Journal: Advanced Science

Article Title: The Evolutionarily Conserved TPM1 Super‐Enhancer Drives Skeletal Muscle Regeneration via Mechanotransduction Signaling

doi: 10.1002/advs.202514271

Figure Lengend Snippet: Deletion of TPM1_SE reduces muscle mass and delays skeletal muscle regeneration. A) Longitudinal measurement of body weight in male mice from 3 to 8 weeks. B) Growth rate comparison between mCRE_26 flox/flox and mCRE_26 cKO mice. C and D) Gross morphology and mass of GAS, TA, SOL, and QUA muscles in adult mCRE_26 flox/flox and mCRE_26 cKO mice. E) TA muscles weight‐to‐length ratios were calculated to assess muscle integrity. F) H&E staining of TA muscles sections revealed structural alterations in mCRE_26 cKO mice (scale bars, 100 µm). G) RT‐qPCR analysis showed decreased expression of TPM1, Pax7, MyOD1, MyOG, and MyHC in knockout mice. H and I) Gross phenotype of TA muscles following cardiotoxin (CTX)‐induced injury in control and knockout groups. Muscles were harvested at day 28 or day 14 after the last CTX injection for secondary or tertiary injury, respectively. J and K) Post‐injury TA muscles weight‐to‐length ratios were compared. L) Histological analysis of regenerating TA muscles via H&E staining; scale bars, 100 µm. M, N) Quantification of myofiber density following injury. O) Representative immunofluorescence images of eMyHC (green, differentiated myofibers), and DAPI (blue, nuclei) in regenerating TA muscle sections. Boxed regions are shown at higher magnification to the right. Scale bars, 20 µm. P and Q) Proportion of regenerated myofibers in CTX‐injured TA muscles cross‐sections quantified from 500 nucleus‐containing fibers using ImageJ and Image‐Pro Plus. R and S) RT‐qPCR validation of muscle regeneration markers Pax7, MyOD1, MyOG, and MyHC. Data are shown as mean ± SEM ( n = 5).

Article Snippet: Immunofluorescence staining of muscle sections was conducted as described by He et al. [ ] After H&E staining, sections were incubated sequentially with primary antibodies against emyhc (1: 200, Servicebio, Wuhan, China), appropriate secondary antibodies, and DAPI at 4 °C.

Techniques: Comparison, Muscles, Staining, Quantitative RT-PCR, Expressing, Knock-Out, Control, Injection, Immunofluorescence, Biomarker Discovery

Journal: Advanced Science

Article Title: The Evolutionarily Conserved TPM1 Super‐Enhancer Drives Skeletal Muscle Regeneration via Mechanotransduction Signaling

doi: 10.1002/advs.202514271

Figure Lengend Snippet: TPM1‐derived RNA transcripts promote myogenic differentiation and skeletal muscle regeneration. A) Overexpression of TPM1 significantly enhances the myogenic differentiation of C2C12 myoblasts, as evidenced by the increased number of MyHC‐positive myotubes visualized via immunofluorescence (DAPI, blue). B) Fusion index. C) WB analysis reveals elevated MyOD1 protein levels following murine TPM1 overexpression. D) Quantification of the WB results in panel C confirms the upregulation of MyOD1. E) Overexpression of CircTPM1 similarly promotes myogenic differentiation in bovine MuSCs, as demonstrated by increased MyHC‐positive myotubes formation (DAPI, blue). F) Fusion index. G) WB analysis shows that CircTPM1 overexpression upregulates MyOD1 and MyHC expression. H) Quantification of WB bands in panel G corroborates this finding. I) In C2C12 myoblasts, CircTPM1 overexpression also facilitates myogenic differentiation, confirmed by a higher number of MyHC‐positive myotubes (DAPI, blue). J) Fusion index. K) WB analysis further indicates increased protein levels of MyOD1 and MyHC. L) Quantification of the WB data in panel K supports these observations. M and N) Cell deformability was assessed in TPM1 ‐overexpressing C2C12 myoblasts (M) and CircTPM1‐overexpressing MuSCs (N). The left panel presents a box chart illustrating the deformation values across these groups. The middle panel shows a scatter plot depicting the relationship between deformability and cell diameter. In the right panel, density contour plots are displayed, with solid and dashed lines representing the 50% and 95% density contours, respectively, for comparisons between different treatment groups. Data are from n ≥ 1000 cells per subgroup. O and P) Measurement of the TA muscles weight‐to‐length ratio revealed a significant increase in the CircTPM1‐treated group. Q) H&E staining of TA muscles cross‐sections demonstrated improved muscle fiber structure following CircTPM1 overexpression. R and S) Quantitative analysis showed that CircTPM1 increased both myofiber density and cross‐sectional area. T) The percentage of regenerated fibers within the TA muscles cross‐sections was determined using ImageJ and Image‐Pro Plus on at least 500 nucleus‐containing myofibers. U) Immunofluorescence analysis confirmed a higher number of eMyHC‐positive myofibers in the CircTPM1‐treated group (DAPI, blue). V) RT‐qPCR also confirmed an increase in murine TPM1 transcript levels. Data are presented as mean ± SEM ( n = 3–5). Scale bars, 200 µm.

Article Snippet: Immunofluorescence staining of muscle sections was conducted as described by He et al. [ ] After H&E staining, sections were incubated sequentially with primary antibodies against emyhc (1: 200, Servicebio, Wuhan, China), appropriate secondary antibodies, and DAPI at 4 °C.

Techniques: Derivative Assay, Cell Characterization, Over Expression, Immunofluorescence, Expressing, Muscles, Staining, Quantitative RT-PCR