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First published online October 11, 2005
doi: 10.1242/10.1242/jcs.02602

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Evidence that satellite cell decrement contributes to preferential decline in nuclear number from large fibres during murine age-related muscle atrophy

Andrew S. Brack^*, Heidi Bildsoe and Simon M. Hughes

MRC Centre for Developmental Neurobiology and Randall Division for Cell and Molecular Biophysics, New Hunt's House, King's College London, London, SE1 1UL, UK

View larger version (49K): [in a new window]   Fig. 1. Isolated single TA muscle fibres lose cytoplasm and nuclei during ageing. (A-D) Micrographs of DAPI-stained nuclei in fibres. At 2 months (A), all fibres show well-spaced oval peripheral nuclei aligned with the fibre axis. At 26-29 months, some small (B) and large (C) fibres have elongated or mis-orientated nuclei, or nuclear chains (D). Vertical bars denote fibre diameter. Horizontal bar, 100 µm. Mean fibre cross-sectional area (E), nuclei per unit fibre length (F), surface area domain size (SADS, G) and volume domain size (VDS, H) in wild-type mice at 2 months (number of animals per group, n=9), 12 months (n=5), 22-24 months (n=5) and 26-29 months (n=8). **P<0.01. Error bars indicate animal to animal s.e.m.

View larger version (42K): [in a new window]   Fig. 2. Nuclear density decreases with age preferentially in large fibres with high domain size. Relationship of nuclei/unit fibre length (A,C,G), SADS (B,D,H) and VDS (I,J) to fibre size in young/adult (A,B) and adult/old (C-J) animals. (A,B) Larger fibres have more nuclei/unit length and each nucleus has a larger nuclear domain than small fibres during adult life (n=253 and 75 fibres in 2-month-old and 12-month-old mice, respectively). (C,D) With ageing, nuclei/unit length decrease and SADS increases in large fibres. Small fibres show no significant change. (E) Fibre type frequency is not significantly altered in superficial TA with age. (F-J) Fibres pooled into small (<1500 µm2), medium (1500-2500 µm2) and large (>2500 µm2) groups emphasize that the fibre population becomes smaller during ageing (F). Number of fibres in small-sized (n=27, 54, 100) at 12 months, 22-24 months and 26-29 months, respectively, in medium-sized (n=37, 37, 45) and large-sized (n=37, 29, 40) fibres. Large fibres have more nuclei per unit length, but lose this advantage with age (G). This change is reflected by an increase in domain size in large fibres (H-J). *P<0.05, **P<0.01, ***P<0.001. Error bars indicate fibre to fibre s.e.m.

View larger version (55K): [in a new window]   Fig. 3. SCs decline with age. (A-C) Syndecan-4 (Syn4, green), MyoD (red) and DAPI (blue) staining of freshly-isolated superficial TA fibres from 2-month-old (A), 12-month-old (B) and 24-month-old (C) mice. Arrows, myonuclei; arrowhead, SCs. Bar, 100 µm. (D,E) Pax7 staining (red) of fibres from 2-month-old (D) and 24-month-old mice (E). (F) Syn4+ SCs/fibre in large (>2500 µm2), medium (1500-2500 µm2) and small (<1500 µm2) fibres reveal that large adult fibres have more SCs and that the age-related decline in SCs is greatest on large fibres. (G) Comparison of the ratio of Syn4+ SCs to myonuclei (calculated as [Syn4+ cells.fibre–1]/[DAPI+ nuclei/500 µm]), showing that large fibres lose their advantage by 24 months. Number of fibres counted are 20, 30 and 27 for small, medium and large fibres at 2 months, and 23, 37 and 38 at 1 year, and 45, 70 and 74 at 24 months.

View larger version (49K): [in a new window]   Fig. 4. Nuclear density declines at all fibre sizes in the absence of myoD. Mean fibre cross-sectional area (A), nuclei per unit fibre length (B), surface area domain size (SADS, C) in wild-type (number of animals per group, n=9, n=5) and myoD-null (D–/–) mice (n=4, n=5) at 2 months and 22-24 months, respectively. **P<0.01. Error bars indicate animal to animal s.e.m. (D) Fibres pooled into small (<1500 µm2), medium (1500-2500 µm2) and large (>2500 µm2) groups show the fibre size is not lower in ageing D–/– than wild-type mice. (E,F) Relationship of nuclei/unit fibre length (E) and SADS (F) to fibre size in 22-24-month-old D–/– and age-matched controls. (G,H) Nuclei/unit fibre length decreases (G) and SADS increases (H) in 22-24-month-old D–/– mice in all fibre size ranges compared with both age-matched controls. Number of fibres in 22-24-month-old D–/– mice are 60, 23, 30 for small, medium and large fibres and 54, 37, 29 for 22-24-month-old controls. *P<0.05, **P<0.01, ***P<0.001.

View larger version (57K): [in a new window]   Fig. 5. MyoD expression rises with age. (A) In situ mRNA hybridization shows myoD mRNA is lower in 2 month (inset shows a rare myoD+ fibre region) than 24 month superficial TA fibres. Bar, 100 µm. (B) Both MyoD protein and mRNA increase in myonuclei with fibre maturation. (C) X-gal reaction of wholemount TA and EDL muscle from MD6.0-lacZ myoD reporter mice is stronger at 24 months than 2 months. (D) Quantified ß-gal activity in TA muscle homogenates increases between 2 months and 12 months of age. *P<0.05, **P<0.01, ***P<0.001.

View larger version (45K): [in a new window]   Fig. 6. Model of damage/repair cycle changes during ageing. In adult muscle, nuclei lost through damage or exercise may lead to transient domain size increase but are rapidly replaced from myogenic cells, returning domain size to the set point, without significant alteration in fibre size. In ageing muscle, reduced SC function might lead to delayed nuclear replacement and the start of domain size increase. In aged muscle, poor SC function, particularly in large fibres, exacerbates the cycle of decline. Over time, the inability of nuclei to support an excessive domain size leads to cytoplasmic atrophy, returning domain size to the normal set point typical of smaller fibres, which have fewer nuclei and smaller domain sizes than large fibres in healthy adults.