Journal: Nature

Article Title: The extracellular matrix protein agrin promotes heart regeneration in mice

doi: 10.1038/nature22978

Figure Lengend Snippet: a, b, Representative fields of heart cultures stained with DAPI (blue), cTnT (green) and Ki67 (red). White arrows indicate Ki67+cTnT+ cells. CMs, cardiomyocytes. c, d, Percentage of proliferating cardiomyocytes from P1 (c) or P7 (d) hearts in response to P1 and P7 ECM particles. n = 2,069 cardiomyocytes from three samples (c); n = 2,221 cardiomyocytes from four samples (d). e, f, Percentage of proliferating cardiomyocytes (Ki67+cTnT+) in response to P1 and P7 ECM in P1 (e) or P7 (f) cultures, with or without the broad MMP inhibitor (Marimastat). n = 3,480 cardiomyocytes from three samples (e); n = 23,445 cardiomyocytes from four samples (f). g, h, Percentage of P1 (g) or P7 (h) proliferating cardiomyocytes in response to MMP9- or MMP12-cleaved ECM fragments. n = 11,820 cardiomyocytes from four samples (g); n = 15,509 cardiomyocytes from four samples (h). i, qPCR of Agrn mRNA in P1 and P7 hearts. n = 8 P1 and 3 P7 hearts. j, Quantification of western blots for agrin from P1, P7 and 12-week-old (12W) adult heart lysates. A.U., arbitrary units. n = 3 samples per group. k, Images of P1 and P7 heart sections stained for agrin (green) and DAPI (blue). n = 3 samples per group. l, qPCR analysis of cardiac populations (FB, fibroblasts; CM, cardiomyocytes; EC, endothelial cells). n = 4 cardiomyocyte, 4 non-cardiomyocyte, 4 fibroblast, 4 non-fibroblast, 7 endothelial cells and 7 non-endothelial cell samples. Scale bars, 40 μm (a) and 10 μm (k). Data are presented as mean ±s.e.m. *P < 0.05, **P< 0.01, ***P< 0.001; statistical significance was calculated using ANOVA followed by a Dunnett’s post hoc test relative to the control group (c–h) or a Tukey’s post hoc test (j), statistical significance was calculated using a one-tailed t-test (i, l).

Article Snippet: The following primary antibodies were used: anti-rat recombinant Agrn (MAB550, R&D systems), anti-α-dystroglycan (sc-28534, Santa Cruz), anti-β-dystroglycan (Mandag2[7d11], DSHB), anti-syntrophin (sc-50460, Santa Cruz), anti-dystrophin (ab15277, Abcam), anti-Grb2 (sc-8034, Santa Cruz), anti-Myh6 (ab50967, Abcam), anti-actin (A2066, Sigma-Aldrich), anti-Actn1 (A2543, Sigma-Aldrich), anti-Des (ab8592, Abcam), anti-Yap (sc-15407, Santa Cruz) and anti-H3f3a (ab62642, Abcam).

Techniques: Staining, Western Blot, Control, One-tailed Test

Journal: Nature

Article Title: The extracellular matrix protein agrin promotes heart regeneration in mice

