Dynamics of muscle fibre growth during postnatal mouse development

Abstract

Background: Postnatal growth in mouse is rapid, with total skeletal muscle mass increasing several-fold in the first few weeks. Muscle growth can be achieved by either an increase in muscle fibre number or an increase in the size of individual myofibres, or a combination of both. Where myofibre hypertrophy during growth requires the addition of new myonuclei, these are supplied by muscle satellite cells, the resident stem cells of skeletal muscle.

Results: Here, we report on the dynamics of postnatal myofibre growth in the mouse extensor digitorum longus (EDL) muscle, which is essentially composed of fast type II fibres in adult. We found that there was no net gain in myofibre number in the EDL between P7 and P56 (adulthood). However, myofibre cross-sectional area increased by 7.6-fold, and length by 1.9-fold between these ages, resulting in an increase in total myofibre volume of 14.1-fold: showing the extent of myofibre hypertrophy during the postnatal period. To determine how the number of myonuclei changes during this period of intense muscle fibre hypertrophy, we used two complementary mouse models: 3F-nlacZ-E mice express nlacZ only in myonuclei, while Myf5nlacZ/+ mice have beta-galactosidase activity in satellite cells. There was a approximately 5-fold increase in myonuclear number per myofibre between P3 and P21. Thus myofibre hypertrophy is initially accompanied by a significant addition of myonuclei. Despite this, the estimated myonuclear domain still doubled between P7 and P21 to 9.2 x 103 microm3. There was no further addition of myonuclei from P21, but myofibre volume continued to increase, resulting in an estimated approximately 3-fold expansion of the myonuclear domain to 26.5 x 103 microm3 by P56. We also used our two mouse models to determine the number of satellite cells per myofibre during postnatal growth. Satellite cell number in EDL was initially approximately 14 satellite cells per myofibre at P7, but then fell to reach the adult level of approximately 5 by P21.

Conclusions: Postnatal fast muscle fibre type growth is divided into distinct phases in mouse EDL: myofibre hypertrophy is initially supported by a rapid increase in the number of myonuclei, but nuclear addition stops around P21. Since the significant myofibre hypertrophy from P21 to adulthood occurs without the net addition of new myonuclei, a considerable expansion of the myonuclear domain results. Satellite cell numbers are initially stable, but then decrease to reach the adult level by P21. Thus the adult number of both myonuclei and satellite cells is already established by three weeks of postnatal growth in mouse.

Figure 1

EDL myofibres from growing and adult mice. Entire EDL muscles from P3, P7 (a), P14, P21 and P56 (b) Myf5^nlacZ/+ mice were cryosectioned and mid-belly sections stained with haematoxylin and eosin to determine total myofibre content and myofibre cross-sectional area. EDL myofibres were isolated from 3F-nlacZ-E mice at P3 (c), P7 (d), P14 (e), P21 (f) and P56 (g), fixed and incubated in X-gal solution to reveal the β-galactosidase activity of myonuclei by the presence of the blue reaction product. Images of representative myofibres were all taken at the same magnification and show that there is a 4.5-fold increase in length between P3 and adulthood (P56). Myonuclei appear to be uniformly distributed along the length of a myofibre at each age examined (c-g). Scale bar equals 100 μm for (a and b) and 1000 μm for (c-g).

Figure 2

Postnatal growth dynamics of EDL myofibres. Measuring myofibre cross-sectional area from mid-belly EDL muscle sections showed that there was a significant (ANOVA: p < 0.0001; F = 850.6) and highly linear (R² = 1.0) increase with age (a), which was accompanied by a significant increase in the length of isolated EDL myofibres (b). Myonuclei were counted on isolated EDL myofibres from Myf5^nlacZ/+ and/or 3F-nlacZ-E mice and there was a 4-fold increase in myonuclei number per myofibre between P3 and P14 from ~50 to ~200 (c). The adult complement of myonuclei (~250) was reached by P21. Mean total myofibre volume was calculated by multiplying mean length by mean cross-sectional area and increased in a linear fashion between P7 to P56 (blue line - d). By then dividing mean total myofibre volume by the mean number of myonuclei per myofibre, we estimated myonuclear domain during postnatal development (dashed red line - d). Data shown are mean ± SEM, except for (d), which is mean; trend line in (c) depicts the moving average [(a) n > 100 myofibres from each of at least 3 mice at each age; (b) n > 19 myofibres from each of at least 3 mice at each age; (c) n > 30 myofibres from each of at least 2 mice at each age, see Tables 1 and 2 for data sets].

Figure 3

Identification of myonuclei and satellite cells from growing 3F-nlacZ-E and Myf5 ^nlacZ/+ mice. Myofibres were isolated from P14 mice, fixed and incubated in X-gal and DAPI. Myonuclei are revealed in 3F-nlacZ-E myofibres by β-galactosidase activity producing a blue reaction product (a), whereas satellite cells are visible as DAPI-positive nuclei, since the X-gal reaction product obscures the DAPI fluorescence in myonuclei (b). This enables clear discrimination between myonuclei and satellite cells, as seen in the merged image (c). By contrast, in Myf5^nlacZ/+ mice, satellite cells are identified by their β-galactosidase activity on isolated myofibres (d), whereas myonuclei are DAPI-positive nuclei that do not contain the blue X-gal reaction product (e and f). Scale bar equals 50 μm.

Figure 4

Satellite cell number steadily decreases from P6, to reach the adult level by P21. Satellite cells were identified and counted on isolated Myf5^nlacZ/+ and 3F-nlacZ-E myofibres and pooled at each age to give a mean ± SEM (a). The number of satellite cells per myofibre decreased by 0.66 satellite cells per day (R² = 0.98) to reach the adult level by P21, when it remained unchanged until 10 months, our oldest age analysed. The same data with satellite cells expressed as a percentage of total myofibre nuclei (myonuclei plus satellite cells) (b). Due to the rapidly increasing myonuclear number, there is an exaggerated fall from P6 until P21. Data shown are mean ± SEM (n > 30 myofibres from at least 6 mice at P7, P14, P21 and P56, and at least 2 mice per age at all other stages).