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. Author manuscript; available in PMC: 2014 Sep 1.
Published in final edited form as: FEBS J. 2013 Apr 12;280(17):4063–4073. doi: 10.1111/febs.12228

Moderate-intensity treadmill running promotes expansion of the satellite cell pool in young and old mice

Gabi Shefer 1,2,3, Gat Rauner 1, Pascal Stuelsatz 2, Dafna Benayahu 1,3, Zipora Yablonka-Reuveni 2,3
PMCID: PMC3711960  NIHMSID: NIHMS453779  PMID: 23464362

Abstract

Satellite cells, myogenic progenitors located at the myofiber surface, are essential for repair of adult skeletal muscle. There is ample evidence for age-linked decline in satellite cell numbers and performance in limb muscles. Hence, effective means to activate and expand the satellite cell pool may enhance muscle maintenance and reduce the impact of age-associated muscle deterioration (sarcopenia). Toward this aim, we explored the potential beneficial effect of endurance exercise on satellite cells in young and old mice. Animals were subjected to an 8-week moderate-intensity treadmill running approach that does not inflict apparent muscle damage (0° inclination, 11.5 meter/min, 30 min/day, 6 days/week). Myofibers of extensor digitorum longus muscles were then isolated from exercised and sedentary mice and used for monitoring satellite cell numbers and for harvesting individual satellite cells for clonal growth assays. We specifically focused on satellite cell pools of single myofibers, with the view that daily ware of muscles is likely inflicting individual myofibers rather than causing overall muscle damage. We found an expansion of the satellite cell pool in the exercised groups compared with the sedentary groups, with the same increase factor (~1.6) in both age groups. Current results accord with our findings with rat gastrocnemius, attesting for the consistent effect of exercise running on satellite cell expansion in limb muscles. The experimental paradigm established here is useful for studying satellite cell dynamic at the myofiber niche and for broader investigation of the impact of physiologically and pathologically relevant factors on adult myogenesis.

Keywords: Skeletal muscle, myofibers, satellite cells, aging, endurance exercise, Nestin-GFP

Introduction

Satellite cells are Pax7-expressing (Pax7+) myogenic progenitors, residing between the myofiber basal and plasma membranes and are essential for repair of adult skeletal muscles [1–4]. There is evidence that at least some of the satellite cells are bona fide stem cells, capable of self-renewing in addition to contributing progeny for myofiber repair, thereby ensuring maintenance of the satellite cell pool [5, 6]. Routine muscle activity, even if involves only subtle myofiber injuries, raises a continuous demand for functioning satellite cells throughout life. Rodent and human studies have shown, within the context of limb muscles, that there is an age-associated decline in satellite cell numbers and performance [7–17]. This diminution may contribute to age-related muscle deterioration, a condition known as sarcopenia. Sarcopenia is characterized by a decline in mass, strength, endurance and repair ability of skeletal muscles, leading to frailty, reduced life quality and even death [18–22]. Multiple factors, ranging from systemic to muscle-intrinsic, are thought to play a role in this process [22–26]. This muscle deterioration involves a reduction in myofiber number, atrophy of remaining myofibers and increased susceptibility to contraction-induced injuries [27]. Combating sarcopenia requires the identification of means to improve myofiber maintenance in aging muscle. Therefore, a better understanding of satellite cell dynamics at their natural niche, the myofiber, is essential.

The vast majority of satellite cells are typically quiescent in adult limb muscles. This inactivity combined with lack of evidence for satellite cell fusion with myofibers in uninjured muscles, has begun to cast doubts on the importance of satellite cells in “baseline” myofiber maintenance (homeostasis) through adult life. However, the sedentary lifestyle of rodents (most frequently used in satellite cell research, typically maintained in standard cage conditions) is not necessarily an optimal environment for promoting recruitment of satellite cell into performance.

