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. Author manuscript; available in PMC: 2016 Dec 23.
Published in final edited form as: Cell Rep. 2016 Nov 22;17(9):2340–2353. doi: 10.1016/j.celrep.2016.11.002

Conditional Loss of Pten in Myogenic Progenitors Leads to Postnatal Skeletal Muscle Hypertrophy But Age-dependent Exhaustion of Satellite Cells

Feng Yue 1, Pengpeng Bi 1, Chao Wang 1, Jie Li 2, Xiaoqi Liu 2,3, Shihuan Kuang 1,3,4,*
PMCID: PMC5181649  NIHMSID: NIHMS828670  PMID: 27880908

SUMMARY

Skeletal muscle stem cells (satellite cells, SCs) are normally maintained in a quiescent (G0) state. Muscle injury not only activates SCs locally, but also alerts SCs in distant uninjured muscles via circulating factors. The resulting GAlert SCs are adapted to regenerative cues and regenerate injured muscles more efficiently, but whether they provide any long-term benefits to SCs is unknown. Here we report that embryonic myogenic progenitors lacking Pten exhibit enhanced proliferation and differentiation, resulting in muscle hypertrophy but fewer SCs in adult muscles. Interestingly, Pten-null SCs are predominantly in the GAlert state even in the absence of injury. The GAlert SCs are deficient in self-renewal and subjected to accelerated depletion during regeneration and aging, and fail to repair muscle injury in old mice. Our findings demonstrate a key requirement of Pten in G0-entry of SCs, and provide functional evidence that prolonged GAlert leads to stem cell depletion and regenerative failure.

Keywords: Pten, skeletal muscle, hypertrophy, stem cells, regeneration, aging

Graphical Abstract

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eTOC Blurb

Yue et al. find that Pten is required for muscle stem cell (satellite cell) homeostasis. Pten deletion in embryonic myogenic progenitors leads to reduction of postnatal satellite cells that are in an “alert” state. These GAlert satellite cells are defective in self-renewal and depleted with age, leading to regenerative failure.

INTRODUCTION

Skeletal muscles are mainly composed of multinucleated muscle cells called myofibers. Generation of myofibers occurs during embryonic and fetal myogenesis through fusion of myogenic progenitors (myoblasts) that express Pax3/7 and MyoD (Gros et al., 2005; Relaix et al., 2005). Meanwhile, a subpopulation of myoblasts is deposited along the newly formed myofibers. These cells lose the expression of MyoD and Pax3 and become Pax7+ quiescent satellite cells (SCs), the resident muscle stem cells (Tajbakhsh, 2009). By perinatal stage, the number of myofibers is determined and remains constant, but the size of myofibers grows robustly by addition of new myonuclei supplied from satellite cells (White et al., 2010; Yin et al., 2013).

Adult resting skeletal muscle is relatively stable but possesses a remarkable ability to regenerate after injury (Brack and Rando, 2012; Cheung and Rando, 2013; Yin et al., 2013). Muscle regeneration is a highly orchestrated process involving crosstalk between tissues, cells and molecules, but satellite cells are absolutely required for skeletal muscle regeneration (Lepper et al., 2011; Murphy et al., 2011; Sambasivan et al., 2011). In adult resting muscle, satellite cells are retained in a quiescent state, characterized by the expression of Pax7 (Kuang and Rudnicki, 2008). Upon muscle injury, quiescent satellite cells are activated and re-express MyoD, subsequently enter the cell cycle and proliferate (Kuang and Rudnicki, 2008). The proliferating satellite cells differentiate and fuse into multinucleated myofibers to repair the damaged muscles, or self-renew to replenish the stem cell pool (Brack and Rando, 2012; Kuang and Rudnicki, 2008). During aging, the regenerative capacity of skeletal muscle declines due to functional declination of systemic environment and satellite cells (Blau et al., 2015). Many intrinsic and extrinsic signals have been shown to govern the function of satellite cells, including Notch, Wnt, p38/MAPK, JNK/STAT, FGF and many others (Almada and Wagers, 2016; Blau et al., 2015; Brack and Muñoz-Cánoves, 2016; Dumont et al., 2015).

Reversible transition of satellite cells between quiescent and activated state is crucial for stem cell maintenance (Cheung and Rando, 2013). Deregulation of this process could result in abnormal growth of myogenic progenitors or depletion of satellite cells (Almada and Wagers, 2016; Cheung and Rando, 2013). Intriguingly, a recent study reports that quiescent satellite cells can be divided into reversible G0 and GAlert phases (Rodgers et al., 2014). In response to circulatory cues generated by injury, G0 satellite cells even in a remote uninjured muscle transit into an alerted “GAlert” state. Compared to G0 satellite cells, the GAlert satellite cells have higher mitochondrial activity and, when activated, enter the cell cycle more quickly and regenerate injured muscles more efficiently (Rodgers et al., 2014). Mechanistically, the cycling and metabolic features of the GAlert state is dependent on mTORC1 downstream of the PI3K signaling pathway (Rodgers et al., 2014). Thus, the G0 to GAlert switch primes satellite cells to enter an adaptive stage, allowing them to respond better to future injuries. However, it is unknown what signal regulates the opposite GAlert to G0 switch and if the GAlert state provides any long-term benefits to stem cell maintenance and tissue homeostasis.

In the present study, using a conditional knockout approach, we specifically deleted the phosphatase and tensin homologue (Pten) in activated myoblasts and their descendants driven by the MyoDCre allele. As Pten is a negative regulator of PI3K, we hypothesized that Pten deletion should lead to activation of the downstream Akt–mTORC1 signaling, and influence the differentiation and bidirectional switch between activation and quiescence of myoblasts. We found that MyoDCre–driven deletion of Pten resulted in a spectrum of phenotypes in muscle tissue mass and satellite cell behavior, thus establishing a key role of Pten in regulating muscle stem cell homeostasis.

