Abstract
Adult skeletal muscle is a highly plastic tissue that readily reduces or gains its mass in response to mechanical and metabolic stimulation; however, the upstream mechanisms that control muscle mass remain unclear. Notch signalling is highly conserved, and regulates many cellular events, including proliferation and differentiation of various types of tissue stem cell via cell–cell contact. Here we reveal that multinucleated myofibres express Notch2, which plays a crucial role in disuse- or diabetes-induced muscle atrophy. Mechanistically, in both atrophic conditions, the microvascular endothelium upregulates and releases the Notch ligand, Dll4, which then activates muscular Notch2 without direct cell–cell contact. Inhibition of the Dll4–Notch2 axis substantively prevents these muscle atrophy and promotes mechanical overloading-induced muscle hypertrophy in mice. Our results illuminate a tissue-specific function of the endothelium in controlling tissue plasticity and highlight the endothelial Dll4–muscular Notch2 axis as a central upstream mechanism that regulates catabolic signals from mechanical and metabolic stimulation, providing a therapeutic target for muscle-wasting diseases.
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Data availability
The RNA-seq data have been deposited in the Gene Expression Omnibus repository under accession codes GSE161693 (RNA-seq) and GSE185678 (snRNA-seq). Uncropped immunoblot images are provided as source data. Other raw data are available on reasonable requests to the corresponding author. Source data are provided with this paper.
Change history
16 March 2022
A Correction to this paper has been published: https://doi.org/10.1038/s42255-022-00560-6
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Acknowledgements
We thank R. Fujita, S. Ogawa, Y. Tsuchiya, K. Yoshioka, Y. Kitajima, D. T. Shima, Y. Kubota and N. Suzuki for sharing materials and technical assistance. We also thank S. Burden for providing Mlc1f-Cre mice; I. Imayoshi and R. Kageyama for providing Hes1-flox mice and T. Honjo for providing RBPJ-flox mice. The Pax7 and MF20 antibodies, developed by A. Kawakami and D.A. Fischman, respectively, were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa. This work was supported by the FOREST program of the Japan Science and Technology Agency (JST, grant no. JPMJFR205C to Y.O.), the Japan Agency for Medical Research and Development (AMED, grant no. JP19bm0704036 to Y.O.) and Grants-in-Aid for Scientific Research KAKENHI (grant nos. 25882024 to S.M.; 15H05368, 18H03193 and 20K21763 to Y.O.; 20K19641 to S.F. and 20J01669 to T.Ma.). This work was also supported, in part, by the Center for Metabolic Regulation of Healthy Aging (to Y.O. and S.F.), the Takeda Science Foundation (to Y.O. and S.F.), the Naito Foundation (to Y.O.), the Uehara Memorial Foundation (to S.F.), NIH R21 (grant no. AR078400 to A.A.) and Regenerative Medicine Minnesota (grant no. 092319 TR010 to A.A.). T.Ma., H.N. and N.H. were funded by a JSPS Research Fellowship.
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S.F. conducted and performed the experiments, interpreted the data, assembled the input data and wrote the paper. T.Ma., H.N., N.H., D.S. and S.M. performed cell culture and animal experiments. M.M. and T.Mi. performed EC-culture and immunohistochemical experiments, interpreted and analysed the data, and wrote the paper. Y.T., Y.M. and T.F. performed electron microscopy and immunohistochemical experiments, interpreted the data and wrote the paper. X.W., Y.A. and A.A. performed the animal experiments, interpreted the data and wrote the paper. S.U. and K.Y. prepared data library for snRNA-seq and analysed the data. Y.K and R.N. generated key materials and interpreted the data. Y.O. designed and performed the experiments, interpreted the data, assembled the input data and wrote the paper. All authors reviewed the paper and approved the final version.