Studies with isolated myofibers, using conditions that retain satellite cells at their niche, have shown that this positional intimacy with the myofiber is essential for satellite cell interplay between progeny expansion versus self-renewal [28–30]. Nevertheless, much of our current understanding of in-vivo myogenesis in adult life is based on models of induced muscle trauma [31, 32]. These studies have typically relied on using agents such as snake venom or barium chloride, or exhaustive eccentric exercise protocols that cause extensive muscle damage [33, 34]. While such muscle injury approaches have provided a wealth of knowledge about muscle regeneration, they do not retain muscle architecture and physiological milieu associated with daily muscle use. Furthermore, daily muscle use inflicts subtle myofiber damage that requires localized myofiber repair. It is reasonable to consider that this process relies on the performance of individual resident satellite cells in few myofibers at a time, rather than on “en masse” satellite activation that is characteristic to regeneration after massive muscle damage. Thus, in order to gain insights into the satellite cell pool through life, we have studied satellite cells at the single-cell level in isolated myofibers, demonstrating a drastic age-associated diminution of the satellite cell pool in mice and rats [9–11]. This decline may represent deterioration in the capacity of all or a sub-set of satellite cells to self-renew [10, 35].

We recently began using endurance exercise for in-vivo rodent satellite cell studies [11, 36], especially because this activity has been considered not to inflict muscle trauma and is even beneficial in certain myopathies [37–40]. While many human and rodent studies have shown enhanced satellite cell numbers following a single bout of eccentric activity [41–43] studies on satellite cells using in-vivo models that do not involve muscle damage are scarce. Endurance exercise is beneficial to many aspects of human health [44, 45]. Likewise, rodent studies have provided evidence for the beneficial effects of endurance exercise, including lifespan and health-span expansion [46–51]. The effect of endurance exercise on satellite cells has been analyzed only in a limited number of studies [11, 52–54]. One detailed study looked at satellite cell numbers in the rat plantaris muscle using voluntary wheel running [52] and we studied the effect of moderate-intensity, treadmill running (0° inclination) on gastrocnemius muscle of young and old rats [11]. Our preference of using treadmill running is based on the high reproducibility among mice subjected to exercise running, while mice subjected to voluntary wheel running vary widely in traveled distance outcomes. Treadmill-running endurance training engages muscle groups linked to toe and ankle functions [11, 55, 56]. These include extensor digitorum longus (EDL), tibialis anterior, flexor digitorum brevis, and gastrocnemius muscles. These fast twitch muscles are affected by aging [57]. Notably, exercise-induced affects on contralateral muscles that have not been engaged directly during physical activity have been reported [58]. Hence, exercise may also have systemic beneficial effects on satellite cells in distant muscles.

In the present study, we have investigated satellite cell pools of individual EDL myofibers, with the view that daily wear of muscles is likely affecting individual myofibers rather than causing overall muscle damage as occurs in response to robust trauma. Specifically, we monitored satellite cell numbers as an overall measure for the satellite cell pool homeostasis and performance, which depends on the dynamic between quiescence, proliferation, renewal, and survival of satellite cells and their progeny [28, 35, 59]. We demonstrate that moderate-intensity treadmill running elevates the number of satellite cells in young and old mice. Current results accord with our findings with rat gastrocnemius, demonstrating the consistent effect of exercise running on satellite cell numbers in limb muscles. The experimental paradigm established here is beneficial for studying satellite cell dynamic at the myofiber niche and for broader investigations on the impact of physiological and pathological relevant factors on adult myogenesis.

Results and Discussion

Experimental approach

  1. In this study, we investigated the effect of moderate-intensity treadmill running on satellite cell number and performance in young (4-month old) and old (16-month old) male mice. Age of mice specified throughout the manuscript reflects their age at the beginning of the 8-weeks period of the experiment (i.e., exercise or sedentary schedule). Hindlimb (EDL) muscles were harvested at the end of this period for single myofiber isolation in order to quantify satellite cells and perform clonal growth analysis.

  2. The exercise groups were subjected to treadmill running (0° inclination) at a speed of 11.5 meters/minute, for 30 minutes/day, 6 days a week for 8 consecutive weeks. The moderate-intensity running conditions used in the present study are comparable to running conditions that were previously described by others for young and old mice [60, 61].

  3. Transgenic mice expressing GFP under the control of nestin gene regulatory elements (NES-GFP) were used throughout the study for detecting satellite cells in isolated myofibers according to GFP expression. As we previously published, NES-GFP+ satellite cells are easily distinguished from myonuclei that are GFP-negative, while both satellite cells and myonuclei are commonly stained with DAPI [62]. Regardless of mouse age, there is an over 95% agreement when satellite cells are detected by the endogenous marker Pax7 versus the transgenic marker NES-GFP [10, 62].