RESULTS

MyoDCre–Driven Deletion of Pten in Mice Results in Postnatal Muscle Hypertrophy

We established the MyoDCre::Ptenf/f (PtenMKO) mouse model to delete Pten specifically in MyoD–expressing embryonic myoblasts and their descendent satellite cells and myofibers. The PtenMKO mice were born at normal Mendalian ratios with normal morphology and body weight. However, the PtenMKO mice outgrew their littermate WT mice during postnatal growth, resulting in heavier body weights and larger body size starting from 10-week-old (Figures S1A–S1C). By contrast, heterozygous MyoDCre::Pten+/f (PtenM+/−) mice were indistinguishable from the WT mice (Figures S1B and S1C).

To determine if the increased body weight is due to increases in muscle mass, we examined various skeletal muscles. The tibialis anterior (TA), digital extensor longus (EDL), soleus (Sol) and gastrocnemius (Gas) muscles of adult PtenMKO mice were larger and heavier than those of age-matched WT and PtenM+/− mice (Figures 1A–1C, S1D and S1E). The increases in muscle size and weight in PtenMKO mice were also apparent in juvenile mice at P7 (Figures S1F and S1G), before manifestation of a significant increase in body weight (Figure S1A). Histologically, PtenMKO myofibers appeared larger in TA, EDL and Sol cross sections (Figure 1D and S1H), and the cross-sectional area (CSA) distribution curve of PtenMKO Sol myofibers showed a right-shift when overlaid to that of the WT mice (Figure 1E), indicating larger myofiber size. In addition, PtenMKO mice had 15% and 10% more myofibers than WT control mice in TA and EDL muscles, respectively (Figure 1F). Moreover, PtenMKO EDL myofibers contained ~30% more myonuclei/myofiber than did the WT and PtenM+/− myofibers (Figure 1G and S1I). Taken together, these results indicate that Pten loss in embryonic myoblasts leads to increases in skeletal muscle mass due to myofiber hypertrophy (increases in size and myonuclei number per myofiber) and hyperplasia (increases in myofiber numbers).

Figure 1. Pten Deletion in Myogenic Progenitors Leads to Postnatal Muscle Hypertrophy.

Figure 1

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(A–B) Representative image of whole hind limb (A) and TA, Sol and Gas muscles (B) in adult WT and PtenMKO mice.

(C) Muscle weight in adult WT and PtenMKO mice (n=7).

(D) Dystrophin staining showing relative size of myofibers in muscle cross sections.

(E) Frequency of distribution for cross-section area (CSA, µm2) of Sol muscle (n=4).

(F) Total myofiber number of TA, EDL and Sol muscles (n=4–8).

(G) Immunofluorescence of DAPI and myonucleus number count in fresh myofibers. (n=4 mice, 20 myofibers per mouse).

Scale bar, 50 µm. Data are shown as mean ± SEM (t-test: *P<0.05; **P<0.01; ***P<0.001).

Also see Figure S1.

PtenMKO Mice Have Improved Skeletal Muscle Function and Are Protected from Denervation-induced Muscle Atrophy

To explore if muscle hypertrophy is associated with functional improvements in the PtenMKO mice, we first examined their exercise performance on treadmill. Both male and female PtenMKO mice outperformed the sex-matched WT littermates in maximum speed, running time and running distance (Figures 2A–2C). We also investigated the retention of muscle mass after denervation, and found that denervation-induced muscle loss was reduced in PtenMKO mice compared to WT control (Figure 2D). At 21-day after denervation, the weights of TA and Gas muscles were reduced by ~50% in control mice, but ~ 40% in PtenMKO mice (Figure 2E). The denervated myofibers were also larger in the PtenMKO mice than in WT mice (Figures 2F and 2G). Importantly, the preservation index (size ratio of denervated to control muscles) in PtenMKO mice was significant higher than that of WT mice (Figure 2H). Thus, loss of Pten improves skeletal muscle function and alleviates denervation-induced atrophy.

Figure 2. Loss of Pten Improves Skeletal Muscle Function and Protects Muscle from Denervation-induced Atrophy.

Figure 2

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(A–C) Maximum speed (A), total running time (B) and running distance (C) of adult WT and PtenMKO female and male mice measured by treadmill (n=3).

(D–E) Representative image (D) and percentage of muscle preservation (E) of TA and Gas muscles 21 days post denervation (n=6).

(F–G) H&E (F) and Immunofluorescence (G) staining of TA muscles 21 days post denervation.

(H) Ratio of myofiber size (denervation/control) 21 days post denervation (n=5).

Scale bar, 50 µm. Data are shown as mean ± SEM (t-test: *P<0.05; **P<0.01; ***P<0.001).

Loss of Pten Accelerates Proliferation and Differentiation of Satellite Cells during Perinatal Muscle Growth

During perinatal development, myofibers grow via nuclei accretion from satellite cells (White et al., 2010; Yin et al., 2013). The finding of increased myonuclei in PtenMKO mice prompted us to hypothesize that Pten deletion promotes the proliferation and differentiation of satellite cells during perinatal muscle growth. To test this, we first examined the abundance of satellite cells in hindlimb muscles of newborn mice (P1) by immunostaining of Pax7. Indeed, we detected more Pax7+ cells per unit area in TA muscles of the PtenMKO mice (Figure 3A), with a 51% increase over the WT control (Figure 3B). The number of Pax7+Ki67+ cells in PtenMKO muscles was doubled, comparing to that of WT control (Figures 3A and 3C), indicating that Pten deletion accelerates the proliferation of satellite cells. Moreover, more MyoG+ cells were observed in muscles of newborn PtenMKO mice (Figure 3D), corresponding to a 68% increase over the WT control (Figure 3E). Consistently, the protein levels of Pax7, MyoG, pAkt and CCND1 were higher in skeletal muscles of P7 PtenMKO mice than those of WT mice (Figure 3F). These results reveal that Pten deletion induces the postnatal skeletal muscle hypertrophy through promoting proliferation and differentiation of satellite cells during perinatal growth.

