Satellite cell-specific deletion of Cipc alleviates myopathy in mdx mice

•CIPC is up-regulated during myoblast differentiation•Specific deletion of Cipc in satellite cells alleviates myopathy in mdx mice•Cipc deficiency leads to activation of SP1 to trigger the transcription of Pax7•Loss of Cipc in satellite cells promotes muscle regeneration Skeletal muscle regeneration relies on satellite cells that can proliferate, differentiate, and form new myofibers upon injury. Emerging evidence suggests that misregulation of satellite cell fate and function influences the severity of Duchenne muscular dystrophy (DMD). The transcription factor Pax7 determines the myogenic identity and maintenance of the pool of satellite cells. The circadian clock regulates satellite cell proliferation and self-renewal. Here, we show that the CLOCK-interacting protein Circadian (CIPC) a negative-feedback regulator of the circadian clock, is up-regulated during myoblast differentiation. Specific deletion of Cipc in satellite cells alleviates myopathy, improves muscle function, and reduces fibrosis in mdx mice. Cipc deficiency leads to activation of the ERK1/2 and JNK1/2 signaling pathways, which activates the transcription factor SP1 to trigger the transcription of Pax7 and MyoD. Therefore, CIPC is a negative regulator of satellite cell function, and loss of Cipc in satellite cells promotes muscle regeneration. Skeletal muscle regeneration relies on satellite cells that can proliferate, differentiate, and form new myofibers upon injury. Emerging evidence suggests that misregulation of satellite cell fate and function influences the severity of Duchenne muscular dystrophy (DMD). The transcription factor Pax7 determines the myogenic identity and maintenance of the pool of satellite cells. The circadian clock regulates satellite cell proliferation and self-renewal. Here, we show that the CLOCK-interacting protein Circadian (CIPC) a negative-feedback regulator of the circadian clock, is up-regulated during myoblast differentiation. Specific deletion of Cipc in satellite cells alleviates myopathy, improves muscle function, and reduces fibrosis in mdx mice. Cipc deficiency leads to activation of the ERK1/2 and JNK1/2 signaling pathways, which activates the transcription factor SP1 to trigger the transcription of Pax7 and MyoD. Therefore, CIPC is a negative regulator of satellite cell function, and loss of Cipc in satellite cells promotes muscle regeneration. Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder caused by mutations of the gene encoding the protein dystrophin, which anchors the extracellular matrix to the cytoskeleton inside a myofiber. Many Duchenne mutations are caused by multiple exon deletions. Some of these deletions (or insertions) can result in frameshift mutations or nonsense mutations, leading to the production of truncated and non-functional dystrophin protein (Duan et al., 2021Duan D. Goemans N. Takeda S. Mercuri E. Aartsma-Rus A. Duchenne muscular dystrophy.Nat. Rev. Dis. Primers. 2021; 7: 13https://doi.org/10.1038/s41572-021-00248-3Crossref PubMed Scopus (100) Google Scholar; Verhaart and Aartsma-Rus, 2019Verhaart I.E.C. Aartsma-Rus A. Therapeutic developments for Duchenne muscular dystrophy.Nat. Rev. Neurol. 2019; 15: 373-386https://doi.org/10.1038/s41582-019-0203-3Crossref PubMed Scopus (166) Google Scholar). Dystrophin is critical for stabilizing the plasma membrane of a myofiber during muscle contraction. In the absence of dystrophin, muscle contraction causes myofiber necrosis, triggering subsequent inflammation and fibrosis, leading to progressive muscle weakness and wasting. 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Overexpression of Cipc in HEK293 cells inhibits cell proliferation through the inactivation of ERK (Matsunaga et al., 2016Matsunaga R. Nishino T. Yokoyama A. Nakashima A. Kikkawa U. Konishi H. Versatile function of the circadian protein CIPC as a regulator of Erk activation.Biochem. Biophys. Res. Commun. 