Running enhances the number of satellite cells in young and old mice

The number of satellite cells (NES-GFP+) per individual EDL myofibers was determined in sedentary and exercised mice of young and old age (Fig. 1). Fig 1A provides a summary, per each group, of total numbers of mice, myofibers and satellite cells analyzed, and average (± SEM) number of satellite cells per myofiber. Further details of satellite cell distribution per individual myofibers are summarized in boxplots (Fig. 1B), depicting quartile analysis and average ± SEM satellite cell number per myofiber. Significant effects of age, exercise and a combination of age and exercise were observed (two-way ANOVA, F1, 569=177.28; 129.2 and 11.793, respectively, p<0.001). The number of satellite cells declined with age, while running enhanced the number of satellite cells per myofiber of both young and old mice. Myofibers from young exercised mice contained the highest mean number of satellite cells compared to young sedentary, old exercised and old sedentary groups (average ± SEM, 14.0±0.5 vs. 8.6±0.3, 7.9±0.3 and 5.0±0.2, respectively).

Fig. 1.

Fig. 1

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Quantification of GFP+ satellite cells in EDL myofibers isolated from sedentary and exercised NES-GFP male mice. Satellite cells were recorded per individual myofibers isolated from young (4 months) and old (16-months) mice. Age of mice specified is the age at the beginning of the 8-week run/no-run period. (A) Summary of experimental details of the current figure. Each mouse was processed separately for satellite cell quantification and data within each group were pooled after establishing (by ANOVA) a common satellite cell distribution pattern across mice. (B) Satellite cell numbers per individual myofibers summarized as boxplots. The box represents the interquartile range (IQR) which spans from the 1st to the 3rd quartile (Q1 and Q3), the dividing line within the box is the median, and the white marks show the average ± SEM. Outliers (red circles, right Y-axis) are defined as being either greater than Q3 + 1.5×IQR or less than Q1 − 1.5×IQR. The whiskers on each side of the box are taken to the minimum and maximum values or when outliers are present to 1.5×IQR from Q1 and Q3 (http://www.physics.csbsju.edu/stats/box2.html). Outliers identified in the young/exercised group were contributed by two mice. (C) Myofiber distribution according to satellite cell numbers. Myofibers are arranged in ascending order according to the number of satellite cells per myofiber (X-axis) versus the number of myofibers containing a given number of satellite cells (Y-axis). The exercise-driven increase in the number of satellite cells per myofiber is also illustrated in the cumulative curves. Each data point (diamond-shaped, right Y-axis) in these cumulative curves shows the percentage of myofibers out of total myofibers analyzed that contain less than, or equal to (≤) the corresponding number of satellite cells per myofiber that is indicated on the X-axis.

Exercise induced a significant increase in average satellite cell numbers in both young and old mice (p<0.005, for both ages). This is reflected in an overall shift toward higher number of satellite cells per myofiber with a noticeable increase in the lower end (from 1 to 6 and from 0 to 3 in young and old exercised mice, respectively). Furthermore, exercise brings the average number of satellite cells in myofibers of old exercised mice to the same level measured in myofibers of young sedentary mice. Remarkably, the increase factor (i.e. ratio between the average number of satellite cells per myofiber in exercised and sedentary mice) is conserved between the young and old group, amounting to 1.55 and 1.58, respectively. This observation suggests that satellite cells from old mice are equally capable of responding to the exercise as their younger counterpart.

Exercise effect on satellite cell numbers is further demonstrated in graphs depicting myofiber distribution according to satellite cell numbers (Fig. 1C). Myofibers are arranged in ascending order of the numbers of satellite cells per myofiber (X-axis) versus the number of myofibers containing a given number of satellite cells (Y-axis). The exercise-driven increase in the number of satellite cells per myofiber in both young and old mice is evident in the shift of the respective cumulative curves. Each data point in these cumulative curves depicts the percentage of myofibers (out of total myofibers analyzed) that contain less than, or equal to (≤) the corresponding number of satellite cells per myofiber that is indicated on the X-axis. These cumulative plots (Fig. 1C) and the boxplots (Fig. 1B) demonstrate the persistent effect of exercise on increasing the relative abundance of myofibers containing high numbers of satellite cells. The median (50% value) for satellite cell number per myofiber rose from 8 (no-run) to 12 (run) for the young group (Fig. 1B & C). Median value for satellite cell numbers per myofiber in the old group rose from 5 (no-run) to 8 (run).