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Extended data
Extended Data Fig. 1 Notch2 is expressed in adult myofibres.
a, Representative images of primary cultures of satellite cells maintained in GM or Diff. M for 4 days (Diff. M 4d). b, qPCR analysis for gene expression of Notch1 and Notch2 during myogenic progression (up to day 5 in Diff. M) (n = 3). Myh3 served as a marker of cell differentiation. c, Immunoblotting analysis for protein levels of Notch1 and Notch2 during myogenic progression (up to day 5 in Diff. M). Upper and lower bands in the images of Notch1 and Notch2 indicate the full-length proteins (FL) and the transmembrane/intracellular region (NTM), respectively. Myosin heavy chain (MyHC) and β-tubulin served as a marker of cell differentiation and an internal control, respectively. d, To determine the expression of Notch1 and Notch2 in terminally differentiated adult myofibres with or without satellite cells, Pax7CreERT2/+;Rosa26LSL-DTA/+ mice that express tamoxifen (TMX)-inducible diphtheria toxin-A (DTA) under the control of the Pax7 promoter were used. TMX was intraperitoneally injected five times into Pax7CreERT2/+;Rosa26LSL-DTA/+ mice to deplete the satellite cell population, and individual myofibres were isolated from EDL muscle (Pax7-DTA myofibre). Non-TMX treated mice were used as a control (Control myofibre). e, qPCR analysis for gene expression of Notch1 and Notch2 in Pax7-DTA myofibres. Pax7 served as a marker of satellite cell depletion in Pax7-DTA myofibres (n = 3). f–j, Satellite cells were isolated from EDL muscle in Pax7CreERT2/+;Rosa26LSL-DTA mice, cultured in Diff. M, and treated with 4OH TMX in order to deplete the self-renewed and undifferentiated satellite cells. f, Time course of culture conditions. g, Representative images of immunostaining for Pax7 (scale bar, 50 µm). Yellow arrows indicate Pax7+ cells. h, Percentage of Pax7+ nuclei in the total DAPI+ nuclei (n = 6). i, qPCR analysis for gene expression of Notch2 in Cont and DTA myotubes (n = 6). j, Immunoblotting analysis for protein levels of Notch2 and Pax7. All qPCR data were normalised to Tbp expression. Data represent the mean ± s.e.m. and points are individual mice.
Extended Data Fig. 2 Constitutive activation of Notch2 induces muscle atrophy.
a–g, To examine the effect of constitutive activation of Notch2 on adult muscle in vivo, HSArtTA/+;TRECre/+;Rosa26LSL-N2ICD/+ mice (N2-mTg) were generated. HSArtTA/+;TRECre/+ or Rosa26LSL-N2ICD/+ mice were used as a control (Cont). a, N2-mTg and Cont mice were treated with DOX (2 mg ml−1 in drinking water) to induce constitutive expression of N2ICD. b, qPCR analysis for confirming the overexpression of Notch2 in GAS (n = 3). c,d, Body weight (c) and muscle weights of GAS, TA, and PLA normalised to body weight (d, n = 3 for Cont and n = 5 for N2-mTg). e, Representative images of immunostaining for Laminin on TA cross-sections (scale bar, 100 µm). f, Myofibre CSA of TA (n = 3). g, Grip strength (n = 4 for Cont and n = 5 for N2-mTg). qPCR data were normalised to Tbp expression. Data represent the means ± s.e.m., and points indicate individual mice. *P < 0.05, **P < 0.01, NS as determined by Student’s two-tailed unpaired t-test (c,d,f,g).
Extended Data Fig. 3 Effects of Notch2 ablation on mouse growth and Akita diabetic mice.
a, qPCR analysis for confirming the deletion of the Notch2 gene in individual myofibres isolated from EDL muscles of Notch2flox/flox (N2f/f) mice and Mlc1fCre/+;Notch2flox/flox (N2−/−) mice (normalised to Tbp expression). b–e, Profiles of N2–/– adult mice with regard to body weight (b), muscle weights normalised to body weight (c), the number of nuclei per myofibre (d), and the number of Pax7+ cells (satellite cells) per myofibre (e) (n = 5). f,g, Akita mice were used as a diabetic hyperglycemic model. Thirteen-week-old male littermates were used. Blood glucose levels (f) and muscle weights normalised to body weight (g) (n = 3). Data represent the means ± s.e.m. and points indicate individual mice. **P < 0.01, ***P < 0.001, NS (not significant) as determined by: two-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (f,g); Student’s two-tailed unpaired t-test (b–e).