doi: 10.1038/nature22978

Figure Lengend Snippet: a, Diagram showing the mesoderm conditional knockout of agrin (agrin-cKO) in mice. b, qPCR of Agrn mRNA in P1 wild-type and agrin-cKO hearts. n = 8 wild-type (WT) and 7 cKO samples. c, Immunofluorescence images of agrin in P1 wild-type and agrin-cKO heart sections. n = 3 samples of each group. d, Quantification of western blots for agrin from wild-type and agrin-cKO mice heart lysates. n = 8 samples of each group. e, Immunofluorescence analysis and Pearson’s correlation coefficient analysis of T-tubules labelled with Cav3 in the z-lines (as indicated by Actn2). n = 4 wild-type and 3 cKO samples. White arrow heads indicate colocalization of T-tubules and z-lines. f, Myh6:Myh7 protein ratio from wild-type and agrin-cKO mice. n = 5 wild-type and 6 agrin-cKO samples. g, Staining and mean pixel intensity quantification of mitochondrial content in cardiomyocytes measured by Tom20 staining. n = 5 wild-type and 3 agrin-cKO samples. h, i, In vivo evaluation of P1 cardiomyocyte cell-cycle markers (Ki67; h) and (Aurkb; i) by immunofluorescence analysis in wild-type and agrin-cKO left ventricle heart sections. n = 41,695 cardiomyocytes from 11 wild-type and 4 agrin-cKO samples (h); n = 3,212 cardiomyocytes from 3 samples per group (i). j, Histological sections of P1 resected wild-type and agrin-cKO mice, 28 days after injury, stained with Masson’s trichrome. Bottom left corner of the cKO (left panel) was cropped to remove the adjacent section. k, l, Scar quantification of heart sections four weeks after resection of wild-type and agrin-cKO hearts. LV, left ventricle; none, 0% of the left ventricular wall; moderated ≤1% of the left ventricular wall; large ≥1% of the left ventricular wall. n = 12 wild-type and 8 cKO mice. m, n, Functional cardiac recovery measurements (ejection fraction (EF) and fractional shortening (FS)) of hearts from agrin-cKO and wild-type mice 28 days after resection. n = 5 wild-type and 3 cKO mice. o, p, In vivo evaluation of cardiomyocyte cell-cycle re-entry in the peri-infarct region by immunofluorescence analysis of Ki67 (o) or Aurkb (p) in heart sections 7 days after resection of wild-type and agrin-cKO hearts. n = 5,556 cardiomyocytes from seven wild-type and six cKO samples (o); n = 2,235 cardiomyocytes from three wild-type and four cKO samples (p). Scale bars, 10 μm (c, e, g–i, p), 20 μm (o), 100 μm (j, right) and 1 mm (j, left). Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; statistical significance was calculated using a one-tailed t-test.

Article Snippet: The following primary antibodies were used: anti-rat recombinant Agrn (MAB550, R&D systems), anti-α-dystroglycan (sc-28534, Santa Cruz), anti-β-dystroglycan (Mandag2[7d11], DSHB), anti-syntrophin (sc-50460, Santa Cruz), anti-dystrophin (ab15277, Abcam), anti-Grb2 (sc-8034, Santa Cruz), anti-Myh6 (ab50967, Abcam), anti-actin (A2066, Sigma-Aldrich), anti-Actn1 (A2543, Sigma-Aldrich), anti-Des (ab8592, Abcam), anti-Yap (sc-15407, Santa Cruz) and anti-H3f3a (ab62642, Abcam).

Techniques: Knock-Out, Immunofluorescence, Western Blot, Staining, In Vivo, Functional Assay, One-tailed Test

Journal: Nature

Article Title: The extracellular matrix protein agrin promotes heart regeneration in mice

doi: 10.1038/nature22978

Figure Lengend Snippet: a–c, In vivo evaluation of cardiomyocyte cell-cycle re-entry in the peri-infarct region seven days after myocardial infarction by Ki67 (a), Aurkb (b) or 21 days after myocardial infarction BrdU (c) n = 1,842 cardiomyocytes from five PBS and six agrin samples; n = 2,259 cardiomyocytes from five samples per group (b); n = 9,307 cardiomyocytes from six samples per group (c). For BrdU pulse-chase experiment see Methods. d, e, Heart section scar assessment following PBS or agrin treatment at indicated days after myocardial infarction (MI). Representative images are shown in d and quantified in e. n = 4 mice per group for day 0, 5 PBS- and 4 agrin-treated mice for day 4, 4 PBS- and 5 agrin-treated mice for day 14, 7 PBS- and 8 agrin-treated mice for day 35. f–h, Serial echocardiographic measurements of ejection fraction, fractional shortening and wall thickness of uninjured and injured hearts treated with PBS or agrin. LVAW, left ventricle anterior wall; LVPW, left ventricle posterior wall. n = 8 baseline, 5 uninjured, 6 PBS- and 8 agrin-treated mice (f, g); n = 2 uninjured, 5 PBS- and 6 agrin-treated mice (h). Scale bars, 10 μm (a, c), 20 μm (b) and 1 mm (d). Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01; statistical significance was calculated using ANOVA followed by Dunnett’s post hoc test relative to control group (f–h) or using a one-tailed t-test relative to PBS (a–e).

Article Snippet: The following primary antibodies were used: anti-rat recombinant Agrn (MAB550, R&D systems), anti-α-dystroglycan (sc-28534, Santa Cruz), anti-β-dystroglycan (Mandag2[7d11], DSHB), anti-syntrophin (sc-50460, Santa Cruz), anti-dystrophin (ab15277, Abcam), anti-Grb2 (sc-8034, Santa Cruz), anti-Myh6 (ab50967, Abcam), anti-actin (A2066, Sigma-Aldrich), anti-Actn1 (A2543, Sigma-Aldrich), anti-Des (ab8592, Abcam), anti-Yap (sc-15407, Santa Cruz) and anti-H3f3a (ab62642, Abcam).

