Jonathan R. Beauchamp

Muscle satellite cells adopt divergent fates a mechanism for self-renewal?

Growth, repair, and regeneration of adult skeletal muscle depends on the persistence of satellite cells: muscle stem cells resident beneath the basal lamina that surrounds each myofiber. However, how the satellite cell compartment is maintained is unclear. Here, we use cultured myofibers to model muscle regeneration and show that satellite cells adopt divergent fates. Quiescent satellite cells are synchronously activated to coexpress the transcription factors Pax7 and MyoD. Most then proliferate, down-regulate Pax7, and differentiate. In contrast, other proliferating cells maintain Pax7 but lose MyoD and withdraw from immediate differentiation. These cells are typically located in clusters, together with Pax7−ve progeny destined for differentiation. Some of the Pax7+ve/MyoD−ve cells then leave the cell cycle, thus regaining the quiescent satellite cell phenotype. Significantly, noncycling cells contained within a cluster can be stimulated to proliferate again. These observations suggest that satellite cells either differentiate or switch from terminal myogenesis to maintain the satellite cell pool.

Pax7 and myogenic progression in skeletal muscle satellite cells

Skeletal muscle growth and regeneration are attributed to satellite cells - muscle stem cells resident beneath the basal lamina that surrounds each myofibre. Quiescent satellite cells express the transcription factor Pax7 and when activated, coexpress Pax7 with MyoD. Most then proliferate, downregulate Pax7 and differentiate. By contrast, others maintain Pax7 but lose MyoD and return to a state resembling quiescence. Here we show that Pax7 is able to drive transcription in quiescent and activated satellite cells, and continues to do so in those cells that subsequently cease proliferation and withdraw from immediate differentiation. We found that constitutive expression of Pax7 in satellite-cell-derived myoblasts did not affect MyoD expression or proliferation. Although maintained expression of Pax7 delayed the onset of myogenin expression it did not prevent, and was compatible with, myogenic differentiation. Constitutive Pax7 expression in a Pax7-null C2C12 subclone increased the proportion of cells expressing MyoD, showing that Pax7 can act genetically upstream of MyoD. However these Pax7-null cells were unable to differentiate into normal myotubes in the presence of Pax7. Therefore Pax7 may be involved in maintaining proliferation and preventing precocious differentiation, but does not promote quiescence.

Dynamics and mediators of acute graft attrition after myoblast transplantation to the heart

Survival and proliferation of skeletal myoblasts within the cardiac environment are crucial to the therapeutic efficacy of myoblast transplantation to the heart. We have analyzed the early dynamics of myoblasts implanted into the myocardium and investigated the mechanisms underlying graft attrition. At 10 min after implantation of [14C]thymidine‐labeled male myoblasts into female mice hearts, 14C measurement showed that 39.2 ± 3.0% of the grafted cells survived, and this steadily decreased to 16.0 ± 1.7% by 24 h and to 7.4 ± 0.9% by 72 h. PCR of male‐ specific Smcy gene calculated that the total (surviving plus proliferated) number of donor‐derived cells was 18.3 ± 1.6 and 23.3 ± 1.3% at 24 and 72 h, respectively, indicating that proliferation of the surviving cells began after 24 h. Acute inflammation became prominent by 24 h and was reduced by 72 h as indicated by myeloperoxidase activity and histological findings. Multiplex RT‐PCR revealed corresponding changes in IL‐1β, TGF‐β, IL‐6, and TNF‐α expression. Treatment with CuZn‐superoxide dismutase attenuated the initial rapid death and resulted in enhanced cell numbers afterward, giving a twofold increased total number at 72 h compared with the nontreatment. This effect was associated with reduced inflammatory response, suggesting a causative role for superoxide in the initial rapid graft death and subsequent inflammation. These data describe the early dynamics of myoblasts implanted into the myocardium and suggest that initial oxidative stress and following inflammatory response may be important mechanisms contributing to acute graft attrition, both of which could be potential therapeutic targets to improve the efficiency of cell transplantation to the heart.

Transplanted primary neonatal myoblasts can give rise to functional satellite cells as identified using the Myf5nlacZl+ mouse.