Extended Data Fig. 4 Notch2-induced muscle atrophy is independent of RBPJ and Hes1.
a–e, Effects of HU or DM on muscles in muscle-specific Rbpj-ablated mice. HSArtTA/+;TRECre/+;Rbpjflox/flox mice were treated with DOX (2 mg ml−1 in drinking water) for 3 weeks to induce deletion of Rbpj in myofibres (Rbpj–/–). Rbpjflox/flox mice were used as control (Rbpjf/f). a, qPCR analysis for confirming the deletion of Rbpj expression in myofibres isolated from EDL (n = 3). b, Rbpjf/f and Rbpj–/– mice started 7 day-HU after 2 week-DOX treatment. Mice maintained in the ground condition were used as ground control (GC). c, Muscle weight of SOL, PLA, and GAS muscles normalised to body weight of Rbpjf/f and Rbpj–/– mice under GC and HU conditions (n = 3). d, Rbpjf/f and Rbpj–/– mice were intraperitoneally injected with STZ (150 mg kg−1 body weight; DM) or vehicle (Veh; citrate buffer) 2 weeks after starting DOX treatment and sacrificed 14 days following STZ infusion. e, Muscle weight of TA, PLA, and GAS muscles normalised to body weight of Rbpjf/f and Rbpj–/– mice under Veh and DM conditions (n = 3). f–j, Effects of HU or DM on muscles in muscle-specific Hes1-ablated mice. Hes1flox/flox (Hes1f/f) and Mlc1fCre/+;Hes1flox/flox (Hes1–/–) mice were used. f, qPCR analysis for confirming the deletion of Hes1 expression in myofibres isolated from EDL (n = 4). g, Time course of the HU experiment. h, Muscle weight of SOL and GAS muscles normalised to body weight of Hes1f/f and Hes1–/– mice (n = 3 for GC and n = 5 for HU). i, Time course of the diabetes experiment. j, Muscle weight of TA muscle normalised to body weight in Hes1f/f and Hes1–/– mice (n = 4, except n = 6 for Hes1–/– DM). k–s, Effects of constitutive activation of Notch2 on muscles of muscle-specific Rbpj–/– mice. k, HSArtTA/+;TRECre/+;Rosa26LSL-N2ICD/+;Rbpjflox/flox mice were treated with DOX for 3 weeks to induce constitutive activation of Notch2 and Rbpj deletion in myofibres (N2-mTg;Rbpj–/–). Rbpjflox/flox mice were used as control (Rbpjf/f). l, qPCR analysis for confirming the overexpression of Notch2 and the deletion of Rbpj in myofirers isolated from EDL (n = 4 for Rbpjf/f and n = 5 for N2-mTg;Rbpj–/–). m,n, Body weight (m) and muscle weights normalised to body weight (GAS, TA, and PLA; n) (n = 6 for Rbpjf/f and n = 7 for N2-mTg;Rbpj–/–). o,p, the number of nuclei per myofibre (o), and the number of Pax7+ cells (satellite cells) per myofibre (p) (n = 4). q–s, Neuromuscular junctions (NMJs) of EDL myofibres isolated from Rbpj–/– and N2-mTg;Rbpj–/– mice were analysed. q, Representative phase-contrast images of NMJs on myofibres (scale bar, 50 µm). r,s, Distribution of myonuclear numbers in NMJ region (r, n = 3) and the ratio of fragmented NMJs (s, n = 3). All qPCR data were normalised to Tbp. Data represent the means ± s.e.m., and points indicate individual mice. *P < 0.05, **P < 0.01, ***P < 0.001, NS (not significant) as determined by: two-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (c,e,h,j); Student’s two-tailed unpaired t-test (m–p,r,s).