Figure 3. Pten Deletion Promotes Satellite Cell Proliferation and Differentiation during Perinatal Muscle Growth.

Figure 3

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(A) Immunofluorescence of Pax7 and Ki67 in cross-sections of hind limb muscles from newborn (P1) WT and PtenMKO mice.

(B–C) Quantification of Pax7+ cell (B) and Pax7+Ki67+ cell (proliferating, C) number in cross-sections of hind limb muscles (n=3).

(D) Immunofluorescence of MyoG and dystrophin in cross-sections of hind limb muscles.

(E) Quantification of MyoG+ cell number in cross-sections of hind limb muscles (n=3).

(F) Western blot analysis of myogenic and proliferating markers in skeletal muscles from P7 mice. Arrow indicates the right band.

Scale bar, 50 µm. Data are shown as mean ± SEM (t-test: *P<0.05; **P<0.01).

Also see Figure S2.

Pten Negatively Regulates Proliferation and Differentiation of Primary Myoblasts in vitro

To confirm the observations in vivo, we investigated the function of Pten in satellite cell-derived primary myoblasts through loss-of-function and gain-of-function approaches. The Pten-deficient primary myoblasts grew significant faster than control myoblasts (Figure S2A). Single cell colony formation assay showed that the colony size of Pten-null primary myoblasts was larger than the control colonies (Figure S2B). The mRNA levels of mitotic related genes CDK2, CCND1 and CCND2 were significantly higher in Pten-null compared to WT myoblasts (Figure S2C). The protein levels of pAkt, pS6 and CCND1 were also increased in primary myoblasts after Pten deletion (Figure S2D). However, the protein levels of Pax7, MyoD and MyoG were comparable in control and Pten KO myoblasts (Figure S2D).

In contrast to Pten-deficient myoblasts, Pten-overexpressing myoblasts exhibited significantly slower growth kinetics that did the control myoblasts infected with Ad-GFP (Figure S2E). Immunostaining confirmed that Pten-overexpressing myoblasts were mostly Ki67−, resulting in a much lower percentage of Ki67− myoblasts compared to control myoblasts (Figure S2F). Moreover, the mRNA levels of MyoD, MyoG and CCND1 were significantly down-regulated by Pten overexpression (Figure S2G), as well as the protein level of MyoG (Figure S2H). Importantly, Pten-overexpressing myoblasts failed to differentiate into myotubes while the Ad-GFP infected control myoblasts differentiated efficiently (Figure S2I). Together, these results demonstrate that Pten negatively regulates the proliferation and differentiation of primary myoblasts.

Satellite Cells in Adult PtenMKO Mice Are Predominantly at the GAlert State

As satellite cells are derived from embryonic MyoD-expressing myoblasts (Kanisicak et al., 2009), we next examined how Pten KO affects satellite cells in adult PtenMKO mice. To determine Pten KO efficiency, we double labeled satellite cells with Pax7 and Pten (Figure 4A). All (100%) Pax7+ satellite cells were Pten+ in WT muscles, but only less than 6% of Pax7+ satellite cells were Pten+ in PtenMKO muscles (Figure 4A), suggesting Pten deletion in >94% of adult satellite cells. Strikingly, the number of Pax7+ satellite cells in adult PtenMKO mice was only ~60% of that in WT mice (Figure 4B). FACS analysis of satellite cells isolated from quadriceps muscles confirmed that both the number (5188/muscle vs 6822/muscle) and percentage (4.3% vs 7.9%) of Pax7+ satellite cells were decreased in adult PtenMKO mice when compared to WT mice (Figure S3A and 3B).

Figure 4. Quiescent Satellite Cells Exist in an “Alert” (GAlert) State in Adult PtenMKO Mice.

Figure 4

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(A) Immunofluorescence of Pax7 and Pten, and percentage of Pten+ satellite cells on fresh myofibers of adult WT and PtenMKO mice (n=4 mice, 20 myofibers per mouse).

(B) Quantification of satellite cell number on fresh myofibers (n=7 mice, 20 myofibers per mouse).

(C) Cell cycle entry of satellite cells analyzed by EdU incorporation in cultured myofibers (n=3 mice, average 100 satellite cells were count per mouse).

(D) Immunofluorescence of Pax7 and EdU, and percentage of proliferating satellite cells in myofibers cultured for 40 h (n=3 mice, 15 myofibers per mouse).

(E) Immunofluorescence of pAkt in Pax7+ satellite cells, and percentages of pAktNeg, pAktLow and pAktHigh satellite cells on fresh myofibers (n=3 mice, 20 myofibers per mouse).

(F) Immunofluorescence of pS6 in Pax7+ satellite cells, and percentages of pS6Neg, pS6Low and pS6High satellite cells on fresh myofibers (n=3 mice, 20 myofibers per mouse).

Scale bar, 20 µm. Data are shown as mean ± SEM (t-test: *P<0.05; **P<0.01; ***P<0.001).

Also see Figure S3.