Collectively, the analysis of satellite cell content in individual myofibers has demonstrated that moderate-intensity treadmill running in both young and old mice leads to an increase in the frequency of myofibers with more satellite cells when compared to their non-exercised counterparts. Such an effect was also observed in our previous rat study where satellite cells were detected based on Pax7 immunostaning [11]. Myofibers lacking satellite cells, typically present in old mice and rats [9–11], are not detected following moderate-intensity treadmill running (Fig. 1C). The absence of such myofibers that lack satellite cells following long-term running could be due to inability of these myofibers to sustain the load/trauma and survive without their supporting satellite cells. We aim to explore this possibility by subjecting mice to the running protocol after inflicting age-specific ablation of satellite cells using Cre-loxP mice based on Pax7-Cre driven activation of diphtheria toxin in satellite cells. Using this paradigm, an absolute requirement for satellite cells in muscle regeneration following major trauma has been confirmed [32, 33]. Nevertheless, satellite cells do not appear to be required for muscle hypertrophy or recovery from unloading-induced atrophy in adult life [63, 64].

Alternatively, the absence of myofibers void of satellite cells in the exercised old mice could be due to the reconstitution of their satellite cell pool through contribution from neighboring progenitors during physical activity. Using a genetic model where ‘cell lineage trees’ can be re-constructed based on the similarity in somatic mutations (in non-coding sequences) between cells, we have previously shown that each myofiber has its own pool of satellite cells [65]. This suggests that during standard cage activity there is no cross over of satellite cells between myofibers [65]. However, due to the fragility of these genetically modified mice, we are unable to age these animals and establish satellite cell numbers in combination with exercise running. Detection of clonal patches of revertant (dystrophin+) myofibers in mdx mice does, in fact, support the existence of pioneer myogenic progenitors that produce progeny, which populate adjacent myofibers during postnatal life of mdx mice [66].

Effects of age and exercise running on clonal performance of satellite cells

As an additional measure for the effect of exercise running on satellite cell performance, we analyzed the number of myogenic clones that developed from individual myofibers (Fig. 2). The same mice analyzed for satellite cell numbers (Fig. 1A) were used for the clonal studies, investigating 3–4 myofibers per mouse (Fig. 2A). Myofibers were individually triturated to strip the satellite cells. The resulting suspension of each myofiber was dispensed into 12 wells and then followed for the development of clones. Clones were identified following the 3rd culture day and defined as myogenic based on the presence of myotubes that are typically found by 7–10 days in culture [10]. Fig 2A provides per each group a summary of total myofibers and myogenic clones analyzed, and average (± SEM) number of myogenic clones per myofiber. There was an overall age effect (F1, 88=23.33, p<0.005) showing a reduction in the number of myogenic clones in older mice, in agreement with the age-associated decline in satellite cells numbers detailed above (Fig. 1). The detection of a lower number of myogenic clones per myofiber compared to the number of satellite cell per myofiber (Fig. 1A vs. 2A) agrees with our early studies that have indicated about 50% satellite cell clonability in young and old mice [10]. The myogenic clone data analyzed in boxplots (Fig. 2B) depict quartile distribution and average ± SEM for the number of clones per myofiber. Myofiber distribution according to their number of myogenic clones and cumulative curves are also shown (Fig. 2C). These data demonstrate a trend toward more myofibers that are capable of generating more clones upon exercise for each age group. This exercise effect follows what we have observed previously with myogenic clones from exercised versus sedentary rats of young and old ages [11]. Noticeably, the 3rd quartile (75th percentile) value for the sedentary group is the median value for the exercised group (i.e., 5 for young and 3 for old mice). Furthermore, the increase factor between the average number of clones per myofiber in exercised and sedentary mice is conserved between the young and old group (1.31 and 1.27, respectively), as observed with the ratios of average number of satellite cells (Fig. 1).

