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Mature Female Muscle

Skeletal muscle contractility and myosin function decline following ovariectomy in mature female mice. In the present study we tested the hypothesis that estradiol replacement can reverse those declines. Four-month-old female C57BL/6 mice (n = 69) were ovariectomized (OVX) or sham operated. Some mice were treated immediately with placebo or 17beta-estradiol (OVX + E(2)) while other mice were treated 30 days postsurgery. Thirty or sixty days postsurgery, soleus muscles were assessed in vitro for contractile function and susceptibility to eccentric contraction-induced injury. Myosin structural dynamics was analyzed in extensor digitorum longus (EDL) muscles by electron paramagnetic resonance spectroscopy. Maximal isometric tetanic force was affected by estradiol status (P or= 0.401) but did restore ovariectomy-induced increases in muscle wet mass caused by fluid accumulation. Collectively, estradiol had a beneficial effect on female mouse skeletal muscle.

mature female muscle

The purposes of this study were to determine the effects of ovarian hormone removal on force-generating capacities and contractile proteins in soleus and extensor digitorum longus (EDL) muscles of mature female mice. Six-month-old female C57BL/6 mice were randomly assigned to either an ovariectomized (OVX; n = 13) or a sham-operated (sham; n = 13) group. In vitro contractile function of soleus and EDL muscles were determined 60 days postsurgery. Total protein and contractile protein contents were quantified, and electron paramagnetic resonance (EPR) spectroscopy was used to determine myosin structural distribution during contraction. OVX mice weighed 15% more than sham mice 60 days postsurgery, and soleus and EDL muscle masses were 19 and 15% greater in OVX mice, respectively (P or = 0.242), indicating that the quantity of contractile machinery was not affected by removing ovarian hormones. EPR spectroscopy showed that the fraction of strong-binding myosin during contraction was 15% lower in EDL muscles from OVX mice compared with shams [0.277 (SD 0.039) vs. 0.325 (SD 0.020); P = 0.004]. These results indicate that the loss of ovarian hormones has detrimental effects on skeletal muscle force-generating capacities that can be explained by altered actin-myosin interactions.

Satellite cells are myogenic cells found between the basal lamina and the sarcolemma of the muscle fibre. Satellite cells are the source of new myofibres; as such, satellite cell transplantation holds promise as a treatment for muscular dystrophies. We have investigated age and sex differences between mouse satellite cells in vitro and assessed the importance of these factors as mediators of donor cell engraftment in an in vivo model of satellite cell transplantation. We found that satellite cell numbers are increased in growing compared to adult and in male compared to female adult mice. We saw no difference in the expression of the myogenic regulatory factors between male and female mice, but distinct profiles were observed according to developmental stage. We show that, in contrast to adult mice, the majority of satellite cells from two week old mice are proliferating to facilitate myofibre growth; however a small proportion of these cells are quiescent and not contributing to this growth programme. Despite observed changes in satellite cell populations, there is no difference in engraftment efficiency either between satellite cells derived from adult or pre-weaned donor mice, male or female donor cells, or between male and female host muscle environments. We suggest there exist two distinct satellite cell populations: one for muscle growth and maintenance and one for muscle regeneration.

Although the role of the satellite cell in healthy adult skeletal muscle remains ambiguous, the satellite cell occupies a relatively undisputed position as a cell that is essential for muscle regeneration [1], [7]. The relative efficiency of male and female satellite cells to regenerate dystrophic muscle remains unexplored. Considering the previously mentioned influence of circulating androgens on satellite cell activity, it is likely that the host environment will mediate donor cell engraftment efficiency. In addition, if there are indeed intrinsic differences in male and female satellite cells, the sex from which donor satellite cells are derived may also affect the extent of donor cell contribution to regeneration.