The reduction in satellite cell numbers in the PtenMKO mice suggests that Pten-null embryonic myoblasts are defective in forming quiescent satellite cells, or the Pten-null satellite cells cannot maintain proper quiescence. To distinguish these possibilities, we investigated the cell cycle status of satellite cells in adult PtenMKO mice. By immunostaining of MyoD (marker of activated satellite cells) and MyoG (marker of differentiating myoblasts) in freshly isolated myofibers, we found that there were essentially no MyoD+ or MyoG+ cells in both WT and PtenMKO mice at 2.5 and 8 months old (Figures S3C and S3D). Consistently, immunostaining of proliferating marker Ki67 confirmed that no satellite cells in PtenMKO and WT mice were Ki67+ (Figure S3E). These results suggest that the Pten-deficient satellite cells in the PtenMKO mice are maintained in a quiescent state.

It has been shown recently that quiescent satellite cells exist at two functional distinct phases: G0 and GAlert (Rodgers et al., 2014). One feature of GAlert satellite cells is their accelerated activation and proliferation kinetics. Based on EdU incorporation in myofibers cultured ex vivo, we found that PtenMKO satellite cells displayed accelerated cell cycle entry compared to WT satellite cells (Figure 4C). Specifically, > 40% of Pten-deficient satellite cells had incorporated EdU (EdU+) within 20 h, comparing to < 20% of EdU+ satellite cells in WT mice at the same time (Figure 4C). At the clonal level, most Pten-deficient satellite cells had already divided 1–2 times in 40 h, forming 2–4 cell clusters, whereas the WT satellite cells had only divided 0–1 times at the same time (Figure 4D). Another feature of GAlert satellite cells are their metabolic activation. Using MitoTracker staining as readout of mitochondria activity, we found that Pten-deficient quiescent satellite cells and primary myoblasts exhibited much more intensive MitoTracker labeling than did the WT cells (Figures S3F and S3G). These results suggest that Pten-null satellite cells are at the GAlert state.

Activation of mTORC1 signaling has been shown to be necessary and sufficient to mediate the G0 to GAlert switch (Rodgers et al., 2014). To further confirm the GAlert state of Pten-deficient satellite cells, we performed co-immunostaining of Pax7 with pAkt and pS6 downstream of mTORC1. Most quiescent satellite cells in WT muscles were negative for pAkt labeling (pAktNeg), whereas the Pten-null satellite cells predominantly pAkt+ with low to high labeling intensity (Figure 4E). More intensive pS6 signals were also observed in the Pten-null compared to WT satellite cells (Figure 4F). Quantitatively, percentages of pAktNeg and pS6Neg satellite cells were remarkably decreased, but percentages of pAktHi and pS6Hi satellite cells were dramatically increased, in PtenMKO mice (Figures 4E and 4F), suggesting activation of mTORC1 pathway in Pten-deficient satellite cells. Together, these results indicate that Pten deletion in embryonic myoblasts renders satellite cell at the GAlert state.

GAlert Satellite Cells Efficiently Regenerate Injury Muscles in Young Mice at the Expense of Self-Renewal

We next asked if the reduced number of GAlert satellite cells in PtenMKO mice could maintain muscle regeneration after CTX-induced muscle injury (Figure 5A). Strikingly, no significant differences were found between WT and PtenMKO mice in gross morphology and muscle weight recovery 7 days after injury (Figures 5B and 5C). In addition, cross sections of both WT and PtenMKO muscles displayed similar levels of inflammatory infiltration, regeneration (central nuclear myofibers) and dystrophin expression (Figures 5D and 5E). At 21 days after injury, regenerated myofibers in PtenMKO mice remained larger than those of WT mice (Figures 5D and 5E). These results suggest that the reduce numbers of GAlert satellite cells in adult PtenMKO mice are sufficient to maintain muscle regeneration after acute injury.

Figure 5. GAlert Satellite Cells Are Able to Maintain Muscle Regeneration at the Expense of Self-Renewal in Young PtenMKO Mice.

Figure 5

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(A) Schematic outline of CTX injection and sample collection.

(B–C) Representative images (B) and recovery rate (C) of TA muscles in young WT and PtenMKO mice at 7 and 21 days after injury (n=3).

(D) H&E staining of TA muscle cross-sections.

(E) Immunofluorescence of dystrophin in TA cross-sections.

(F) Immunofluorescence of Pax7 and dystrophin, and satellite cell number per area of TA crosssections (n=3–6).

Scale bar, 50 µm. Data are shown as mean ± SEM (t-test: *P<0.05; **P<0.01; ***P<0.001; NS: no significant difference).

Also see Figures S4 and S5.

We also examined the dynamics of the GAlert satellite cells during muscle regeneration. Prior to muscle injury (Day 0), the number of Pax7+ satellite cells in PtenMKO mice was only ~50% of that in WT mice, but there were significantly more satellite cells in PtenMKO than in WT muscles at Day 7 after injury (Figure 5F). This result suggests that the PtenMKO satellite cells responded more quickly to injury and proliferate faster than do the WT satellite cells. At Day 17 and 21 when muscle regeneration gradually finishes, proliferating satellite cells exit the cell cycle and return to quiescence (Yin et al., 2013). Intriguingly, significantly fewer satellite cells were found in PtenMKO than in WT muscles at Day 17 and 21 after injury, with only 14% of that in WT muscles at Day 21 (Figure 5F). This observation indicates that initial faster proliferation of the GAlert satellite cells is associated with reduced self-renewal, and therefore exacerbating satellite cell loss in PtenMKO mice after regeneration.