Myoblast transplantation is a potential therapeutic approach for the genetic modification of host skeletal muscle tissue. To be considered an effective, long-lived method of delivery, however, it is essential that at least a proportion of the transplanted cells also retain their proliferative potential. We sought to investigate whether transplanted neonatal myoblasts can contribute to the satellite cell compartment of adult skeletal muscle by using the Myf5nlacZ/+ mouse. The Myf5nlacZ/+ mouse has nlacZ targeted to the Myf5 locus resulting in β-galactosidase activity in quiescent satellite cells. Following transplantation, β-galactosidase-labelled nuclei were detected in host muscles, showing that donor cells had been incorporated. Significantly, β-galactosidase-positive, and therefore donor-derived, satellite cells were detected. When placed in culture, β-galactosidase marked myogenic cells emanated from the parent fibre. These observations demonstrate that cell transplantation not only results in the incorporation of donor nuclei into the host muscle syncytia, but also that the donor cells can become functional satellite cells. The Myf5nlacZ/+ mouse therefore provides a novel and specific marker for determining the contribution of transplanted cells to the satellite cell pool.

Transplantation of skeletal myoblasts secreting an IL-1 inhibitor modulates adverse remodeling in infarcted murine myocardium

After myocardial infarction (MI), adverse remodeling with left ventricular (LV) dilatation is a major determinant of poor outcome. Skeletal myoblast (SkM) implantation improves cardiac function post-MI, although the mechanism is unclear. IL-1 influences post-MI hypertrophy and collagen turnover and is implicated in SkM death after grafting. We hypothesized that SkM expressing secretory IL-1 receptor antagonist (sIL-1ra) at MI border zones would specifically attenuate adverse remodeling and exhibit improved graft cell number. Stable murine male SkM lines (5 × 105 cells), expressing or nonexpressing (cont) for sIL-1ra, were implanted into infarct border zones of female nude mice immediately after left coronary artery occlusion. LV ejection fraction (LVEF), end-diastolic diameter, and transmitral peak early/late (E/A) flow velocity ratio were determined by echocardiography. Cardiac myocyte hypertrophy and fibrosis were assessed by morphometry, picrosirius red staining, and hydroxyproline assay. At 3 weeks, cont-SkM-engrafted hearts showed reduced hypertrophy, improved LVEF (55.7 ± 1.2% vs. MI-only: 40.3 ± 2.9%), and preserved E/A ratios. sIL-1ra-SkM implantation enhanced these effects (LVEF, 67.0 ± 2.3%) and significantly attenuated LV dilatation (LV end-diastolic diameter, 4.0 ± 1.1 mm vs. cont-SkM, 4.5 ± 1.2 mm vs. MI-only, 4.8 ± 1.8 mm); this was associated with greater graft numbers, as shown by PCR for male-specific smcy gene. Enzyme zymography showed attenuated matrix metalloproteinase-2 and -9 up-regulation post-MI by either donor SkM type, although infarct-remote zone collagen was reduced only with sIL-1ra-SkM. These results suggest that SkM implantation improves cardiac function post-MI by modulation of adverse remodeling, and that this effect can be significantly enhanced by targeting IL-1 as a key upstream regulator of both adverse remodeling and graft cell death.

Role of Interleukin-1β in Acute Inflammation and Graft Death After Cell Transplantation to the Heart

Background—Poor survival of grafted cells is a major factor hindering the therapeutic effect of cell transplantation; however, the causes of cell death remain unclear. We hypothesized that interleukin-1&bgr; (IL-1&bgr;) might play a role in the acute inflammatory response and graft death after cell transplantation and that inhibition of IL-1&bgr; might improve graft survival. Methods and Results—14C-labeled male skeletal muscle precursor cells were implanted into female mouse hearts by direct intramuscular injection. The amount of 14C-label provides an estimate of the surviving cell number, whereas the amount of male-specific Smcy gene measured by polymerase chain reaction indicates the total (surviving+proliferated) number of donor-derived cells. At 10 minutes after implantation, 44.8±2.4% of the grafted cells survived and this steadily decreased to 14.6±1.1% by 24 hours, and to 7.9±0.6% by 72 hours (n=6 in each point). Proliferation of the surviving cells, which began after 24 hours, resulted in an increase in the total cell number from 15.5±0.8% at 24 hours to 24.4±1.6% at 72 hours. Acute inflammation was prominent at 24 hours and was reduced by 72 hours, in parallel with IL-1&bgr; expression. Administration of anti–IL-1&bgr; antibody improved graft survival at both 24 (25.6±1.6%) and 72 hours (14.8±1.1%) and resulted in a 2-fold increase in the total cell number at 72 hours (45.8±2.4%). The effects of IL-1&bgr; inhibition corresponded with a reduced inflammatory response. Conclusion—IL-1&bgr; is involved in acute inflammation and graft death after direct intramyocardial cell transplantation. Targeted inhibition of IL-1&bgr; may be a useful strategy to improve graft survival.

