation in vitro (24) and in vivo (43). In human skeletal muscle, Hydahl and colleagues (20) did not observe any alteration in NG2+ and ALP+ pericyte quantity 3 h post-ECC. However, this may be due to the early time point of the muscle biopsy.

Serum response factor (SRF) is a transcription factor that is sensitive to mechanical loading (16). A recent study demonstrated that deletion of SRF from myofibers can reduce interleukin-6 (IL-6) gene expression and subsequently inhibit satellite cell proliferation and hypertrophy associated with overload (16). The extent to which SRF is increased in the myonuclei or pericyte for the purpose of driving synthesis of paracrine factors, such as IL-6, post-ECC has not been determined.

High-quality protein consumption following resistance exercise can augment myofiber protein synthesis and enhance muscle growth (37), yet its effect on the muscle regenerative response is not well understood. Some studies have observed that following acute eccentric exercise, consumption of whey protein can enhance maximal force recovery in young adults (7, 10). Additionally, leucine supplementation has been observed to attenuate the decline in peak force following ECC (22). In support of a role for whey in muscle repair, we have recently demonstrated that whey protein supplementation can augment the increase in satellite cell quantity in young, healthy subjects after eccentric exercise-induced muscle damage (14). The effects of whey protein supplementation on other cell types in the muscle microenvironment have yet to be determined but could provide insight into whether protein supplementation influences satellite cell activity directly or indirectly.

In the current study, we use a model of high-volume maximal eccentric muscle contractions (ECC), an established model of muscle damage in humans (19, 20, 29, 39), to evaluate the regenerative response in human muscle. Specifically, we evaluate integrin subunit content, pericyte quantity, and expression of SRF in both myonuclei and pericytes over multiple time points. Furthermore, we evaluate the pericyte response to ECC in the absence and presence of whey protein ingestion. We hypothesized that ECC would increase integrin subunit protein expression, as well as acutely and transiently increase pericyte quantity and coexpession with SRF. Additionally, similar to its effects on satellite cells, we speculated that whey protein supplementation would augment pericyte quantity.

MATERIALS AND METHODS Participants. A detailed account of the participant population has been provided previously, and these data represent new data from a study partially published elsewhere (14). Briefly, twenty-four recreationally active, healthy male volunteers (23 ± 0.5 yr; height 182 ± 1.3 cm; weight 76 ± 1.9 kg; body fat 13 ± 0.9%) were included. Participants were excluded if they 1) participated in a planned resistance or high-intensity training program within the past 6 mo, 2) had participated in strenuous physical activity 48 h prior to testing, or 3) had a history of past lower limb musculoskeletal injury, 4) were vegans, 5) consumed dietary supplements (protein supplements, antioxidants), 6) had taken nonsteroidal anti-inflammatory drugs (NSAIDs) or prescription medications (angiotensin-converting enzyme inhibitors), or 7) consumed alcohol during the experimental time period. Participants were informed of the procedures and potential risks associated with participating in the study and provided written informed consent prior to participation. All protocols were approved by The Central Denmark Region Committees on Health Research Ethics (Ref No.: M-20110179) and conformed to the guidelines of the use of human subjects in research as outline in the Declaration of Helsinki. Study design. The study used an unpaired, randomized, double-blinded design. Participants were divided into one of two groups: one that received a whey protein supplement (WHY) containing 28 g of whey protein hydrolysate (4%) + 28 g of carbohydrate (4%), and one that received a carbohydrate placebo (CHO) containing 56 g of carbohydrate (8%). Both groups reported to the lab after an overnight fast fourteen days prior to performing a single exercise bout for obtaining basal muscle biopsies. Subjects were instructed not to perform intense recreational activities involving eccentric muscle actions during the following 14-day period; however, nonintense activities (e.g., biking, walking involved in daily self-transportation) were allowed. Fourteen days following the initial biopsy, subjects returned to the lab to complete the muscle-damaging