2016; 469: 377-383https://doi.org/10.1016/j.bbrc.2015.11.117Crossref PubMed Scopus (2) Google Scholar). This study demonstrated that myopathy in mdx mice can be alleviated by deleting Cipc in satellite cells. Using a satellite-cell-specific Cipc knockout approach, we demonstrate that CIPC inhibits Pax7 expression and regenerative myogenesis. Our results show that deletion of Cipc in satellite cells activates ERK1/2 and JNK1/2 signaling pathways, leading to phosphorylation of the transcription factor SP1, which augments the gene expression of Pax7 and MyoD. To determine the role of CIPC in satellite cell function, we generated satellite-cell-specific Cipc knockout (Cipcscko) mice by crossing Cipcflox/flox mice with Pax7-Cre mice (Figure S1A), and the genotype was identified by PCR (Figure S1B). The efficiency of Cipc deletion in satellite cells was determined by qRT-PCR (Figure S1C). We then isolated primary myoblasts from 8-week-old wild-type (WT) mice and Cipcflox/flox and Cipcscko mice and investigated whether myoblasts of adult mice express CIPC by immunostaining and western blot. To verify the purity of primary myoblasts, we performed Pax7 staining, and the results showed that most cells were Pax7+ (Figures S2A and S2B). Confocal microscopy of myoblasts revealed co-localization of CIPC and Pax7 in primary myoblasts, and CIPC was expressed in the cytoplasm of myoblasts in WT and Cipcflox/flox mice but was not expressed in myoblasts from Cipcscko mice (Figure 1A ). Western blot results showed that CIPC was expressed in myoblasts of Cipcflox/flox mice, but not in myoblasts of Cipcscko mice (Figures 1B and 1C). Myoblasts undergo differentiation when cultured in the differentiation medium (DM) containing 2% horse serum. The differentiation is characterized by gradual reduction of Pax7 and increased myogenic markers, such as MyoD, myogenin, myosin heavy chain (MyHC) (Olguin and Olwin, 2004Olguin H.C. Olwin B.B. Pax-7 up-regulation inhibits myogenesis and cell cycle progression in satellite cells: a potential mechanism for self-renewal.Dev. Biol. 2004; 275: 375-388https://doi.org/10.1016/j.ydbio.2004.08.015Crossref PubMed Scopus (372) Google Scholar). To find out the relationship between CIPC and myogenic markers, we performed western blot using the cell lysates of WT myoblasts. We analyzed the levels of CIPC, Pax7, MyoD, and MyHC at different time points after switching the cells to DM. The results showed that CIPC, MyoD, and MyHC levels were increased during differentiation, whereas the level of Pax7 was decreased (Figures 1D and 1E). Immunostaining revealed that CIPC is expressed in the differentiated myotubes (Figure S2C). qRT-PCR also confirmed the inverse relationship between Pax7 and Cipc expression during differentiation (Figures S2D and S2E). To determine whether CIPC negatively regulates Pax7, we overexpressed Cipc in myoblasts and analyzed the expression of Pax7 by qRT-PCR. The results showed that Pax7 expression was reduced when Cipc was overexpressed (Figures S2F and S2G). Collectively, the results suggest that CIPC is negatively correlated with Pax7 expression. To assess the impact of Cipc deletion on muscle regeneration, BaCl2 solution was locally injected into the tibial anterior (TA) muscle of adult Cipcflox/flox and Cipcscko mice. The TA muscle was isolated at different time points after injury. Muscle weight was decreased in Cipcflox/flox after injury at day 5, while that of injured Cipcscko mice did not change significantly compared with uninjured mice (Figures S1D and S1E). H&E staining was performed to analyze muscle morphology and the numbers of centronucleated fibers (CNFs) myofibers. The number of newly formed CNFs and cross-sectional area (CSA) of CNFs was higher in TA muscle of Cipcscko mice than that of littermate Cipcflox/flox mice after BaCl2 injection (Figures 1F–1I). In Cipcscko mice, reconstitution of muscle structure was completed at day 10 post-injury, where muscle sections of the Cipcflox/flox mice or the Pax7-Cre mice still showed areas of regeneration (Figures 1F and S1F). These data indicate that the deletion of Cipc enhances muscle regeneration. The expression of the embryonic isoform of MyHC (eMyHC) is a hallmark of muscle regeneration (Addicks et al., 2019Addicks G.C. Brun C.E. Sincennes M.C. Saber J. Porter C.J. Francis Stewart A. Ernst P. Rudnicki M.A. MLL1 is required for PAX7 expression and satellite cell self-renewal in mice.Nat. Commun. 