Pax7 distribution in human skeletal muscle biopsies and myogenic tissue cultures

Demonstration of the importance of the paired box transcription factor Pax7 for the murine myosatellite cell population, with persistent expression in mature skeletal muscle, prompted us to investigate the distribution of Pax7 protein in biopsy samples of normal and pathological human skeletal limb muscle. Immunostaining for M-cadherin, an adhesion molecule present at the interface between myofibre and satellite cell, and the characteristic position adjacent to the muscle fibre and beneath the fibre’s basement membrane were used to identify satellite cells. Anti-Pax7 reactivity was found in the majority of satellite cells but a small population was Pax7 negative. Neither could we identify Pax7-positive nuclei in freshly regenerating myotubes or in presumed myoblasts in these biopsies. Similarly, in myogenic cell cultures derived from the explantation of human foetal muscle Pax7 expression was low or undetectable at the proliferative myoblast stage but it became prominent in an increasing proportion of mononucleate cells after the induction of differentiation. This expression was, however, restricted to mononucleate cells; it did not persist into the differentiation stage of newly formed multinucleate myotubes. Despite this, in the biopsy samples, we occasionally found Pax7-positive nuclei in muscle fibres that seemed to be undergoing degenerative changes. Most of these were found to be the nuclei of cells engaged in focal regenerative processes, but Pax7 re-expression by myonuclei “in distress” cannot be ruled out entirely.

Myogenic cell proliferation and generation of a reversible tumorigenic phenotype are triggered by preirradiation of the recipient site

Environmental influences have profound yet reversible effects on the behavior of resident cells. Earlier data have indicated that the amount of muscle formed from implanted myogenic cells is greatly augmented by prior irradiation (18 Gy) of the host mouse muscle. Here we confirm this phenomenon, showing that it varies between host mouse strains. However, it is unclear whether it is due to secretion of proliferative factors or reduction of antiproliferative agents. To investigate this further, we have exploited the observation that the immortal myogenic C2 C12 cell line forms tumors far more rapidly in irradiated than in nonirradiated host muscle. We show that the effect of preirradiation on tumor formation is persistent and dose dependent. However, C2 C12 cells are not irreversibly compelled to form undifferentiated tumor cells by the irradiated muscle environment and are still capable of forming large amounts of muscle when reimplanted into a nonirradiated muscle. In a clonal analysis of this effect, we discovered that C2 C12 cells have a bimodal propensity to form tumors; some clones form no tumors even after extensive periods in irradiated graft sites, whereas others rapidly form extensive tumors. This illustrates the subtle interplay between the phenotype of implanted cells and the factors in the muscle environment.

A dual-marker system for quantitative studies of myoblast transplantation in the mouse.

BACKGROUND Myoblast transplantation (MT) is a potential approach for gene transfer into skeletal muscle, the efficiency of which depends upon the number of copies of donor genome incorporated into the host tissue. We have developed a system for quantitative studies of MT that measures amounts of donor-derived genome in host muscles and estimates the contributions of donor cell survival and proliferation in vivo. METHODS [14C]thymidine-labeled, male myoblasts were transplanted into female muscles, providing two donor cell markers, Y chromosome and [14C]. The markers were measured in muscle extracts by slot blotting and scintillation counting, respectively. RESULTS In each extract, the amount of Y chromosome was used to quantify donor-derived genome, whereas the radiolabel provided an estimate of cell survival. Furthermore, the different modes of inheritance of the markers meant that proliferation of surviving donor cells was detected as a change in marker ratio. CONCLUSIONS This system provides a method for assessing potential improvements of MT.

Mouse myotomes pairs exhibit left-right asymmetric expression of MLC3F and α-skeletal actin

Most muscle originates from the myotomal compartment of the somites, paired structures flanking the neural tube. Whereas vertebrate embryos show molecular and morphological asymmetry about the left–right body axis, somitic myogenesis is thought to occur symmetrically. Here, we provide the first evidence that myotome pairs are transiently left–right asymmetric, with higher expression of α‐skeletal actin and myosin light chain 3F (MLC3F) on the left side between embryonic day 9.5–10.25. In iv mutants with situs inversus, the asymmetric expression of α‐skeletal actin and MLC3F was inverted, showing that this process is regulated by global left–right axis cues, initiated before gastrulation. However, although left–sided identity is later maintained by Pitx2 genes, we found that Pitx2c null embryos have normal left‐biased expression of α‐skeletal actin and MLC3F. Myotome asymmetry, therefore, is downstream of the iv mutation but upstream of, or unrelated to, the Pitx2c pathway. Developmental Dynamics 231:795–800, 2004.