Rapamycin Rescues Age-Related Changes in Muscle-Derived Stem/Progenitor Cells from Progeroid Mice

Abstract

Aging-related loss of adult stem cell function contributes to impaired tissue regeneration. Mice deficient in zinc metalloproteinase STE24 (Zmpste24 ^-/-) exhibit premature age-related musculoskeletal pathologies similar to those observed in children with Hutchinson-Gilford progeria syndrome (HGPS). We have reported that muscle-derived stem/progenitor cells (MDSPCs) isolated from Zmpste24 ^-/- mice are defective in their proliferation and differentiation capabilities in culture and during tissue regeneration. The mechanistic target of rapamycin complex 1 (mTORC1) regulates cell growth, and inhibition of the mTORC1 pathway extends the lifespan of several animal species. We therefore hypothesized that inhibition of mTORC1 signaling would rescue the differentiation defects observed in progeroid MDSPCs. MDSPCs were isolated from Zmpste24 ^-/- mice, and the effects of mTORC1 on MDSPC differentiation and function were examined. We found that mTORC1 signaling was increased in senescent Zmpste24 ^-/- MDSPCs, along with impaired chondrogenic, osteogenic, and myogenic differentiation capacity versus wild-type MDSPCs. Interestingly, we observed that mTORC1 inhibition with rapamycin improved myogenic and chondrogenic differentiation and reduced levels of apoptosis and senescence in Zmpste24 ^-/- MDSPCs. Our results demonstrate that age-related adult stem/progenitor cell dysfunction contributes to impaired regenerative capacities and that mTORC1 inhibition may represent a potential therapeutic strategy for improving differentiation capacities of senescent stem and muscle progenitor cells.

Keywords: MDSPC; mTORC1; multipotent differentiation; muscle regeneration; rapamycin; vaging.

Figure 1

Prelamin A Processing and Gross Musculoskeletal Pathology in Zmpste24^−/− MDSPCs (A) Schematic representation of the mTORC1 pathway and its regulation via rapamycin treatment in Zmpste24^−/− MDSPCs. Arrows indicate activating events; perpendicular lines indicate inhibitory events. mTOR exists in two complexes: mTORC1 containing RAPTOR and mTORC2 containing RICTOR. Down-stream effectors of mTORC1 include p70/S6K regulation of cell proliferation, mRNA translation, and protein synthesis. mTOR is regulated by PI3K and AKT. (B) Representative immunohistochemical staining with anti-lamin A antibodies. Original magnification, ×400. Scale bar, 10 μm. Arrowhead, abnormality in the nuclear membrane. (C) Percentage of abnormal nuclei among cells (*p < 0.05, **p < 0.001). (D) Western blot analysis to measure the expression of the C-terminal prelamin A and lamin A in WT and Zmpste24^−/− MDSPCs, relative to the expression of β-actin or vinculin. (E) Representative micro-CT images of bone microarchitecture in knee joint of 8-week-old Zmpste24^−/− mice. (F and G) Skeletal preparations of 8-week-old (F) and postnatal day 2 (G) Zmpste24^−/− mice double stained with Alizarin red (bone) and Alcian blue (cartilage). Scale bars, 8 mm. (H and I) Masson’s-trichrome-stained gastrocnemius muscle (H) indicating significant levels of fibrosis in 8-week-old Zmpste24^−/− mice versus age-matched WT animals (I) (*p < 0.05). Scale bar, 100 μm.

Figure 2

Elevated mTORC1 Activity in Zmpste24^−/− MDSPCs (A) Representative western blots indicating mTORC1, p-mTORC1, and p-4E-BP1 expression levels in MDSPCs isolated from Zmpste24^−/− and WT mice, cultured with and without rapamycin. (B) Quantification of western blots (##p < 0.001 versus WT DMSO; *p < 0.05, **p < 0.001 versus Zmpste24^−/− DMSO). (C) Representative image of western blots indicating p-AMPK and p-AKT expression in MDSPCs isolated from Zmpste24^−/− and WT mice, cultured with and without rapamycin. (D) Quantification of p-AMPK and p-AKT expression relative to the expression of β-actin (##p < 0.001 versus WT DMSO). All immunoblots are representative of experiments with similar results (n = 4).

Figure 3

Rapamycin’s Effect on Apoptosis in Zmpste24^−/− MDSPCs (A) Representative TUNEL images indicating apoptotic cell death of MDSPCs isolated from Zmpste24^−/− and WT mice, cultured with and without rapamycin. Scale bar, 50 μm. Arrow, TUNEL-positive, apoptotic (green) cells. (B) Percentage of TUNEL-positive cells reported from images obtained from four independent MDSPC populations (##p < 0.001 versus WT DMSO; **p < 0.001 versus Zmpste24^−/− DMSO). (C) Representative western blots showing expression of the apoptosis-related marker, cleaved PARP, in MDSPCs isolated from Zmpste24^−/− and WT mice, cultured with and without rapamycin. (D) Quantification of cleaved PARP levels relative to β-actin (n = 4; ##p < 0.001 versus WT DMSO; **p < 0.001 versus Zmpste24^−/− DMSO).