exercise protocol, which consisted of 15 sets of 10 repetitions of unilateral maximal eccentric muscle contractions of the quadriceps femoris using an isokinetic dynamometer (Humac Norm, CSMI, Stoughton) at 30°/s, with 70° range of motion and 1 min rest between sets. For each subject, one leg was selected at random to undergo the exercise protocol. A detailed figure of the study design has been previously published (14). Subjects were restrained at the chest, hips, and thighs to isolate the leg extensors and were provided verbal encouragement during each set. Data and methods pertaining to indexes of muscle damage following ECC in this same set of participants has been previously reported (14). Volunteers randomized into WHY or CHO groups were told to consume the beverage three times per day on the exercise day as well as on days 1 and 2 post-ECC (3 days total). On the exercise day, the supplements were ingested immediately postexercise (approximately 10 am), and 3 (1 pm), and 6 (4 pm) h postexercise. On days 1 and 2, the supplements were ingested at the same time of the day corresponding to the exercise day. Participants recorded all energy-containing food and drinks on the exercise day and days 1 and 2 post-ECC, while maintaining their habitual food intake. Participants received a login to an online food registration software program (Madlog.dk Aps, Kolding, DK) and received verbal and written information on the usage. During the later off-line analysis, the dietary intake was analyzed for macronutrient distribution as well as total energy intake. Muscle biopsies and tissue preservation. Muscle biopsies were obtained from the midportion of the vastus lateralis 14 days prior to the eccentric exercise protocol (Pre), and 3, 24, 48, and 168 h post. The prebiopsy was obtained from the nonexercise leg to reduce the number of biopsies in the exercise leg, similar to previous studies (29). The 3- and 24-h time points were selected to evaluate the immediate changes in integrin protein expression (20). Since the pericyte response to acute exercise has never previously been examined in human skeletal muscle, we based our selection of time points on previous literature examining a different muscle progenitor cell population, satellite cells, to eccentric exercise. To evaluate acute changes in cell quantity, the 24- and 48-h time points were selected as these have previously been shown to correspond to the earliest increase in satellite cell quantity following eccentric exercise (29, 30, 39). Finally, the 168-h time point was selected because by this time the muscle repair process (20) and satellite cell quantity following eccentric exercise-induced muscle damage have been shown to be returning to baseline (29). Samples were obtained under local anesthetic (10 mg/ml lidocaine) using the Bergstrom needle technique with manual suction as previously described (36). Each incision was separated by ∼3 cm and muscle tissue was freed of any visible blood, fat, or connective tissue. A portion of the muscle biopsy was immediately flash frozen in liquid nitrogen, then stored at −80°C for later analysis of protein content by Western blotting. A separate portion of the biopsy was immediately mounted in optimal cutting temperature (Tissue-Tek, Qiagen) and frozen in liquid nitrogen-cooled isopentane, then stored at −80°C for immunofluorescence analysis. With the exception of the 3-h biopsy, all biopsies were collected at the same time of day to avoid any effects of diurnal variation, and given a unique identifier so that investigators were blinded during analysis. Western blotting. After crushing in liquid nitrogen in a mortar and pestle, samples were homogenized in ice-cold homogenization buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) with phosphatase inhibitor (Roche) and protease inhibitor plus EDTA (Roche) by vortexing and incubating with rotation at 4°C for 90 min. Samples were centrifuged at 15,000 rpm for 15 min to separate the soluble fraction which was collected and stored at −80°C until use. An aliquot of the supernatant was used for determination of protein concentration by Bradford assay (Bio-Rad). Equal amounts of protein (45 μg for α7 and 15 μg for β1A and β1D) were loaded in 8% gels, separated by SDS-PAGE, and transferred to nitrocellulose membranes. After blocking in 5% milk/TBST for 1 h at room temperature, membranes were incubated overnight in primary antibodies at 4°C. After several washes, membranes were incubated in secondary antibody for 1 h at room temperature, and proteins were detected using ECL (SuperSignal) and Bio-Rad ChemiDoc imaging