Bendi Gong

Temporal and spatial mRNA expression patterns of TGF-β1, 2, 3 and TβRI, II, III in skeletal muscles of mdx mice

Abstract To address potential regulatory roles of TGF-β1 in muscle inflammation and fibrosis associated with dystrophin deficiency, we performed quantitative RT-PCR and in situ hybridization to characterize the temporal and spatial mRNA expression patterns of TGF-β1 and other TGF-β subfamily members, TGF-β2 and TGF-β3, as well as their receptors, in quadriceps and diaphragm muscles of mdx mice. TGF-β1 mRNA was markedly upregulated in the endomysial inflammatory cells and regenerating fibers of mdx quadriceps and diaphragm, with the mRNA levels correlated with the degree of endomysial inflammation. Upregulation of TGF-β2, β3, and their receptors was also appreciated but to a much lesser degree. While high levels of TGF-β1 mRNA remained in the aging mdx quadriceps but not the diaphragm, progressive fibrosis only occurred in the diaphragm. Our data support a regulatory role for TGF-β1 in muscle inflammation in mdx mice. It also suggests different susceptibility of quadriceps and diaphragm muscles to fibrosis induced by TGF-β1 signaling pathway.

Comprehensive expression profiling by muscle tissue class and identification of the molecular niche of extraocular muscle

Muscle tissue is an elegant model for biologic integration of structure with function and is frequently affected by a variety of inherited diseases. Traditional muscle classes‐‐skeletal, cardiac, and smooth‐‐share basic aspects of contractile and energetics mechanisms but also have distinctive role‐specific adaptations. We used large‐scale oligonucleotide microarrays to broaden knowledge of the adaptive expression patterns underlying muscle tissue differences and to identify transcript subsets that are most likely to represent candidate disease genes. Using stringent analysis criteria, we found ≥95 transcripts, which were preferentially expressed by each muscle class and were validated by inclusion of known muscle class‐specific and inherited disease‐related genes. Differentially expressed transcripts not previously identified as class‐specific extend understanding of muscle class transcriptomes and may represent novel muscle‐specific disease genes. We also analyzed the expression profile of extraocular muscle, which is divergent from other skeletal muscles, in the broader context of all major muscle classes. Data show that the extraocular muscle phenotype results from the combination of tissue‐specific transcripts, novel expression levels of skeletal muscle transcripts, and partial sharing of gene expression patterns with cardiac and smooth muscle. These, and additional proteomic data, establish that extraocular muscle does not constitute a distinctive muscle class but that it does occupy a novel niche within the skeletal muscle class.

Analysis of gene expression differences between utrophin/dystrophin-deficient vs mdx skeletal muscles reveals a specific upregulation of slow muscle genes in limb muscles

Dystrophin deficiency leads to the progressive muscle wasting disease Duchenne muscular dystrophy (DMD). Dystrophin-deficient mdx mice are characterized by skeletal muscle weakness and degeneration but they appear outwardly normal in contrast to DMD patients. Mice lacking both dystrophin and the dystrophin homolog utrophin [double knockout (dko)] have muscle degeneration similar to mdx mice, but they display clinical features similar to DMD patients. Dko limb muscles also lack postsynaptic membrane folding and display fiber-type abnormalities including an abundance of phenotypically oxidative muscle fibers. Extraocular muscles, which are spared in mdx mice, show a significant pathology in dko mice. In this study, microarray analysis was used to characterize gene expression differences between mdx and dko tibialis anterior and extraocular skeletal muscles in an effort to understand the phenotypic differences between these two dystrophic mouse models. Analysis of gene expression differences showed that upregulation of slow muscle genes specifically characterizes dko limb muscle and suggests that upregulation of these genes may directly account for the more severe phenotype of dko mice. To investigate whether any upregulation of slow genes is retained in vitro, independent of postsynaptic membrane abnormalities, we derived mdx and dko primary myogenic cultures and analyzed the expression of Myh7 and Myl2. Real-time reverse transcriptase-polymerase chain reaction analysis demonstrates that transcription of these slow genes is also upregulated in dko vs mdx myotubes. This data suggests that at least part of the fiber-type abnormality is due directly to the combined absence of utrophin and dystrophin and is not an indirect effect of the postsynaptic membrane abnormalities.

Postnatal suppression of myomesin, muscle creatine kinase and the M-line in rat extraocular muscle

SUMMARY The M-line and its associated creatine kinase (CK) M-isoform (CK-M) are ubiquitous features of skeletal and cardiac muscle. The M-line maintains myosin myofilaments in register, links the contractile apparatus to the cytoskeleton for external force transfer and localizes CK-based energy storage and transfer to the site of highest ATP demand. We establish here that the muscle group responsible for movements of the eye, extraocular muscle (EOM), is divergent from other striated muscles in lacking both an M-line and its associated CK-M. Although an M-line forms during myogenesis, both in vivo and in vitro, it is actively repressed after birth. Transcripts of the major M-line structural proteins, myomesin 1 and myomesin 2, follow the same pattern of postnatal downregulation, while the embryonic heart-specific EH-myomesin 1 transcript is expressed early and retained in adult eye muscle. By immunocytochemistry, myomesin protein is absent from adult EOM sarcomeres. M-line suppression does not occur in organotypic co-culture with oculomotor motoneurons, suggesting that the mechanism for suppression may lie in muscle group-specific activation or workload patterns experienced only in vivo. The M-line is, however, still lost in dark-reared rats, despite the developmental delay this paradigm produces in the visuomotor system and EOMs. EOM was low in all CK isoform transcripts except for the sarcomeric mitochondrial (Ckmt2) isoform. Total CK enzyme activity of EOM was one-third that of hindlimb muscle. These findings are singularly unique among fast-twitch skeletal muscles. Since EOM exhibits isoform diversity for other sarcomeric proteins, the M-line/CK-M divergence probably represents a key physiological adaptation for the unique energetics and functional demands placed on this muscle group in voluntary and reflexive eye movements.