Figure 4

Rapamycin’s Effect on Senescence Markers in Zmpste24^−/− MDSPCs (A) Representative western blots showing expression of senescence-related markers p21 and p16 in MDSPCs isolated from Zmpste24^−/− and WT mice, cultured with and without rapamycin. (B) Quantification p21 and p16 expression relative to β-actin (n = 4; ##p < 0.001 versus WT DMSO; **p < 0.001 versus Zmpste24^−/− DMSO; *p < 0.05 versus Zmpste24^−/− DMSO). (C) Proliferation of Zmpste24^−/− and WT MDSPCs, with or without rapamycin expressed as optical density (490 nm). (D) Representative images of SA-β-gal assay for labeling senescence of MDSPCs isolated from Zmpste24^−/− and WT mice, cultured with and without rapamycin. Scale, 50 μm. Arrow, SA-β-gal-positive (blue) cells. Percentage of SA-β-gal-positive cells from four independent MDSPC populations (#p < 0.05, ##p < 0.001 versus WT DMSO; **p < 0.001 versus Zmpste24^−/− DMSO).

Figure 5

Myogenic Differentiation Potential of MDSPCs Isolated from Progeroid Zmpste24^−/− Mice (A) Representative images of in vitro myogenic differentiation of MDSPCs isolated from Zmpste24^−/− and WT mice, cultured with and without rapamycin. Cells were immunostained for the terminal differentiation marker f-My-HC (red) and counterstained with DAPI for visualizing nuclei (blue). Original magnification, ×200. Scale bar, 50 μm. (B) Quantification of myogenic differentiation was calculated as the fraction of cells (DAPI, blue) expressing f-My-HC (red) from four independent MDSPC populations (p < 0.001 versus WT DMSO; **p < 0.001 versus Zmpste24^−/− DMSO). (C and D) qPCR results indicating mRNA expression levels of terminal myogenic differentiation markers, desmin (C) and MyHC (D), after myogenic differentiation of MDSPCs isolated from Zmpste24^−/− and WT mice, cultured with and without rapamycin treatment (n = 4; ##p < 0.001 versus WT DMSO; *p < 0.05 versus Zmpste24^−/− DMSO).

Figure 6

Chondrogenic Differentiation Potential of MDSPCs Isolated from Progeroid Zmpste24^−/− Mice (A) Representative images of in vitro chondrogenic pellets (ruler is in mm) from MDSPCs isolated from Zmpste24^−/− and WT mice, cultured with and without rapamycin. (B) Representative images of Alcian Blue staining of each pellet (blue, chondrogenesis). Scale bar, 500 μm. Original magnification, ×200, ×40 in inset. (C) Measurement of diameter of chondrogenic pellets (##p < 0.001 versus WT DMSO; *p < 0.05 versus Zmpste24^−/− DMSO). (D and E) qPCR results indicating mRNA expression levels of the chondrogenic differentiation markers, Col2a1 (D) and aggrecan (E), after chondrogenic differentiation of MDSPCs isolated from Zmpste24^−/− and WT mice, cultured with and without rapamycin treatment (n = 4; ##p < 0.001 versus WT DMSO; *p < 0.05, **p < 0.001 versus Zmpste24^−/− DMSO).

Figure 7

Osteogenic Differentiation Potential of MDSPCs Isolated from Progeroid Zmpste2^−/− Mice (A) Representative images of in vitro Alizarin Red staining of MDSPCs isolated from Zmpste24^−/− and WT mice, cultured with and without rapamycin. Left upper panels in each image represent whole images. Scale bar, 50 μm. (B) Quantification of alizarin red staining by photometric analysis. The extracted alizarin red dye was measured with an absorption spectrometer at 415 nm wavelength. The amount of alizarin red dye was expressed as an optical density (#p < 0.05 versus WT DMSO). (C and D) qPCR results indicating mRNA expression levels of osteogenic differentiation markers, Col1A1 (C) and osteocalcin (D), after osteogenic differentiation of MDSPCs isolated from Zmpste24^−/− and WT mice, cultured with and without rapamycin treatment (n = 4; #p < 0.05, ##p < 0.001 versus WT DMSO; *p < 0.05 versus Zmpste24^−/− DMSO).

Figure 8

Adipogenic Differentiation Potential of MDSPCs Isolated from Progeroid Zmpste24^−/− Mice (A) Representative images of Oil Red O (lipid) staining of MDSPCs isolated from Zmpste24^−/− and WT mice, cultured with and without rapamycin. Right upper panels in each image indicate high magnification of the area surrounded by a dotted square. Arrows indicate positive Oil Red O staining. Scale bar, 50 μm. (B) Quantification of Oil Red O staining by photometric analysis. Absorbance of Oil Red O was measured at a wavelength of 510 nm (##p < 0.001 versus WT DMSO; *p < 0.05 versus Zmpste24^−/− DMSO). (C and D) qPCR results indicating mRNA expression levels of adipogenic differentiation markers, PPARγ (C) and LPL (D), after adipogenic differentiation of MDSPCs isolated from Zmpste24^−/− and WT mice, cultured with and without rapamycin treatment (n = 4; ##p < 0.001 versus WT DMSO; **p < 0.001 versus Zmpste24^−/−DMSO).