system. Quantification was conducted relative to Ponceau (α7) or β-actin (β1A and β1D) using Quantity One software (Bio-Rad) with background correction. Data are expressed as fold change relative to Pre values, and any samples giving negative optical density values (below background) were changed to 0. The following primary and secondary antibodies were used: β1A and β1D (1:1000, kind gift from Woo Keun Song, Kwangju Institute of Science and Technology, Korea), α7 (1:1000, Abcam), β-actin (1:2000; Cell Signaling Technology), and anti-rabbit horseradish peroxidase (1:25,000; ImmunoResearch Laboratories). Immunofluorescence. Immunofluorescence staining was conducted on transverse sections (10 μm) as previously described (18, 20). For NG2 immunofluorescence, samples were fixed in ice-cold acetone for 10 min, washed, then blocked in 5% BSA. Samples were incubated in primary antibodies for mouse anti-dystrophin (Sigma) for 30 min at 37°C, then rabbit anti-NG2 (Millipore) for 1 h at room temperature. After multiple washes, sections were successively incubated in appropriate secondary antibodies: TRITC anti-mouse and FITC anti-rabbit (Jackson ImmunoResearch) for 1 h each at room temperature. Sections were washed, stained with DAPI to label nuclei; a coverslip was applied with Vectashield mounting media (Vector Laboratories), and then stored at 4°C until imaging. Images were acquired with a Zeiss AxioCam digital camera and OpenLab software (Zeiss). For ALP immunofluorescence, slides were fixed for 10 min in 2% paraformaldehyde (PFA), then blocked in blocking buffer (2% BSA, 5% FBS, 2% goat serum, 0.2% Triton X-100, 0.1% sodium azide) for 90 min at room temperature. Sections were incubated in mouse anti-ALP (TRA-2-49/6E, Developmental Studies Hybridoma Bank) and rabbit anti-serum response factor (SRF; Santa Cruz Biotechnology) overnight at 4°C. After several washes, sections were incubated in appropriate secondary antibodies: FITC anti-mouse (Jackson ImmunoResearch) and Alexaflour 633 anti-rabbit (Life Technologies) for 90 min at room temperature. Sections were washed, refixed in 2% PFA to prevent antibody migration, then reblocked in 5% BSA for 1 h at room temperature before incubation in mouse anti-dystrophin (Sigma) for 30 min at 37°C. After several washes, sections were incubated in TRITC anti-mouse secondary antibody for 60 min at room temperature. Sections were washed, stained with DAPI to label nuclei, coverslipped with Vectashield mounting media (Vector Laboratories), and then stored at 4°C until imaging. Secondary only controls and isotype controls were used when appropriate to confirm specificity of staining. NG2/dystrophin/DAPI images were examined with a Leica DMRXA2 microscope and acquired using a Zeiss AxioCam digital camera and OpenLab software (Zeiss). All other immunofluorescence images were acquired using a Hamamatsu NanoZoomer (Digital Slide Scanner NanoZoomer, Hamamatsu). Image analysis. Images were converted to .TIFF files and analyzed offline using ImageJ software (NIH). All analysis was conducted by investigators blinded to condition and time. For NG2 and ALP analyses, only NG2/ALP positive cells that c-localized with DAPI and were located in the interstitial space as determined by dystrophin staining were quantified. For SRF stains, all SRF that colocalized with myonuclei were counted as well as SRF that colocalized with ALP to determine the quantity of SRF positive pericytes. Since ImageJ only has capacity for three different color fields, SRF and dystrophin were both analyzed in the red channel. Punctate red staining that colocalized with nuclei were counted as SRF positive as punctate nuclear colocalization was not expected with dystrophin staining. It was necessary to include dystrophin staining in these analyses to accurately identify pericytes in the interstitium. All ALP analyses were conducted by at least two blinded investigators and their results were averaged to acquire final numbers. ALP, ALP/SRF, and NG2 were quantified as a percentage of total myonuclei as well as per myofiber. Pericytes were quantified from an average of 304.7 fibers. Statistical analysis. Data are presented as means ± SE with P < 0.05 considered significant. Data were analyzed via two-factor (supplement condition × time) ANOVA with repeated measures on time. Significant main effects or interactions were further tested with Tukey's post hoc test. Data sets that failed to meet the assumptions of normality and/or equal variance were log transformed prior to analysis. All statistical analyses were conducted with SigmaPlot 12.5 (Systat Software, San Jose, CA).