Extended Data Fig. 5 Notch2-induced muscle atrophy is mediated through FoxO signalling pathway.
a–d, Pathway analysis using RNA-Seq data sets that were significantly upregulated in N2f/f muscle but not in N2–/– muscle, under hindlimb unloading or diabetic conditions, corresponding to Fig. 1o-q. a, Venn diagram showing DEGs obtained from the comparison between the HU and Cont conditions in GAS muscles from N2f/f (red circle) and N2–/– mice (blue circle) (genes with fold change >2.0, q < 0.05, and TPM > 500 were analysed). b, Upregulated pathways obtained from KEGG pathway analysis using genes upregulated in unloaded GAS muscle of N2f/f but not N2–/– mice. c, Venn diagram showing DEGs obtained from the comparison between the DM and Cont conditions in GAS muscles from N2f/f mice (red circle) and N2–/– mice (blue circle) (genes with fold change >2.0, q < 0.05, and TPM > 500 were analysed). d, Upregulated pathways obtained from KEGG pathway analysis using genes upregulated in diabetic GAS muscle of N2f/f but not N2–/– mice. e–g, Effect of constitutive activation of Notch2 with Rbpj ablation on the expressions of atrogenes and FoxO target genes in myofibres. e, HSArtTA/+;TRECre/+;Rosa26LSL-N2ICD/+;Rbpjflox/flox mice were treated with DOX for 1 week to induce constitutive activation of Notch2 and Rbpj deletion in myofibres (N2-mTg;Rbpj–/–). Rbpjflox/flox mice were used as control (Rbpjf/f). f, qPCR analysis for confirming the overexpression of Notch2 and its target gene Hes1, and the deletion of Rbpj in GAS muscles (n = 4). g. Expressions of FoxO target genes and atrogenes in GAS muscles (n = 4). h,i, Assessment of direct interaction between Notch2 and FoxO transcription factors. h, Satellite cells isolated from EDL muscles of WT mice were transfected with 3xFLAG-N2ICD plasmid by electroporation. Cells were induced to undergo myogenic differentiation for 5 days and harvested as IP samples. i, Immunoblotting analysis for Notch2 (NTM), FoxO1, FoxO3a, and Akt using plasmid-transfected (TF) and non-transfected (Cont) samples immunoprecipitated using FLAG antibody. Total cell lysates were used as input controls. j–l, Effect of Notch2 deficiency on FoxO phosphorylation and localization under atrophic conditions. j, Protein accumulation of phospho-Akt (Ser473), total-Akt, phospho-FoxO1/3a (Thr24/Thr32), total-FoxO3a in GAS muscle of N2f/f and N2−/− mice under control, HU, and DM conditions. k,l, Protein accumulation of FoxO1 and FoxO3a in nuclear fractions of GAS muscles from N2f/f and N2−/− mice under control and DM conditions. Representative blot patterns (k) and quantitative data (l) were shown. Histone H3 served as an internal control. m–r, Effects of Notch2 deficiency on FoxO1-induced muscle atrophy. m, Satellite cells were isolated from N2f/f or N2−/− and transfected with plasmid encoding constitutively active form of human FOXO1 (caFOXO1) or empty vector (Cont) by electroporation. Cells were induced to undergo myogenic differentiation for 4 days and then harvested. n, Gene expressions of Notch2, FoxO1, and FoxO1-target genes including Ccng2 and Gadd45 in myotubes (n = 4). o, Representative images of MyHC immunostaining (scale bars, 50 µm). p,q, Quantitative data of the myotube diameter (p) and the fusion index, which is a proportion of the number of nuclei within MyHC+ myotubes (containing more than two nuclei) relative to the total number of nuclei (q) (n = 4). r, Gene expressions of atrogenes. All qPCR data were normalised to Tbp (n = 4). Data represent the means ± s.e.m., and points indicate individual mice. *P < 0.05, **P < 0.01, ***P < 0.001, NS (not significant) as determined by: two-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (l,n,p–r); Student’s two-tailed unpaired t-test (g).