Fig. 2.

Fig. 2

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Quantification of myogenic clones derived from individual EDL myofibers of young and old mice. (A) Summary of the number of myofibers and clones analyzed for the current figure. Clones were prepared from myofibers isolated from mice detailed in Fig. 1, investigating 3–4 myofibers per mouse and data were then pulled within each group. (B) Myogenic clone data are illustrated in boxplots, depicting quartile distribution, average ± SEM (white marks) and outliers (red circles) for the number of clones per myofiber, as detailed in Fig. 1B. Right Y-axis provides the scale of outlier data points. Outliers identified in the young/sedentary group were contributed by two mice. (C) Myofiber distribution according to the number of myogenic clones per each myofiber and cumulative curves. Myofibers are arranged in ascending order according to the number of myogenic clones per myofiber (X-axis) versus the number of myofibers containing a given number of myogenic clones (Y-axis). In the cumulative curves, each data point (diamond-shaped, right Y-axis) shows the percentage of myofibers out of total myofibers analyzed that contain less than, or equal to (≤) the corresponding number of myogenic clones per myofiber that is indicated on the X-axis. (D–G) Phases images of myogenic clones developed from individual myofibers isolated from exercised young (D and E) and old (F and G) mice. Images were taken at 2-weeks and depict for each age group representative lower (D and F) and higher (E and G) density clones (10x objective).

At the morphological level, there was no apparent difference between the myogenic clones from the four groups analyzed. Noticeably, myofibers from both young and old exercised mice produced typical large myogenic clones depicting lower and higher cell densities (Fig. 2D–G) similar to the sedentary groups and in accordance with previous observations [10]. Hence, exercise-induced satellite cell expansion in vivo (Fig. 1) does not seem to be exhausting the post-exercise proliferative potential of satellite cells. Occasional non-myogenic clones were observed arising from a small number of the myofibers processed for satellite cell clonal analysis. Such non-myogenic clones (typically no more than 1 per myofiber) amounted to 2.3% and 2.9% of total clones for no-run and run groups, respectively in the young mice, and 14.8% and 8.8% for no-run and run groups, respectively in the old mice. These clones, which were void of myotubes, were comprised of distinctive large and flat cells negative for myogenic markers such as MyoD and sarcomeric myosin when analyzed by immunostaining. These non-myogenic clones have likely been derived from interstitial cells that are occasionally co-isolated with myofibers and their presence increases with age [10]. Future running studies with stable lineage marking of satellite cells such as MyoDCre-driven reporter expression [67, 68] can further assist in validating the non-satellite cell origin of the non-myogenic clones.

Concluding Remarks

In light of the importance of satellite cells in muscle maintenance and the possibility that reduced satellite cell performance constitutes an underlying factor of sarcopenia, we investigated the effects of aging on the number and function of satellite cells and their progeny in rodents [9–11]. Here, we focused on the individual and combined effects of exercise running and age on mouse satellite cells. The specific running approach applied in the present study (i.e. long-term moderate-intensity) has been considered not to inflict apparent muscle damage and even be beneficial in certain myopathies [37–40]. The positive effects of endurance exercise on satellite cell numbers parallel our previous work where we inspected the gastrocnemius muscle of young and old rats [11], attesting for the general effect of moderate-intensity treadmill running on satellite cell numbers, irrespective of the animal species or muscle type.

The current study raises a number of questions worthy of future investigation. Do the satellite cells proliferate throughout the running period, or only during the initial days? Do the satellite cells or their progeny fuse with myofibers during running? Is it possible that the expansion of the satellite cell pool in exercised animals is due to Do some of the satellite cells observed at the end of the 8-week period represent the original satellite cells or do all cells go through at least one proliferative round giving rise to differentiated progeny and replenishing also the reserve pool of satellite cells? Is the proliferative potential of single satellite cells enhanced/reduced after the 8-week running? Our hypothesis is that there is no decline in satellite cell proliferative potential as a result of the running, a proposition supported by the clonal performance of the satellite cells and the large number of cells that developed in the myogenic clones from myofibers of exercised mice, regardless of age.