DISCUSSION Elucidating the regenerative response to physiological muscle damage can provide insight regarding defective repair in pathological conditions and provide an improved understanding of beneficial adaptations to exercise. In this study, we examined pericyte quantity across a variety of time points following a single bout of muscle-damaging ECC. Responses were investigated under conditions with or without protein supplementation. As confirmation that our ECC protocol did indeed induce muscle damage, we have previously reported a decrease in muscle strength, an increase in serum creatine kinase, and an increase in muscle soreness in these same participants (14). In the current study, we provide the first evidence in human muscle that α7 and β1(A and D) integrin subunit protein expression is increased within 24 h of ECC. Despite prior and current verification of damage and subsequent myofiber response to acute, high-intensity ECC, pericyte quantity remained unaltered at 24, 48, or 168 h postexercise. Furthermore, myonuclear or pericyte expression of SRF, involved in the production of IL-6 as well as other paracrine factors, did not change using this protocol. Finally, the effects of supplementation with whey protein were minimal. These data suggest that integrins are important proteins involved in the acute repair/adaptive response to ECC, while the role of pericytes in the acute phase remains to be determined. Muscle-damaging ECC induces a well-characterized repair and regenerative response characterized by membrane damage, inflammatory cell infiltration, and initiation of repair mechanisms including cell signaling and satellite cell activation (19). An early response to ECC-induced muscle damage is detachment of myofibers from the ECM (38). In support of this idea, Mackey and colleagues (26) recently demonstrated that tenascin C, a protein involved in disassembly of focal adhesions (15), is increased acutely in response to muscle damage induced by electrically-stimulated isometric contractions. Integrins are a primary family of transmembrane proteins involved in myocellular adhesion to ECM and mechanotransduction in skeletal muscle (8). In the present study, we observed a robust increase in the quantity of α7, β1A, and β1D integrin protein expression 24 h following ECC. These data support findings in mice of increased α7 integrin mRNA and protein expression 1 day following an acute bout of eccentric exercise (4, 6). We interpret these findings as an indication of an acute adaptive response to ECC to reestablish connections between the ECM and myofiber and to protect fibers from similar stress in the future. Indeed, increased myofiber coexpression of α7 integrin and tenascin-C was observed in mice following eccentric exercise (6), supporting the concept that mechanisms involved in myofiber-ECM disassembly and reassembly are simultaneously activated by eccentric exercise. In this manner, integrins may act as key players in muscle regeneration and the repeated bout effect in humans (9). We have previously established that both the α7 integrin subunit protein expression (6) and mesenchymal stem cell (Sca-1+CD45−) content (42) increase in skeletal muscle of mice following an acute bout of ECC. Furthermore, overexpression the α7 integrin subunit resulted in an accumulation of Sca-1+CD45− cells following an acute bout of ECC (42). The majority of the Sca-1+CD45− cells in muscle postexercise express pericyte markers, including NG2, PDGFRβ, and CD146 (42). Based on this observation, we speculated that similar changes would occur in human skeletal muscle post-ECC. Contrary to our original hypothesis, we did not observe an increase in pericyte quantity at 24, 48, or 168 h following ECC, potentially suggesting that α7 integrin expression and pericyte quantity may either not be related in regenerating adult human muscle, or that prior elevation in the expression and localization of α7 integrin at the myofiber membrane may be a prerequisite for an increase in pericyte number in response to ECC. To our knowledge, this is the first report of the time course of the pericyte response to physiological muscle damage in humans. We used two established phenotypic markers of human skeletal muscle pericytes, NG2 and ALP (11, 20), and found no difference in the response of pericytes to ECC using either marker for quantification. The lack of increase in pericyte quantity following ECC was similar to the findings of Hyldahl and colleagues (20) who also failed to observe any changes in pericyte quantity following ECC, although they only evaluated pericyte quantity at a single time point, 3 h post-ECC, when an increase in cell quantity would not be expected. Total NG2 and ALP expression was divergent in our samples, with a higher percentage of cells identified by NG2. We cannot be certain if this finding is consistent with those of Hyldahl and colleagues (20) as they present their pericyte quantity data as an average of results obtained using each marker. However, if