TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration.

Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-kappaB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.

Diseased muscles that lack dystrophin or laminin-alpha2 have altered compositions and proliferation of mononuclear cell populations.

BACKGROUND: Multiple types of mononucleate cells reside among the multinucleate myofibers in skeletal muscles and these mononucleate cells function in muscle maintenance and repair. How neuromuscular disease might affect different types of muscle mononucleate cells had not been determined. In this study, therefore, we examined how two neuromuscular diseases, dystrophin-deficiency and laminin-alpha2-deficiency, altered the proliferation and composition of different subsets of muscle-derived mononucleate cells. METHODS: We used fluorescence-activated cell sorting combined with bromodeoxyuridine labeling to examine proliferation rates and compositions of mononuclear cells in diseased and healthy mouse skeletal muscle. We prepared mononucleate cells from muscles of mdx (dystrophin-deficient) or Lama2-/- (laminin-alpha2-deficient) mice and compared them to cells from healthy control muscles. We enumerated subsets of resident muscle cells based on Sca-1 and CD45 expression patterns and determined the proliferation of each cell subset in vivo by BrdU incorporation. RESULTS: We found that the proliferation and composition of the mononucleate cells in dystrophin-deficient and laminin-alpha2-deficient diseased muscles are different than in healthy muscle. The mdx and Lama2-/- muscles showed similar significant increases in CD45+ cells compared to healthy muscle. Changes in proliferation, however, differed between the two diseases with proliferation increased in mdx and decreased in Lama2-/- muscles compared to healthy muscles. In particular, the most abundant Sca-1-/CD45- subset, which contains muscle precursor cells, had increased proliferation in mdx muscle but decreased proliferation in Lama2-/- muscles. CONCLUSION: The similar increases in CD45+ cells, but opposite changes in proliferation of muscle precursor cells, may underlie aspects of the distinct pathologies in the two diseases.

Muscle-specific BCL2 expression ameliorates muscle disease in laminin {alpha}2-deficient, but not in dystrophin-deficient, mice.

To examine the role of apoptosis in neuromuscular disease progression, we have determined whether pathogenesis in dystrophin-deficient (mdx) and laminin alpha2-deficient (Lama2-null) mice is ameliorated by overexpression of the anti-apoptosis protein BCL2 in diseased muscles. The mdx mice are a model for the human disease, Duchenne muscular dystrophy (DMD), and the Lama2-null mice are a model for human congenital muscular dystrophy type 1A (MDC1A). For these studies, we generated transgenic mice that overexpressed human BCL2 under control of muscle-specific MyoD or MRF4 promoter fragments. We then used cross-breeding to introduce the transgenes into diseased mdx or Lama2-null mice. In mdx mice, we found that overexpression of BCL2 failed to produce any significant differences in muscle pathology. In contrast, in the Lama2-null mice, we found that muscle-specific expression of BCL2 led to a several-fold increase in lifespan and an increased growth rate. Thus, BCL2-mediated apoptosis appears to play a significant role in pathogenesis of laminin alpha2 deficiency, but not of dystrophin deficiency, suggesting that therapies designed to ameliorate disease by inhibition of apoptosis are more likely to succeed in MDC1A than in DMD.

Inhibition of apoptosis improves outcome in a model of congenital muscular dystrophy.

The most common form of human congenital muscular dystrophy (CMD) is caused by mutations in the laminin-alpha2 gene. Loss of laminin-alpha2 function in this autosomal recessive type 1A form of CMD results in neuromuscular dysfunction and, often, early death. Laminin-alpha2-deficient skeletal muscles in both humans and mice show signs of muscle cell death by apoptosis. To examine the significance of apoptosis in CMD1A pathogenesis, we determined whether pathogenesis in laminin-alpha2-deficient (Lama2(-/-)) mice could be ameliorated by inhibiting apoptosis through either (a) inactivation of the proapoptosis protein Bax or (b) overexpression of the antiapoptosis protein Bcl-2 from a muscle-specific transgene. We found that both of these genetic interventions produced a several-fold increase in the lifespan of Lama2(-/-) mice. Bax inactivation also improved postnatal growth rate and myofiber histology and decreased fixed contractures of Lama2(-/-) mice. Thus, Bcl-2 family-mediated apoptosis contributes significantly to pathogenesis in the mouse model of CMD1A, and antiapoptosis therapy may be a possible route to amelioration of neuromuscular dysfunction due to laminin-alpha2 deficiency in humans.

Immortalization of mouse myogenic cells can occur without loss of p16INK4a, p19ARF, or p53 and is accelerated by inactivation of Bax.

BACKGROUND: Upon serial passaging of mouse skeletal muscle cells, a small number of cells will spontaneously develop the ability to proliferate indefinitely while retaining the ability to differentiate into multinucleate myotubes. Possible gene changes that could underlie myogenic cell immortalization and their possible effects on myogenesis had not been examined. RESULTS: We found that immortalization occurred earlier and more frequently when the myogenic cells lacked the pro-apoptotic protein Bax. Furthermore, myogenesis was altered by Bax inactivation as Bax-null cells produced muscle colonies with more nuclei than wild-type cells, though a lower percentage of the Bax-null nuclei were incorporated into multinucleate myotubes. In vivo, both the fast and slow myofibers in Bax-null muscles had smaller cross-sectional areas than those in wild-type muscles. After immortalization, both Bax-null and Bax-positive myogenic cells expressed desmin, retained the capacity to form multinucleate myotubes, expressed p19ARF protein, and retained p53 functions. Expression of p16INK4a, however, was found in only about half of the immortalized myogenic cell lines. CONCLUSIONS: Mouse myogenic cells can undergo spontaneous immortalization via a mechanism that can include, but does not require, loss of p16INK4a, and also does not require inactivation of p19ARF or p53. Furthermore, loss of Bax, which appears to be a downstream effector of p53, accelerates immortalization of myogenic cells and alters myogenesis.

Up-regulation of MHC class I expression accompanies but is not required for spontaneous myopathy in dysferlin-deficient SJL/J mice.

We found that up-regulation of major histocompatibility complex (MHC) class I expression accompanies, but is not required for, appearance of spontaneous myopathy in SJL/J mice. In some neuromuscular diseases, MHC class I expression is markedly up-regulated in muscles, though the consequences of this up-regulation for pathology are not clear. To study MHC class I in myopathy, we compared muscles of SJL/J mice to muscles of SJL/J mice that were also MHC class I-deficient due to targeted mutation in the beta-2-microglobulin gene (SJL/J B2m (-/-) mice). SJL/J mice show spontaneous myopathy and have a mutation in the dysferlin gene, a gene which is also mutated in human limb-girdle muscular dystrophy type 2B (LGMD2B). Muscles of eight-month-old SJL/J mice had higher levels of MHC class I expression than muscles of either C57BL/6J (wild-type) or SJL/J B2m (-/-) mice. In contrast, the percentage of abnormal muscle fibers was similar in SJL/J and SJL/J B2m (-/-) muscles. Invading Mac-1(+) cells were most abundant in SJL/J B2m (-/-) muscles, moderately abundant in SJL/J muscles, and rare in C57BL/6J muscles. Thus, MHC class I was markedly up-regulated in SJL/J muscles, but this high level of MHC class I was not necessary for the appearance of myopathy.