To exclude the effect of hypertrophic myofiber-niche on the response of satellite cells to injury, we generated Pax7CreER/Ptenf/f mice to achieve acute deletion of Pten in adult satellite cells. At 12 days after tamoxifen-induced deletion of Pten, the number of satellite cells in TA muscles was decreased by 58%, (Figures S4A and S4B), suggesting that Pten is required for maintaining quiescent satellite cells in resting muscles. We also examined satellite cell self-renew and muscle repair during injury (Figure S4C). Similar to what we found in the PtenMKO mice, TA muscles of Pax7CreER/Ptenf/f mice regenerated normally despite the nearly 60% loss of satellite cells prior to the injury (Figures S4D and S4E). Notably, a more pronounced reduction of satellite cells in injured TA muscles of Pax7CreER/Ptenf/f mice was observed 7 days after CTX injection (Figure S4F), a pattern similar to the loss of satellite cells during muscle regeneration in the PtenMKO mice. These results indicate that Pten is required for the self-renew of activated satellite cells in a cell-autonomous manner independent of the hypertrophic myofiber-niche.

Disruption of Pten Affects the Reversible Quiescence of Satellite Cell Progenitors

A failure of the Pten-null satellite cells to replenish their pool after muscle regeneration might be due to programmed cell death. To test this possibility, we used terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay to detect apoptotic cells. TUNEL+ cells were observed in injured TA muscles in both WT and PtenMKO mice (Figure S5A). However, none of them were Pax7+ in WT (n=245 cells) and PtenMKO (n=298 cells) mice (Figure S5A). Consistently, no expression of active caspase3 was found in Pax7+ cells from WT (n=95 cells) and PtenMKO (n=82 cells) mice (Figure S5B). These results demonstrated that the depletion of satellite cells after regeneration was not due to cell apoptosis.

Alternatively, Pten might be required for activated satellite cells to return to quiescence and the lack of Pten might have blocked their re-entry into the quiescent state. To examine this possibility, we analyzed the number of Pax7+MyoD− quiescent satellite cells in TA muscles 10 days post muscle injury. As expected, the number of quiescent satellite cells was significantly reduced in the PtenMKO mice compared to WT control, with a concomitant increase in the number of Pax7+MyoD+ activated myoblasts (Figure 6A). To confirm the in vivo results, we next performed ‘reserve’ cell analysis in culture using low passage primary myoblasts from Pax7CreER/Ptenf/f mice (Shea et al., 2010). Compared to the control media treatment, 4-OH-Tamoxifen induced Pten KO significantly reduced the percentage of Pax7+MyoD–Ki67− reserved cells (Figure 6B), and increased the percentages of MyoG+ cells and nuclei in MF20+ myotubes (Figures 6C and 6D). In addition, the expression of differentiation-related genes MyoG, MYH8, Cadh15 and FNDC5 was up-regulated after Pten deletion (Figure 6E). These observations demonstrate that lack of Pten blocks the re-entry of activated satellite cells into quiescence and promotes their terminal differentiation.

Figure 6. Pten KO Affects the Reversible Quiescence of Satellite Cell Progenitors.

Figure 6

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(A) Immunofluorescence of Pax7 and MyoD, and percentage of Pax7+MyoD− and Pax7+MyoD+ SCs in TA muscles of adult WT and PtenMKO mice after 10 days of CTX injury (n=3 mice).

(B) Immunofluorescence of Pax7, MyoD and Ki67, and percentage of Pax7+MyoD−Ki67− cells in cultures of control and Pten KO primary myoblasts after 2.5 days differentiation to show reserved quiescent Pax7+ cells (n=6).

(C) Immunofluorescence of MyoG and MF20, and percentage of MyoG+ nuclei in cultures (n=4).

(D) Quantification of the percentage of nuclei in myotube and nuclei in MF20− cells in cultures (n=4).

(E) RT-PCR analysis of gene expression in cultures (n=3).

Scale bar, 50 µm. Data are shown as mean ± SEM (t-test: *P<0.05; **P<0.01; ***P<0.001).

Also see Figure S6.

We further asked whether the failure of cycling Pten-null satellite cell progenitors to re-enter quiescence is due to the activation of Akt/mTOR signaling. To test this, we treated Pten KO and control myoblasts with LY294002 (a potent PI3K inhibitor) or rapamycin (a specific inhibitor of mTOR), in the low serum ‘reserve’ cell culture condition. LY294002 and rapamycin treatment inhibited the differentiation and fusion of both control and Pten KO myoblasts (Figure S6A). Notably, LY294002 treatment increased the percentages of reserve cells (Pax7+MyoD− Ki67−) in both control and Pten KO group, and rescued the defect of quiescence re-entry of Pten KO myoblasts (Figure S6B). Rapamycin similarly normalized the percentage of reserve cells in Pten KO cells to a level comparable to the control, though it decreased the overall percentage of reserve cells in both control and Pten KO myoblasts (Figure S6B). These observations suggest that inhibition of Akt/mTOR pathway rescues the deficiency of Pten-null myoblasts to re-enter quiescence. Collectively, these results indicate that Pten is required for the reversible quiescence of satellite cells.

Accelerated Depletion of GAlert Satellite Cells during Aging Leads to Regenerative Failure

During aging, the number and function of satellite cells declines (Chakkalakal et al., 2012; Collins et al., 2007; Shefer et al., 2006). The observation that accelerated depletion of GAlert satellite cells during muscle regeneration in young mice prompted us to ask whether the GAlert satellite cells in PtenMKO mice could maintain the stem cell pool and regenerative capacity with age. To determine this, we first enumerated the number of satellite cells in resting muscles of 19 months old WT and PtenMKO mice. Consistent with previous reports, decline of satellite cells was observed in aged muscles (~2.5 satellite cells/myofiber) compared to young muscles (~6.2 satellite cells/myofiber) in the WT mice (Figure 7A). Strikingly, there were almost no satellite cells (<1 cell/myofiber) in the 19 months old PtenMKO mice (Figure 7A). Notably, ~25% of the satellite cells observed in the PtenMKO mice were Pten positive (Figures S7A and S7B), suggesting that they were escapers of Pten deletion. Moreover, when cultured ex vivo for 3 days, clusters of proliferating satellite cell were observed on myofibers of WT mice, but rarely on myofibers of PtenMKO mice (Figure 7B). Consistently, dramatically reduced number of satellite cells was observed in TA muscle cross sections (Figure 7C). These results are indicative of accelerated depletion of Pten-deficient satellite cells with aging.