we similarly average NG2 and ALP, the number of pericytes per myonuclei is comparable. Despite evaluating multiple time points after eccentric exercise, we have considered the possibility that pericyte quantity may have increased between 48 and 168 h post-ECC. Indeed, the quantity of other non-myogenic cell types believed to be involved in muscle regeneration, such as FAPs, fibroblasts and vascular cells, peaks between 3 and 7 days in mice (1, 33). Recently, it was suggested that pericyte subpopulations exist in muscle that have preferential capacity for either adipogenic/fibrogenic (Type 1) or myogenic (Type 2) differentiation (2, 3). It has yet to be determined if similar pericyte subpopulations exist in human skeletal muscle and the extent to which ECC can influence pericyte differentiation and function. We recognize that ECC may exert a more predominant effect on pericyte paracrine factor secretion, rather than proliferation, and this may be the basis for the lack of increase in pericyte quantity observed in the current study. The small but significant effect of protein supplementation on ALP+ pericyte cell quantity was an interesting finding that was contrary to our original hypothesis. We observed a significant decrease in ALP+ pericyte quantity with whey supplementation that seemed to be mostly a consequence of a downward trend in pericyte quantity following ECC in the WHY group concomitant with a slight upward trend in the CHO group. The decrease in pericyte quantity in the WHY group was opposite to the previously reported effects of whey, as well as whey combined with training, on satellite cell accretion (14, 34). The interpretation and significance of a decline in pericyte quantity with whey protein supplementation is not clear and should be addressed in future studies. Muscle-resident pericytes are primarily located in the vascular niche of the perimysium (11, 20, 40), and secrete paracrine factor necessary for ECM remodeling and repair (5, 24). In this study, we attempted to evaluate pericyte function, in vivo, based on SRF expression. We did not observe any change in SRF expression in pericytes or myonuclei following ECC. We may have missed the optimal time point for detection of SRF activity, as our first biopsy for immunohistochemical staining was collected at 24 h post-ECC and we would expect changes in transcription factor translocation and binding to occur immediately postexercise. Although SRF expression was not altered in myonuclei or ALP+ pericytes in response to ECC, SRF was expressed in a small proportion of myonuclei (∼5%), and ∼65% of pericytes. Given the role for SRF in promoting myogenic differentiation, it is interesting to speculate that the SRF+/ALP+ pericytes quantified in the present study are consistent with the type 2 pericytes observed in mouse models (2, 3). A strength of the present study is the use of two separate, previously reported, human pericyte markers, NG2 and ALP (20), to quantify pericytes. The effect of ECC was similar whether NG2 or ALP was used, suggesting that these markers either identify the same cell population or that they are separate but overlapping populations that respond in the same way to ECC. The lack of unique cell surface markers to identify distinct populations of muscle-resident stromal cells presents a significant challenge to the evaluation of the stem/stromal cell response to exercise. For example, PDGFRα+ mesenchymal progenitors have been observed in human muscle that exhibit enhanced fibroadipogenic potential (49), yet it is not clear if these cells are distinct from pericytes or are pericyte descendants that share marker expression. Ideally, lineage tracing experiments will clarify the mononuclear cell composition in muscle, and future studies are needed that make use of multicolor flow cytometry strategies to simultaneously evaluate the response of multiple sub-populations to exercise. In summary, our examination of the acute response to eccentric exercise with or without protein supplementation demonstrated that α7, β1A and β1D integrin subunit protein expression is robustly increased in response to ECC. Total pericyte quantity was not increased 24, 48, or 168 h post-ECC, and a small but significant suppression of pericyte quantity was detected with protein supplementation. Finally, SRF expression within pericytes or myonuclei did not differ with eccentric exercise or with supplementation. The results from this study suggest that paracrine factor release, rather than quantity, may provide the basis for pericyte contribution to repair in human skeletal muscle following eccentric exercise.

GRANTS This research was supported by grants from Abbott Nutrition and the Center for Nutrition, Learning, and Memory at the University of Illinois (392 Abbott CNLM ZA68 to M.D. Boppart) and ARLA Foods (to K. Vissing) . Granting agencies had no input into the design, conduct, analysis or dissemination of research findings.

DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s).