2019; 10: 4256https://doi.org/10.1038/s41467-019-12086-9Crossref PubMed Scopus (17) Google Scholar; Liu et al., 2020Liu Z. Zhang X. Lei H. Lam N. Carter S. Yockey O. Xu M. Mendoza A. Hernandez E.R. Wei J.S. et al.CASZ1 induces skeletal muscle and rhabdomyosarcoma differentiation through a feed-forward loop with MYOD and MYOG.Nat. Commun. 2020; 11: 911https://doi.org/10.1038/s41467-020-14684-4Crossref PubMed Scopus (13) Google Scholar; Schiaffino et al., 2015Schiaffino S. Rossi A.C. Smerdu V. Leinwand L.A. Reggiani C. Developmental myosins: expression patterns and functional significance.Skelet. Muscle. 2015; 5: 22https://doi.org/10.1186/s13395-015-0046-6Crossref PubMed Scopus (210) Google Scholar). Immunofluorescence staining showed that the number of eMyHC+ fibers was increased in injured muscles from Cipcscko mice compared with Cipcflox/flox mice up to 5 days post-injury. Then the number of eMyHC+ fibers gradually decreased as muscle regeneration neared completion (Figures S3A, S3B, and S3G). The CSA of eMyHC+ fibers followed a similar pattern (Figures S3A, S3C, and S3G). qRT-PCR confirmed that eMyHC mRNA (Myh3) expression in Cipcscko muscles was higher than that of Cipcflox/flox muscles (Figure S3D). Western blot analysis showed that the expression of eMyHC protein peaked on day 5 after injury in Cipcscko muscles. However, eMyHC protein did not reach the peak until day 7 in Cipcflox/flox muscles (Figures S3E and S3F). These findings suggest that deletion of Cipc in satellite cells promotes myogenic regeneration. The increased expression of eMyHC in the injured muscle of Cipcscko mice suggests that Cipc deletion led to an increased number of Pax7+ satellite cells, which subsequently differentiated and formed new myofibers. To determine whether CIPC affects satellite cell proliferation and quiescence, we performed immunofluorescence staining on muscle sections from uninjured or injured Cipcscko and Cipcflox/flox mice using antibodies against Pax7 and laminin (Figure 2A ). The number of Pax7+ cells was increased 2-fold in the uninjured muscles of Cipcscko mice compared with that of Cipcflox/flox mice (Figures 2A and 2B), and this increase was even more evident on day 5 post-injury (4-fold) (Figures 2A and 2C). By the end of the regeneration process at day 21 after injury, satellite cells in the TA muscle have returned to a quiescent state, and the number of Pax7+ cells in TA muscle of Cipcscko is still higher than that of Cipcflox/flox mice (Figures 2A and 2D). These results indicate that loss of Cipc increases the number of quiescent satellite cells in vivo and promotes their proliferation upon injury. To further investigate whether loss of Cipc affects the differentiation of satellite cells, we performed immunofluorescence staining on muscle sections from BaCl2-injured Cipcflox/flox mice and Cipcscko mice using antibodies against myogenin (Myog) and laminin (Figure 2E). On day 5 post-injury, Cipcscko mice exhibited a 3-fold increase in the numbers of myogenin-expressing cells compared with Cipcflox/flox mice (Figures 2E and 2F). The increased number of Myog+ cells could be a consequence of the increased number of satellite cells. Alternatively, it could suggest that loss of Cipc promotes differentiation. BaCl2 solution was locally injected into the TA muscles of adult WT and mdx mice to assess the expression level of Cipc during myogenesis. The mRNA levels of Cipc and Pax7 in TA muscle of WT and mdx mice after 0, 3, 5, 10, 15, and 21 days of injury were determined with qRT-PCR. Although the expression pattern of Pax7 is similar in the injured TA muscle of mdx mice, its expression level is significantly lower than WT mice at day 3 post-injury (Figure S4A). The mRNA levels of Cipc decreased at day 3 post-injury in TA muscles of WT mice compared with day 0. Then its expression level gradually increased and reached