Our recent studies where we monitored the number of cells in day 10 myogenic clones, demonstrated an average of ~2100 (±260 SEM, n=54) cells per clone when satellite cells were isolated from old mice, similar to what we measured for young mice (Stuelsatz P, Shearer A, Yablonka-Reuveni Z, unpublished). However, the production of satellite cell-like, reserve cells (Pax7+/MyoD−/NES-GFP+) declined in clones from old age [10]. Hence, it would be of interest to investigate satellite cell dynamics in a paradigm of a long running period extending the duration of the exercise routine of the young group all the way to the age of the old group, and sampling myofibers and muscle sections for satellite cell numbers and expression of satellite cell proliferation/differentiation distinctive markers (i.e., Pax7, MyoD, myogenin, Ki67) at different time points throughout this extended running period. We hypothesize that such long-term running may support maintenance of satellite cell numbers in the old age group at a similar level to that of the young group, following the ‘use it or lose it’ concept [69, 70].

In particular, the use of the NES-GFP transgenic mouse to monitor satellite cells has introduced additional aspects that could be pursued in the future. Typically, when we have followed proliferation of NES-GFP+ satellite cells in isolated myofibers or primary cultures, the GFP signal was still evident, albeit at lower intensity, in proliferating (BrdU+) progeny by 3rd culture day, but diminished on day 4 [62]. While most proliferating cells (Pax7+/MyoD+) transited into the differentiating, myogenin+ state, some cells entered the renewal state (Pax7+/MyoD−), which also entailed cell cycle withdrawal and re-appearance of NES-GFP signal [10, 62]. At the conclusion of 8-week running essentially all satellite cells were positive for NES- GFP. Hence, satellite cells either underwent only a minimal number of proliferative rounds during the running period (and therefore, retained a detectable GFP level), or some of the GFP+ cells were renewal cells that re-expressed NES-GFP, or possibly GFP signal was not lost when satellite cells remained at the fiber niche, even if the cells might have proliferated multiple times.

Myofiber damage has been recognized as a trigger of satellite cell activation, leading to myonuclear fortification or formation of new myofibers. However, the current moderate-intensity treadmill running is considered not to induce muscle damage. Thus, it is important to determine in future studies whether the increased satellite cell pool detected herein does involve fortification of myofiber nuclei. It is attractive to consider the possibility that this satellite cell expansion may also play a “non-classical” role in muscle homeostasis, independent of myofiber repair. For example, the described cross talk between satellite cells and the microvasculature [71], could be in further demand during chronic treadmill exercise. It is also of interest to consider what might be the key factors involved in satellite cell pool expansion during moderate-intensity treadmill running, assuming that routine tissue removal and repair mechanism following damage are not occurring. The production of myokines (i.e., cytokines and growth factor produced by the muscle itself) appears to be enhanced following physical activity [72]. For example, IL-6 is produced by muscle cells during exercise, and considering its positively affects satellite cell proliferation, could represent a regulator of the exercise-induced satellite cell expansion observed in the current study [73, 74]. Other candidate myokines that could be involved in exercise-induced satellite cell expansion might include hepatocyte growth factor (HGF) and some of the fibroblast growth factors (FGFs) [75–77]. These factors are not only present in skeletal muscle, but promote satellite cell activation and expansion at the myofiber niche as shown in culture conditions where the satellite cells were retained by the parent myofiber [9, 28, 78–80].

In all, we established an experimental paradigm optimal for investigating the regulation of satellite cell performance in vivo in the context of muscle maintenance through life. This moderate-intensity endurance exercise approach can be used with Cre-loxP systems of knockout and overexpressing mice to study the impact of physiological relevance factors on satellite cell dynamics through life.

Materials and Methods

Animals

Transgenic male mice expressing GFP under nestin promoter (NES-GFP, heterozygous, C57Bl/6 strain background) [10, 62, 81] were used for experimental and control run/no-run groups. Animal procedures were conducted according to Tel Aviv University Institutional Animal Care and Use Committee (permit number M-06-095 and M-06-014).

Treadmill running procedure

Moderate-intensity treadmill running was performed using Horizon ID 100 treadmill custom adjusted for rodent experiments (running area= 41 × 114 cm) as previously described [11]. For the present mouse study, treadmill speed was adjusted to 0.69 kph (11.5 meters per minute) and mice ran for 30 minutes a day, 6 times a week, for 8 weeks. The age of young and old mice as specified throughout this study is the age at the beginning of the 8-week run/no-run period.