Figure 7. Depletion of GAlert Satellite Cells in Aged Mice Impairs Muscle Regeneration.

Figure 7

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(A) Immunofluorescence of Pax7, and satellite cell number in fresh myofibers isolated from old WT and PtenMKO mice (19 months, n=3).

(B) Immunofluorescence of Pax7 in fresh isolated myofibers cultured for 3 days (n=3).

(C) Immunofluorescence of Pax7 and dystrophin, and satellite cell number per area of TA muscle cross-sections (n=3).

(D) Schematic outline of CTX injection and sample collection.

(E–F) Representative images (E) and recovery rate (F) of TA muscles in old WT and PtenMKO mice 14 days post injury (n=3).

(G–H) H&E staining (G), and cross-section area (CSA) per myofiber (H) of TA muscle cross-sections (n=3).

Scale bar, 50 µm. Data are shown as mean ± SEM (t-test: *P<0.05; **P<0.01).

Also see Figure S7.

We next evaluated the regenerative capacity of old PtenMKO mice after CTX injury (Figure 7D). Even though the non-injured PtenMKO TA muscles remained larger than WT muscles, the regenerated PtenMKO muscles were smaller than the WT muscles (Figure 7E). While TA muscles of WT mice regenerated effectively with ~80% of recovery rate after injury, the PtenMKO muscles only recovered ~56% of their mass (Figure 7F). H&E staining revealed that whereas the WT muscle regenerated efficiently with homogeneous centrally nucleated myofibers, the PtenMKO muscles contained fewer regenerated myofibers and numerous densely packed nuclei indicative of inflammatory infiltration (Figure 7G). Quantitatively, the average sizes of myofibers were significantly reduced in the PtenMKO muscles (Figure 7H). Labeling of myofiber boundary with dystrophin confirms the existence of pockets of dystrophin negative nuclei in the PtenMKO but not WT muscles (Figure S7C). These data indicate that accelerated depletion of Pten-deficient GAlert satellite cells during aging leads to regenerative defects.

DISCUSSION

Satellite cell dysfunction leads to failures of muscle repair, but the molecular mechanisms regulating satellite cell homeostasis remain poorly understood. Using conditional knockout approach, our current study demonstrates that loss of Pten in embryonic myoblasts results in postnatal skeletal muscle hypertrophy, better motor function and resistance to denervation-induced muscle atrophy. However, the Pten KO induced muscle hypertrophy is at the expense of disrupting the homeostasis of satellite cells by rendering them at the GAlert state. This transiently promotes early postnatal muscle growth but accelerates satellite cell exhaustion during aging. These findings point to a novel role of Pten in muscle progenitors, and provide functional evidence that prolonged GAlert leads to stem cell depletion and regenerative failure.

Activation of PI3K/Akt pathway induces skeletal muscle hypertrophy by increasing protein synthesis (Bodine et al., 2001; Lai et al., 2004; Rommel et al., 2001). Thus, inactivation of Pten, which inhibits PI3K, should activate Akt and promotes muscle hypertrophy. However, myofiber-specific Pten KO driven by muscle creatine kinase (MCK)-Cre fails to induce muscle hypertrophy under normal conditions (Hamilton et al., 2010; Wijesekara et al., 2005). By contrast, we show that MyoDCre-driven Pten KO in myoblasts induces robust muscle hypertrophy. These observations suggest that muscle hypertrophy in our PtenMKO mice is mainly due to Pten deficiency in myoblasts but not in myofibers. Surprisingly, Pten KO driven by muscle/brown fat progenitor-specific Myf5Cre fails to elicit any muscle phenotypes (Sanchez-Gurmaches et al., 2012). Conversely, systemic overexpression of Pten increases energy expenditure due to improvements in brown adipose function, but the skeletal muscle phenotype of that mouse model is unclear (Ortega-Molina et al., 2012). Thus, the role of Pten in myogenic progenitors remained unknown. Our results demonstrate that Pten KO accelerates myoblast proliferation and differentiation during postnatal muscle growth, promotes myonuclei accretion and consequently muscle hypertrophy. Consistently, increased proliferation and fusion of satellite cells have been shown to cause adult muscle hypertrophy via increasing the number of myonuclei (Serrano et al., 2008). Our results provide evidence that an adaptive increase of myonuclei number by Pten inactivation may preserve muscle mass under disease conditions such as muscle atrophy.

A recent study reported that stem cell quiescence is composed of two distinct phases, G0 and GAlert (Rodgers et al., 2014). Stem cells undergo dynamic transitions from G0 to GAlert state, in response to injury and stress. We show that Pten-null satellite cells have elevated levels of pAkt and pS6, resembling the adaptive GAlert stem cells. Supporting our results, activation of mTORC1 due to TSC1 KO is sufficient to induce the G0 to GAlert switch in quiescent satellite cells (Rodgers et al., 2014). As MyoDCre induces Pten deletion in cycling (G1/M/G2 phase) embryonic myoblasts prior to the formation of quiescent (G0) satellite cells, our results further indicate that Pten is necessary for G0 entry of activated myoblasts during development. Our study and Rodgers et al (2014) together demonstrate that Akt/mTORC1 pathway is crucial for the bidirectional G0/GAlert switches. Whereas the previous study illustrates the mechanism for G0 to GAlert transition, our study now demonstrates that Pten is necessary for GAlert to G0 re-entry.