Extended Data Fig. 6 Inhibition of γ-secretase activity attenuates muscle atrophy in unloading and diabetic conditions.
a,b, Wild-type mice were intraperitoneally injected with a γ-secretase inhibitor DAPT (10 mg kg−1 body weight) or corn oil (Cont) every other day during the HU period. a, Time course. b, Muscle weight of SOL, PLA, and GAS (n = 4 for GC and n = 7 for HU). c–e, Wild-type mice were intraperitoneally injected with STZ (150 mg kg−1 body weight; DM) or vehicle (Veh). Mice were then injected with DAPT or corn oil (Cont) every other day from day 3 following STZ infusion. c, Time course. d,e, Blood glucose levels 14 days after STZ infusion (d) and muscle weight of TA, PLA, and GAS (e) (n = 6 for Veh and n = 8 for DM). Muscle weights were normalised to body weight. Data represent the means ± s.e.m. and points indicate individual mice. *P < 0.05, **P < 0.01, ***P < 0.001, NS (not significant) as determined by: two-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (b,d,e).
Extended Data Fig. 7 Dll4 expression is restricted to ECs in adult muscle tissue.
a–e, snRNA-Seq of mouse GAS and PLA muscles at 8 weeks of age. a, Schematic of nuclei isolation and sequencing. b, UMAP plot from unbiased clustering of 16,159 single nuclei isolated from GAS and PLA muscles. c, UMAP plots showing marker gene expression for nuclear populations, including Ttn (myonuclei), Colq (neuromuscular junctions, NMJs), Tigd4 (myotendinous junctions, MTJs), Pax7 (satellite cells, SCs), Pecam1 (endothelial cells, ECs), Ptprc (immune cells), Myh11 (smooth muscle cells), Pdgfra (Fibro-adipogenic progenitors, FAPs), Mkx (tenocytes), and Plin1 (adipocytes). d, UMAP plot showing Dll4 expression with magnified plot of the EC cluster. e, UMAP plots showing Notch1 and Notch2 expressions. f, Immunoblotting for Dll4 protein using GAS muscle from EC-specific homozygous Dll4 knockout mice (Cdh5CreERT2/+;Dll4flox/flox, Dll4−/−), heterozygous Dll4 knockout mice (Cdh5CreERT2/+;Dll4flox/+, Dll4+/−), and control mice (Dll4flox/flox, Dll4+/+). To induce Cre recombination, mice were intraperitoneally injected with tamoxifen (75 mg kg−1 body weight, 4 times) 1 week before analysis. GAPDH served as the internal control. g, Gene expression of Notch ligands (Dll1, Dll4, Jag1, and Jag2) normalised to Tbp expression in myofibres isolated from wild-type PLA or EDL muscle in the HU or DM conditions, respectively (n = 4 mice per condition). h, Representative TEM images of immunogold labelling for Dll4 protein in GAS muscle isolated from diabetic mice. Gold particles were localized on the surface of myofibres (left), collagen-like fibres (middle), and extracellular vesicles (right) in the interstitial spaces between ECs and myofibres (scale bar, 200 nm). Data represent the means ± s.e.m. and points indicate individual mice. *P < 0.05, ***P < 0.001 as determined by two-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (g).
Extended Data Fig. 8 DLL4 blockade prevents atrophy of human skeletal myotubes.
a–i, Effect of coculture of human skeletal muscle myotubes with human umbilical vein endothelial cells (HUVECs) in the transwell culture system. a, Schematic of the transwell culture system. HUVECs were transfected with control siRNA (si-Cont) or siRNA against DLL4 (si-DLL4) 1 day before coculture with myotubes. b, DLL4 protein expression in si-Cont- and si-DLL4-transfected HUVEC lysates and supernatants (Sup) of those culture media. Immunodetection for β-tubulin and ponceau staining served as internal controls. c, Representative images of MyHC-positive myotubes (scale bar, 50 µm). d,e, Quantitative data of myotube diameters (d) and fusion index (e) (n = 4 well per condition, myotubes in more than 3 field of view (10×)/well were measured). f, Effects of treatment with 500 ng ml−1 anti-DLL4 neutralizing antibody (αDLL4) or control antibody (αCont) on human myotubes–HUVECs co-culture. g, Representative images of MyHC-positive myotubes (scale bar, 50 µm). h,i, Quantitative data of myotube diameters (h) and fusion index (i) (n = 4 well per condition, myotubes in more than 3 field of view (10×)/well were measured). Data represent the mean ± s.e.m., and points are individual mice. ***P < 0.001, NS (not significant) as determined by two-way repeated-measures ANOVA followed by Tukey’s multiple comparisons post-hoc test (d,h).