In single fibres from 2 week old mice analysed immediately after isolation, the majority (91% males, 92% females) of satellite cells were shown to co-express Pax7 and MyoD (Fig. 2Di) and are thus actively proliferating. This is to be expected from a fibre that, within the next 10 weeks, will almost double its number of myonuclei (Fig. 1C). Intriguingly, however, not all satellite cells are recruited into this growth program. We observed a rare few quiescent satellite cells, MyoD- Pax7+ (Fig. 2Dii). It was not possible to assess the fate of these cells on a single fibre in culture, as 2 week old fibres could rarely be cultured successfully for any length of time. Fibres that could be analysed had no associated satellite cells by 48 hours. We hypothesise that this is due to changes in the basal lamina between 2 week and 3 month old mice. Fibres of 2 week old mice are more fragile and the basal lamina more easily damaged, such that the satellite cells may not remain associated with the fibre and/or the fibre contracts.

Thus far, single fibre analyses represent myofibre and satellite cell dynamics within a sedentary model where satellite cells are not challenged beyond the requirements of normal growth. It is possible that male and female satellite cells have functional differences with regards to their ability to survive transplantation and regenerate muscle after severe injury. The significantly greater number of satellite cells found in male compared to female mice may also suggest that male donor mice provide a greater pool of satellite cells and will therefore produce greater numbers of donor derived fibres, making them a preferable choice of donors in current models of satellite cell transplantation.

(A) Representative image of donor derived fibres in mdx-nude TA muscle sections. X-gal staining shows the presence of donor-derived fibres co-localised with dystrophin positive fibres. Scale bar 50 µm. (B) Donor cells were injected into 4 week old mdx-nude mice TA muscles. TA muscles were harvested 4 weeks after cell injection and the number of dystrophin positive fibres quantified. Graph shows no significant difference in the amount of donor muscle produced per injected between male and female 3F-nLacZ-2E donors. Blue data points indicate a male host, green indicates a female host. (C) shows no significant difference in the number of dystrophin positive fibres per injected cell between male and female mdx-nude hosts injected with satellite cells from male 3F-nLacZ-2E donors.

Our experiments have demonstrated that there is no intrinsic difference between male and female satellite cells in their ability to regenerate host muscle after transplantation. However, it is possible that sex differences in muscle regeneration may occur, and are governed by environmental, rather than via satellite cell intrinsic mediators e.g. circulating androgens. To investigate this, we isolated satellite cells from 3 month old male 3F-nLacZ-2E mice and injected them into the TA muscles of male and female mdx-nude hosts. We found no difference between the number of donor-derived fibres at 4 weeks post injection between male and female hosts (Fig. 4C). Together these data demonstrate that in our model of satellite cell transplantation, donor derived muscle formation is not altered either by the sex of the host or the sex of the donor.

In vitro analysis of single fibres showed that the age at which the fibre is isolated has the greatest effect on all variables measured. However, previous research has shown that aged and adult donor satellite cells regenerate host muscle equally well [10]. The regenerative capacity of satellite cells isolated from fibres derived from muscles that are actively growing is unknown.

Donor satellite cells were injected into 4 week old mdx-nude mice TA muscles which were harvested 4 weeks later. Donor contribution to muscle regeneration was quantified by counting the number of dystrophin positive fibres and normalizing for the number of cells injected. (A) Representative sections of mdx-nude host TA muscles grafted with donor satellite cells 4 weeks previously. Donor contribution to muscle is shown by β-gal and dystrophin expression. Scale bar 50 µm. (B) There was no difference in the number of donor-derived fibres per injected cell between muscles grafted with cells from 3 month old or 2 week old 3F-nLacZ-2E donors either in male or female mdx-nude hosts.

These data represent a comprehensive investigation of the satellite cell population, its relationship to myonuclei and its regenerative ability after transplantation across the life span of male and female mice. We show that a small percentage of satellite cells in growing muscle are not actively contributing to myonuclear addition. The satellite cell population changes in abundance as a function of age and sex, yet these changes do not relate to regenerative capacity post transplantation. We hypothesise that the satellite cells that are responsible for host muscle regeneration after transplantation are a distinct population from the more numerous satellite cells responsible for muscle growth and maintenance in situ. 041b061a72


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