Dual role of the caspase enzymes in satellite cells from aged and young subjects

Abstract

Satellite cell (SC) proliferation and differentiation have critical roles in skeletal muscle recovery after injury and adaptation in response to hypertrophic stimuli. Normal ageing hinders SC proliferation and differentiation, and is associated with increased expression of a number of pro-apoptotic factors in skeletal muscle. In light of previous studies that have demonstrated age-related altered expression of genes involved in SC antioxidant and repair activity, this investigation was aimed at evaluating the incidence of apoptotic features in human SCs. Primary cells were obtained from vastus lateralis of nine young (27.3±2.0 years old) and nine old (71.1±1.8 years old) subjects, and cultured in complete medium for analyses at 4, 24, 48, and 72 h. Apoptosis was assessed using AnnexinV/propidium iodide staining, the terminal deoxynucleotidyl transferase dUTP nick-end labelling technique, RT-PCR, DNA microarrays, flow cytometry, and immunofluorescence analysis. There was an increased rate of apoptotic cells in aged subjects at all of the experimental time points, with no direct correlation between AnnexinV-positive cells and caspase-8 activity. On the other hand, CASP2, CASP6, CASP7, and CASP9 and a number of cell death genes were upregulated in the aged SCs. Altogether, our data show age-related enhanced susceptibility of human SCs to apoptosis, which might be responsible for their reduced response to muscle damage.

Figure 1

Flow cytometry dot plots of different cell populations in young SCs (left) and aged SCs (right) cultured for 4, 24, 48, and 72 h, as indicated. Early apoptotic cells (AnnV⁺/PI⁻, quadrant D4) can be discriminated from viable cells (AnnV⁻/PI⁻, quadrant D3), late apoptotic cells (AnnV⁺/PI⁺, quadrant D2), and necrotic cells (AnnV⁻/PI⁺, quadrant D1), according to their fluorescence emission

Figure 2

Fluorescence images of young SCs (a) and aged SCs (b) assayed with the TUNEL technique at different time intervals of in vitro culture, as indicated. Nuclei were counterstained with DAPI (blue fluorescence). Green (TUNEL) and blue fluorescence (DAPI) single emissions are shown in the left and right panels, respectively. Representative fields from a representative experiment of the three independent experiments are shown. Original magnification: × 40. Scale bar: 10 μm. (c) Cell cycle distributions of young SCs and aged SCs at different time intervals of in vitro culture, as indicated. Data are means±S.E. of three independent experiments. Significant differences are seen for aged SCs versus young SCs: for G0/G1 at 24 h (P<0.05); for S at 24 h (P<0.05); for S at 48 h (P<0.05); for G2/M at 72 h (P<0.05). (d) Cell cycle profiles of young SCs and aged SCs at 4, 24, 48, and 72 h of in vitro culture, as indicated, showing absence of any hypo-diploid peak. A representative experiment of three independent experiments is shown

Figure 3

(a) Caspase-8 activation in young SCs and aged SCs at different time intervals of in vitro culture, as indicated. Data are means±S.E. of three independent experiments. A significant difference is seen for aged SCs versus young SCs at 24 h (P<0.01). (b) Fold-increase in AnnV/PI-labelled young SCs and aged SCs with and without administration of a caspase-8-specific inhibitor, as indicated. The data were obtained at 4 and 24 h and were normalised to the AnnV/PI-labelled cell levels without the inhibitor, as means±S.E. of three independent experiments. A significant difference is seen for aged SCs versus young SCs at 24 h (P<0.05). (c) Cell cycle distributions of young SCs and aged SCs at different time intervals of in vitro culture without and with administration of a caspase-8-specific inhibitor, as indicated. A representative experiment of three independent experiments is shown. (d) Fold-increase in AnnV/PI-labelled young SCs and aged SCs with and without administration of a caspase-9-specific inhibitor. The data were obtained at 4 and 24 h and were normalised to the AnnV/PI-labelled cell levels without the inhibitor, as means±S.E. of three independent experiments. A significant difference is seen for aged SCs versus young SCs at 4 h (P<0.05)

Figure 4

Expression levels of genes representative of the apoptosis pathway, analysed with RT-PCR using TaqMan low density arrays. Data from the log₁₀ of relative quantifications of the transcripts for the target genes versus GAPDH gene expression are represented as the aged SCs to young SCs ratios, as indicated, following in vitro culture for 4 h (a), 24 h (b), 48 h (c) and 72 h (d)

Figure 5

(a) Immunofluorescence images of cleaved caspase-3/myogenin detection in young SCs (left) and aged SCs (right) following in vitro culture for 72 h. Green (cleaved caspase-3), red (myogenin), and blue (DAPI) fluorescence single emissions are shown, as indicated. Of note, the nuclear and cytoplasmic location of caspase-3 is appreciable only in the aged SCs. Representative fields from a representative experiment of the three independent experiments is shown. Magnification: × 20. Scale bar: 20 μm. (b) Fold-increase in AnnV/PI-labelled young SCs and aged SCs with and without administration of the broad caspase inhibitor z-VAD-fmk. Data were obtained at 4 and 24 h and were normalised to the AnnV/PI-labelled cell levels without the inhibitor, as means±S.E. of three independent experiments. A significant difference is seen for aged SCs versus young SCs at 24 h (P<0.001). (c) Cell cycle distributions of young SCs and aged SCs at different time intervals of in vitro culture with and without administration of the broad caspase inhibitor z-VAD-fmk, as indicated. Data are means±S.E. of three independent experiments. Significant differences are seen for aged SCs versus young SCs: for G0/G1 at 4 h (P<0.05); for S at 4 h (P<0.05); for G2/M at 24 h (P<0.05)

Figure 6

Functional analysis of the 0 h data set of genes with IPA software. (a and b) Key biological functions associated with genes that were selectively upregulated (n=52) and downregulated (n=1) in aged SCs versus young SCs before in vitro culture (0 h). The analysis revealed that the top function involved is represented by cell death. (c–e) The three top networks generated by the IPA software, showing genes selectively dysregulated in the proliferating SCs at 0 h, as shown in a and b (score range, 15–38). Genes in red show increased expression in aged SCs, whereas genes in green show decreased expression in aged SCs, when compared with young SCs. Genes in white represent the transcripts not modulated under the different conditions. Arrows indicate that a molecule acts on another, whereas lines indicate that a molecule binds to another. The gene networks show the nodes of genes involved in cell death (SMAD4, MMP1) (c), in cell death (MCL1), actin stress fibre formation (RBBP4, ITGAV), and muscle development and morphogenesis (DLG1, SHC1) (d), and in apoptotic pathways (F2R, GNB2, DEGS1) (e)