Dynamics of muscle fibre growth during postnatal mouse development.

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.

The fate of implanted syngenic muscle precursor cells in injured striated urethral sphincter of female rats.

We studied the outcome of syngenic skeletal muscle precursor cells (MPCs) implanted in the striated urethral sphincter of the female rat. These cells were injected at the site of a longitudinal sphincterotomy performed 21 days before implantation. MPCs were isolated from the striated hindlimb muscles of syngenic adult rats and were infected with a retrovirus carrying the gene for either the green fluorescent protein (GFP) or the beta-galactosidase enzyme (beta-gal). MPCs (2 x 10(5)) were injected longitudinally at the site of the lesion in 48 animals using a 10-microl Hamilton syringe. Then the whole urethras were excised from 2 h up to 90 days for cross section immunocytochemistry analysis. All the urethras exhibited connective tissue in place of the injury of the striated fibers. Two hours after injection a cluster of small round basophilic cells was observable at the site of injection and some of them expressed GFP or beta-gal. A few GFP- and beta-gal-positive cells were already detectable 7 days after injection. A large amount of injected cells probably died after injection. Many striated fibers of the urethra became GFP positive from day 7 until day 21, suggesting that few MPCs were allowed to incorporate the divided extremities of the striated fibers from day 7. Unfortunately, we did not observe centronucleated regenerated fibers in this experiment.

Mouse sectioned muscle regenerates following auto-grafting with muscle fragments: a new muscle precursor cells transfer?

It was discovered fifty years ago that a minced skeletal muscle replaced in its bed is able to regenerate. This regeneration is due to the presence of quiescent muscle precursor cells so-called satellite cells in the adult muscle which proliferate and fuse to regenerate new centronucleated fibres when the muscle is damaged. These observations open therapeutic perspectives and, in this study, we attempted to test in the mouse whether fragments of minced muscle regenerate new fibres to fill the gap resulting from the trans-section and retraction of the extensor digitorum longus muscle (EDL). When untreated this gap never regenerates. In agreement with Studitsky, we observed that a minced EDL replaced in its bed regenerates fibres that are spatially disorganised. Minced fragments of abdominus rectus muscle placed in the gap resulting of the trans-section of the EDL regenerate muscle fibres in the gap with a better organisation that in the whole minced muscle. These results could have putative clinical applications, for instance in the prevention of incontinence following prostatectomy which implies removal excision of a large part of the striated urethral sphincter.

Improvement of urethral sphincter deficiency in female rats following autologous skeletal muscle myoblasts grafting.

Sphincteric deficiency is the most common cause of urinary incontinence in humans. Various treatments have lead to disappointing results due to a temporary benefit. Recent studies raised the possibility that sphincteric deficiency could be treated by implanting skeletal myoblasts. In the present study, we developed in the female rat a model of chronic sphincteric defect to assess the benefit of myoblast injection. Sphincter deficiency was induced by freezing, longitudinal sphincterotomy, and notexin injection, respectively, to obtain a reproducible and irreversible incontinence. Autologous tibialis anteriors were cultured to be injected in the best model. Functional results were evaluated by measuring the urethral pressure with an open catheter. Histology was performed in the excised urethras. Of the three techniques, only longitudinal sphincterotomy caused definitive incontinence by irreversibly destroying the striated sphincter muscle fibers: a 45% decrease of the closure pressure was observed 21 days after the sphincterotomy. At this time, we injected myoblasts at the sphincterotomy site. In the sham-injected group (n = 18), the closure pressure decrease was not significantly modified 21 days after injection. By comparison, a return to near normal value was observed after cell grafting (n = 21). These results and those obtained by others strongly suggest that the use of myoblasts could be a potential innovative therapy for urethral deficiencies leading to incontinence.

The urethral striated sphincter in adult male rat.

This study reports the morphology of the urethral sphincter in adult male rats, mainly the histological aspects, the features of the endplates, and the heavy myosin chain distribution in the striated fibres. First, the prostate is entirely out of the striated sphincter, which is surprising when compared to man. Second, the urethral striated sphincter consists of two lateral fascicles separated by an anterior and a posterior strip of connective tissue, which extend from the prostatic urethra (i.e. the part of the urethra which runs though the prostate) to the bulb of the penis. An additional third fascicle of striated muscle (SM) covers the caudal part of the anterior connective strip of the membranous urethra (i.e. the urethra which extends from its prostatic part to the bulb of the penis). In the membranous urethra, the striated sphincter surrounds directly the urethral lumen without intercalated smooth muscle. In urethral cross sections, the endplates detected by alpha-bungarotoxin, which binds to nicotinic receptors, are clustered in the postero-lateral part of the lateral fascicles. The cross-sectional area of the urethral striated fibres shows a bimodal distribution: the largest fibres are located at the periphery of the sphincter and these fibres express only fast myosin heavy chains (MHC) as shown by immunochemistry. The smallest fibres are less numerous and are situated near the lumen co-expressing fast and slow MHC. All the striated fibres express desmin and dystrophin as SM fibres do. Taken together, these results suggest that the urethral striated fibres in male rat present the same characteristics as those of the skeletal muscles. The predominance of fast fibres is consistent with phasic contractions playing a role not only during micturition and urinary continence but also probably during ejaculation.