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

Abstract

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 G_Alert 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 the phosphatase and tensin homolog (Pten) exhibit enhanced proliferation and differentiation, resulting in muscle hypertrophy but fewer SCs in adult muscles. Interestingly, Pten null SCs are predominantly in the G_Alert state, even in the absence of an injury. The G_Alert 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 G_Alert leads to stem cell depletion and regenerative failure.

Keywords: Pten; aging; hypertrophy; regeneration; skeletal muscle; stem cells.

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

(A–B) Representative image of whole hind limb (A) and TA, Sol and Gas muscles (B) in adult WT and Pten^MKO mice. (C) Muscle weight in adult WT and Pten^MKO 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.

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

(A–C) Maximum speed (A), total running time (B) and running distance (C) of adult WT and Pten^MKO 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).

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

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

Figure 4. Quiescent Satellite Cells Exist in an “Alert” (G_Alert) State in Adult Pten^MKO Mice

(A) Immunofluorescence of Pax7 and Pten, and percentage of Pten⁺ satellite cells on fresh myofibers of adult WT and Pten^MKO 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 pAkt^Neg, pAkt^Low and pAkt^High satellite cells on fresh myofibers (n=3 mice, 20 myofibers per mouse). (F) Immunofluorescence of pS6 in Pax7⁺ satellite cells, and percentages of pS6^Neg, pS6^Low and pS6^High 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.

Figure 5. G_Alert Satellite Cells Are Able to Maintain Muscle Regeneration at the Expense of Self-Renewal in Young Pten^MKO Mice

(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 Pten^MKO 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.

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

(A) Immunofluorescence of Pax7 and MyoD, and percentage of Pax7⁺MyoD⁻ and Pax7⁺MyoD⁺ SCs in TA muscles of adult WT and Pten^MKO 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.

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

(A) Immunofluorescence of Pax7, and satellite cell number in fresh myofibers isolated from old WT and Pten^MKO 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 Pten^MKO 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.