Despite the enhanced proliferative and regenerative capacity of GAlert satellite cells, they surprisingly are depleted much more rapidly than G0 satellite cells during regeneration and aging, resulting in regenerative failure in old mice. This observation indicates that GAlert satellite cells have a shorter lifespan. Age-related depletion of stem cell pool mainly results from deregulated stem cell homeostasis due to the loss of self-renewal ability and activation of senescence pathways (Brack and Muñoz-Cánoves, 2016; Chakkalakal et al., 2012; Liu and Rando, 2011; Signer and Morrison, 2013; Sousa-Victor et al., 2015). Indeed, we find that GAlert satellite cells have reduced self-renewal ability. PI3K and its downstream mTORC1 pathways are essential regulators of stem cell aging and mammalian longevity (Johnson et al., 2013; Signer and Morrison, 2013). Hyperactivation of mTORC1 pathway leads to enhanced mitochondrial activity, resulting in the increase of intracellular generation of reactive oxygen species, which cause DNA damage and cell senescence (Chen et al., 2008; Iglesias-Bartolome et al., 2012). Additionally, a recent study demonstrates that autophagy, a cellular process inhibited by mTORC1, is required for satellite cell maintenance by preventing senescence (García-Prat et al., 2016). As the Pten-null satellite cells experience persist activation of Akt/mTORC1 pathway, one would predict that they should exhibit accelerated senescence during aging.

Interestingly, functions of Pten in satellite cells are similar to those of Spry1, an inhibitor of FGF signaling (Chakkalakal et al., 2012). Both Spry1 KO and Pten KO in satellite cells result in the loss of quiescence, age-dependent depletion and diminished regenerative capacity. Spry1 antagonizes FGF2 signaling, which is upregulated in the aged niche to disrupt muscle stem cell quiescence (Chakkalakal et al., 2012). As Pten function as an antagonist of the IGF signaling, these studies together highlight the role of inhibitors of growth factor signaling pathways in stem cell maintenance. Intrinsic molecular effectors such as Spry1, Pten and p38αβ/MAPK may function as mediators of extrinsic signaling to control satellite cell homeostasis (Bernet et al., 2014; Brack and Muñoz-Cánoves, 2016; Chakkalakal et al., 2012; Shea et al., 2010).

As a tumor suppressor, mutation of PTEN in stem cells or its progenitors enforces proliferation and tumorigenesis (Hollander et al., 2011; Wang et al., 2006; Zhang et al., 2006). Rhabdomyosarcoma (RMS), a highly aggressive soft-tissue sarcoma in children, arises from abnormal growth of myogenic stem and progenitor cells (Blum et al., 2013; Rubin et al., 2011). A recent study uncovers a high frequency of PTEN mutations and methylation in embryonal RMS (Seki et al., 2015), thus pointing to a potential role of PTEN in regulating the growth and tumorigenic transformation of myogenic progenitors. However, RMS was never detected in our PtenMKO mice, not even in the aged mice. Although Pten-deficient myogenic progenitors exhibit accelerated proliferation, they subsequently differentiate and fuse into myofibers normally. Therefore, combined Pten loss-of-function and blockage of differentiation may be necessary for the pathogenesis of RMS.

Collectively, our study indicates that Pten-deficient myoblasts divide rapidly during perinatal growth, resulting in muscle hypertrophy. On the other hand, Pten KO also accelerates age-dependent depletion of satellite cells. These observations point out a compensatory effect of intrinsic signaling on regulating stem cell homeostasis and tissue growth. That is, robust tissue growth at the expense of accelerated senescence of stem cells. Several factors such as telomere length, mitochondrial activity, metabolic rates and proliferative history are known to influence stem cell aging (Beerman et al., 2013; Cerletti et al., 2012; Oh et al., 2014; Sahin and DePinho, 2010; Signer and Morrison, 2013). Therefore, it’s interesting in the future to determine the exact cellular mechanism governed by Pten in regulating this compensatory effect of stem cell homeostasis and tissue growth.

EXPERIMENTAL PROCEDURES

Mice

All procedures involving mice were guided by Purdue University Animal Care and Use Committee. All mouse strains were obtained from Jackson Laboratory (Bar Harbor, ME) under following stock numbers: MyoDCre (#014140), Pax7CreER (#012476) and Ptenf/f (#006440). The genotypes of experimental knockout and associated control animals are as follows: PtenMKO (MyoDCre::Ptenf/f), PtenM+/− (MyoDCre::Pten+/f) and wild type (Ptenf/f). For conditional Pten knockout in adult satellite cells, Pax7CreER/Ptenf/f and Ptenf/f mice were injected intraperitoneally with 2 mg tamoxifen (TMX, Calbiochem) per day per 20 g body weight for 5 days to induce Cre-mediated deletion. Mice were housed and maintained in the animal facility with free access to standard rodent chow and water.

Muscle Injury and Regeneration

Muscle regeneration was induced by cardiotoxin (CTX) injection. Adult mice were first anesthetized using a ketamine-xylazine cocktail and CTX was injected (50 µl of 10 µM solution, Sigma) into tibialis anterior (TA) muscle. Muscles were then harvested at indicated days post injection to assess the completion of regeneration and repair.

Treadmill Measurement

Mouse was trained on treadmill (Eco3/6 treadmill; Columbus Instruments, Columbus, OH, USA) with a fixed 10% slope, at constant 10 m/minute speed for 5 minutes daily for consecutively 3 days before test. On the exercise testing day, animals ran on the treadmill at 10 m/minute for five minutes and the speed was increased by 2 m/minute every two minutes until they were exhausted or a maximal speed of 46 m/minute was achieved. The exhaustion was defined as the inability of the animal to run on the treadmill for 10 seconds despite mechanical prodding. Running time and maximum speed achieved was measured whereas running distance was calculated.