Extended Data Fig. 9 Effects of αDll4 or Fc-fused Dll4 recombinant protein on muscle atrophy.
a–c, Effect of treatment with EC-conditioned media (EC-CM) on myofibres. a, Primary ECs were isolated from hindlimb muscle tissues. Concentrated EC-CM were added into culture media containing wild-type EDL myofibres. Myofibres were then cultured with the addition of 500 ng ml−1 anti-Dll4 neutralising antibody (αDll4) or control antibody (αCont) for 48 hours. b, Representative images of myofibres (scale bar, 50 µm). c, Quantitative data of myofibre diameters (n = 4 mice per condition, >15 individual myofibres per mouse were measured). d–f, Effect of treatment with Fc-fused Dll4 recombinant protein (rDll4-Fc) on myofibres co-cultured with primary ECs. d, Wild-type EDL myofibres were co-cultured with ECs isolated from hindlimb muscle tissues with the addition of 500 ng ml−1 rDll4-Fc for 48 hours. e, Representative images of myofibres (scale bar, 50 µm). f, Quantitative data of myofibre diameters (n = 4 mice per condition, >15 individual myofibres per mouse were measured). g–i, Effect of rDll4-Fc administration on diabetic muscle atrophy in vivo. g, Wild-type mice were intraperitoneally injected with STZ (150 mg kg−1 body weight; DM) or vehicle (Veh) and analysed 14 days after the injection. Mice were administered with rDll4-Fc at day 7, 9, 11, and 13 following the STZ infusion. PBS was used as a control. h, Blood glucose levels 14 days after STZ infusion (n = 5). i, Muscle weight normalised to body weight. Data represent the means ± s.e.m., and points indicate individual mice. *P < 0.05, **P < 0.01, ***P < 0.001 NS (not significant) as determined by two-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (c,f,h,i).
Extended Data Fig. 10 Inhibition of the Dll4-Notch2 axis promotes hypertrophic response.
a, Gene expression of Notch ligands (Dll1, Dll4, Jag1, and Jag2) in CD31+ ECs and myofibres isolated from overloaded or control PLA muscle (n = 4 mice per condition). qPCR data of ECs and myofibres were normalised to Pecam1 and GAPDH, respectively. b–d, To assess the hypertrophic response of muscle, N2f/f and N2–/– mice were overloaded (OL) by tenotomy for 14 days. b, Time course. c,d, Muscle weight (c) and OL-induced weight gain of PLA muscle (d), PLA muscle weight/body weight of OL vs. sedentary control (n = 6-8). e–g, Wild-type mice were intraperitoneally administered with DAPT (10 mg kg−1 body weight) following tenotomy. e, Time course. f,g, Muscle weight (f) and OL-induced PLA weight gain (g) (n = 6). h–j, Effects of EC-specific conditional deletion of Dll4 on muscle hypertrophy. h, Experimental scheme for assessing the hypertrophic response of EC-specific Dll4-deficient mice after overloading. Dll4flox/flox (Dll4+/+) and Cdh5CreERT2/+;Dll4f/f (Dll4–/–) mice were overloaded (OL) after TMX was intraperitoneally injected four times. i,j, Muscle weight (i) and OL-induced weight gain of PLA muscle (j) (n = 5). Muscle weights were normalised to body weight. Data represent the means ± s.e.m. and points represent individual mice. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by: two-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (c,f,i); Student’s two-tailed unpaired t-test (a,d,g,j).
Supplementary information
Supplementary Information
Supplementary Fig. 1 Gating strategy. Supplementary Table 1 Antibody list. Supplementary Table 2 Primer sequences.
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Source Data Extended Data Fig. 1
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Source Data Extended Data Fig. 8
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Fujimaki, S., Matsumoto, T., Muramatsu, M. et al. The endothelial Dll4–muscular Notch2 axis regulates skeletal muscle mass. Nat Metab 4, 180–189 (2022). https://doi.org/10.1038/s42255-022-00533-9
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DOI: https://doi.org/10.1038/s42255-022-00533-9
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