Treadmill acclimation conditions prior to the 8-week experimental period lasted about 2 weeks. At first, all mice were subjected to two 15-minute sessions (1 a day on consecutive days) in which the treadmill was turned on, while mice were kept in their cages, this was done in order to familiarize mice to the treadmill noise. For the next 3 days mice assigned to the exercise group were placed in the treadmill for 15–20 minutes without turning the treadmill on. Finally, mice assigned to the exercise group were allowed to run for 3 minutes for 2 days and then 1 or 2 minutes/day were added to the running period until all mice were able to run 30 minutes straight.

Satellite cell quantification in isolated myofibers

Satellite cells were quantified based on the number of NES-GFP+ cells in individual myofibers isolated from the hindlimb EDL muscles. As we previously demonstrated, there is 95–100% agreement when monitoring satellite cells by NES-GFP in live or fixed myofibers versus Pax7 immunostaining using fixed myofibers [10, 62]. Each mouse was processed separately for myofiber isolation and satellite cell quantification using our standard protocols for EDL myofiber isolation from young and old mice. Myofibers were released by repetitive gentle trituration of the EDLs after digestion with 0.2% collagenase type I (Sigma-Aldrich, St. Louis, Missouri) for 90–120 minutes [9, 82] and the released myofibers were rinsed extensively as detailed in our recent protocol updates to eliminate residual interstitial cells released during the procedure [10, 83]. GFP+ satellite cells were counted on live myofibers using fluorescent microscope [10]. Per each group, data from individual mice were pooled after establishing a common satellite cell distribution pattern across mice using ANOVA.

Satellite cell clonal analysis

Cloning of satellite cells from individual EDL myofibers of NES-GFP mice was performed following our published procedure [9]. The growth medium consisted of DMEM (high glucose, with l-glutamine, 110 mg/L sodium pyruvate, and pyridoxine hydrochloride supplemented with 50 U/ml penicillin and 50 μg/ml streptomycin (Invitrogen) supplemented with 10% horse serum, 20% fetal bovine serum and 1% chicken embryo extract (Biological Industries, Beit Haemek, Israel). Individual myofibers were transferred into 1.2 ml of warm growth medium in individual tube and then triturated to strip satellite cells from the parent myofiber. Each triturated suspension was dispensed in 100-μl aliquots into 12 Matrigel-coated wells in 24-well tissue culture trays. Trays were left undisturbed for 4 days to allow cell adherence and initiation of clonal growth. Each well was then fortified with 250 μl of fresh growth medium followed by replacement of the culture medium with fresh 500 μl growth medium every other day, starting on culture day 5. Clonal growth was first analyzed on culture days 4–5 and all wells were monitored for clonal growth for a total of 14 days. Myogenic clones were identified morphologically by the presence of myotubes, typically found by 7–10 days in culture. Some clones were defined as non-myogenic by the absence of myotubes and the presence of distinctive large and flat morphology of the cells. These non-myogenic clones were negative for myogenic markers such as MyoD and sarcomeric myosin when analyzed by immunostaining.

Microscopy

Observations were done with an inverted fluorescent microscope (Zeiss, Axiovert 200M), controlled by Axiovision 4.4 Imaging System. Images were acquired with an Axiocam MRm monochrome CCD camera and composites of digitized images were assembled using Adobe Photoshop software.

Statistics

Statistical analyses of satellite cell numbers per myofiber were performed using Statistica 9 software. Analysis of variance was tested with the parametric MANOVA (multiple analyses of variance) test. When significant differences were found they were followed by post-hoc Fisher LSD test for comparisons. For all tests, P values less than 0.05 were considered significant. Descriptive statistics of the data are depicted in boxplots. Data are organized in quartiles with outlier (defined in the legend to Fig. 1) depicted in circles

Acknowledgments

This study was supported by grants from the US-Israel Binational Science Foundation (BSF, 2005132) to DB and ZYR, and from the National Institute on Aging (AG021566) to ZYR.

Abbreviations

NES-GFP

Nestin-GFP

EDL

extensor digitorum longus

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