Sciatic Nerve Denervation

For sciatic nerve denervation, mice were anesthetized with ketamine-xylazine cocktail. The right hindlimb was prepared for surgery. Briefly a 0.5-cm incision was made in the skin along with the axis of the femur, and the sciatic nerve was isolated. To prevent reinnervation, 3–5 mm section of sciatic nerve was cut and removed. Mice were sacrificed 3 weeks after denervation and muscle samples were collected.

Primary Myoblast Isolation, Culture and Differentiation

Satellite cell-derived primary myoblasts were isolated from hindlimb skeletal muscles of Pax7CreER/Ptenf/f mice at the age of 6–8 weeks. Muscles were minced and digested in type I collagenase and Dispase B mixture (Roche). The digestions were stopped with F-10 Ham’s medium containing 20% FBS. Cells were then filtered from debris, centrifuged, and cultured in growth medium (F-10 Ham's medium supplemented with 20% FBS, 4 ng/ml basic FGF, and 1% penicillin-streptomycin) on collagen-coated cell culture plates at 37°C, 5% CO2. For in vitro genetic deletion, Pax7CreER/Ptenf/f primary myoblasts was induced by 2 days of 4-OH tamoxifen (0.4 µM, Calbiochem), and the primary myoblasts treated with vehicle were set as control.

For differentiation, primary myoblasts were seeded on BD Matrigel-coated cell culture plates and induced to differentiate in low serum medium (DMEM supplemented with 2% horse serum and 1% penicillin-streptomycin). For reserve cell culture assay, low passage primary myoblasts isolated from adult Pax7CreER/Ptenf/f mice were treated with control media or 4-OH-Tamoxifen for 48 h and then induced to differentiation at low serum medium condition for 2.5 or 3.5 days with or without LY294002 (a potent PI3K inhibitor, 10µM) or rapamycin (mTOR inhibitor, 25nM).

Single Myofiber Isolation and Culture

Single myofibers were isolated from extensor digitorum longus (EDL) muscles of adult mice (Pasut et al., 2013). Briefly, EDL muscles were dissected carefully and subjected to digestion with collagenase I (2 mg/ml, Sigma) in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma) for 1 h at 37 °C. Digestion was stopped by carefully transferring EDL muscles to a pre-warmed Petri dish (60-mm) with 6 ml of DMEM and single myofibers were released by gently flushing muscles with large bore glass pipette. Released single myofibers were then transferred and cultured in horse serum-coated Petri dish (60-mm) in DMEM supplemented with 20% fetal bovine serum (FBS, HyClone), 4 ng/ml basic fibroblast growth factor (FGF, Promega), and 1% penicillin-streptomycin (HyClone) at 37 °C.

Cell Cycle Entry Assay

Single myofibers isolated from WT and PtenMKO mice were cultured in horse serum-coated Petri dish (60-mm) in DMEM supplemented with 20% fetal bovine serum (FBS, HyClone), 4 ng/ml basic fibroblast growth factor (FGF, Promega), and 1% penicillin-streptomycin (HyClone) at 37 °C. To monitor the cell cycle entry of satellite cells, EdU (5-ethynyl-2’-deoxyuridine, Carbosynth) was added at a concentration of 1 µM into the culture medium. Cultured single myofibers were sampled at indicated time.

Histology and Immunofluorescence Staining

Whole muscle tissues from the WT and PtenMKO mice were dissected and frozen immediately in OCT compound. Frozen muscles were cross sectioned (10 µm) using a Leica CM1850 cryostat. The slides were subjected to histological hematoxylin and eosin (H&E) staining or immunofluorescence staining. For immunofluorescence staining, cross-sections, single myofibers, or cultured cells were fixed in 4% PFA in PBS for 10 min, quenched with 100 mM glycine for 10 min, and incubated in blocking buffer (5% goat serum, 2% bovine serum albumin, 0.1% Triton X-100, and 0.1% sodium azide in PBS) for at least 1 h. Samples were then incubated with primary antibodies and then secondary antibodies and DAPI (See Supplemental Information for more details).

All images were captured using a Leica DM 6000B microscope and images for WT and KO samples were captured using identical parameters. All images shown are representative results of at least three biological replicates.

Statistical Analysis

The number of regenerated myofibers per mm2 and the average cross-sectional area (CSA) of regenerated fibers were calculated using Adobe Photoshop Software. Muscle recovery rate was defined as the ratio of injured muscle to non-injured control muscle in the same mouse. All analyses were conducted with Student's t test with a two-tail distribution. All experimental data are represented as mean ± SEM. Comparisons with P values <0.05 were considered significant.

Supplementary Material

Highlights.

Loss of Pten in myoblasts leads to postnatal muscle hypertrophy

Pten-null satellite cells are maintained in an “alert” state in adult resting muscle

Pten-null satellite cells are defective in self-renewal and cannot reenter quiescence

Pten-null GAlert satellite cells are depleted with age, leading to regenerative failure

Acknowledgments

This work was supported by a grant from the US National Institutes of Health (R01AR060652 to S.K.) and Purdue incentive grant from Purdue University Office of Vice President for Research (OVPR) to S.K. We thank Dr. YongXu Wang (University of Massachusetts Medical School) for generous present of Adenovirus-AdEasy overexpression system, Jun Wu for mouse colony maintenance and members of the Kuang laboratory for valuable comments.

Footnotes

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SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures and seven figures.

AUTHOR CONTRIBUTIONS

F.Y. and S.K. conceived the project, designed the experiments and prepared the manuscript. F.Y., P.B., C.W., J.L., performed the experiments. J.L. and X.L. provided reagents. F.Y. and S.K. analyzed the data.

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