Techniques: In Vivo, Pulse Chase, Control, One-tailed Test

Journal: Nature

Article Title: The extracellular matrix protein agrin promotes heart regeneration in mice

doi: 10.1038/nature22978

Figure Lengend Snippet: a, Western blot of Dag1 and MuSK from P1 and P7 heart lysates. n = 3 samples per group for MuSK, n = 6 samples per group for Dag1. Skeletal muscle extract was used as positive control (PC) for MuSK expression (lower panel). b, c, P7 Dag1 expression by qPCR (b) or membrane staining (c) in cardiomyocytes relative to non-cardiomyocytes (z–x plane presented). WGA, wheat-germ agglutinin. n = 2 cardiomyocyte and 2 non-cardiomyocyte RNA samples; n = 17,012 cardiomyocytes from four samples per group (c). d, e, Western blots of phosphorylated ERK (pERK) and total ERK (tERK) in P7 cultures with the indicated treatments. Ab, antibody. n = 5 samples (d); n = 3 samples (e). f, g, P7 cardiomyocytes cell-cycle analysis by immunofluorescence staining following agrin treatment with a MEK inhibitor (f) or Dag1 inhibitory antibody (g). Ctrl, control; PD, PD0325901. n = 2,743 cardiomyocytes from six samples (f); n = 6,649 cardiomyocytes from four samples (g). h, Glycerol-gradient fractionation of whole-cell extracts from P7 cells with or without (time 0) agrin treatment for 2 and 48 h. Fractions were analysed by SDS–PAGE and immunoblotting (IB) with indicated antibodies. n = 3 samples. Rec-agrin, recombinant agrin. Arrowheads indicate glycosylated form of β-dystroglycan. i, Isolated myofibrillar pellets and cytosol from P7 cells treated with agrin for 48 h analysed by SDS–PAGE and immunoblotting. n = 3 samples. j, Quantification of cardiomyocyte dedifferentiation using Myh6-lineage-derived cardiomyocytes (red mostly nuclear staining) stained with cTnT (green). Arrows indicate cardiomyocytes that have lost cTnT expression. n = 2,869 cardiomyocytes from five samples per group. k, Co-immunoprecipitation (IP) assay of Yap, agrin and various DGC proteins immunoprecipitated with syntrophin. n = 3 samples.l, Co-immunoprecipitation assay from 0, 2, 24 and 48 h agrin-treated cell membranes immunoprecipitated by syntrophin and blotted for Yap, agrin and other DGC proteins. n = 3 samples. m, In vivo quantification of nuclear Yap of heart sections from 12-week-old mice with PBS or agrin treatments one day after myocardial infarction. Representative images are shown. n = 1,167 cardiomyocytes from four mice per group. n, Cardiomyocyte proliferation assay of P7 heart cultures treated with agrin and the Yap–TEAD inhibitor. VP, verteporfin. n = 13,680 cardiomyocytes from eight samples. Scale bars, 10 μm (j) and 20 μm (m). Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; statistical significance was calculated using a one-tailed t-test (b, c, j, m) or ANOVA with Dunnett’s post hoc test (f, g, n).

Article Snippet: The following primary antibodies were used: anti-rat recombinant Agrn (MAB550, R&D systems), anti-α-dystroglycan (sc-28534, Santa Cruz), anti-β-dystroglycan (Mandag2[7d11], DSHB), anti-syntrophin (sc-50460, Santa Cruz), anti-dystrophin (ab15277, Abcam), anti-Grb2 (sc-8034, Santa Cruz), anti-Myh6 (ab50967, Abcam), anti-actin (A2066, Sigma-Aldrich), anti-Actn1 (A2543, Sigma-Aldrich), anti-Des (ab8592, Abcam), anti-Yap (sc-15407, Santa Cruz) and anti-H3f3a (ab62642, Abcam).

Techniques: Western Blot, Positive Control, Expressing, Membrane, Staining, Cell Cycle Assay, Immunofluorescence, Control, Fractionation, SDS Page, Recombinant, Isolation, Derivative Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, In Vivo, Proliferation Assay, One-tailed Test

Journal: Nature

Article Title: The extracellular matrix protein agrin promotes heart regeneration in mice

doi: 10.1038/nature22978

Figure Lengend Snippet: a, b, Immunofluorescence of iPSC–CM cell-cycle activity by pH3 (a) or AURKB (b) in response to rat and human agrin administration (10–1,000 ng ml−1). Representative images of cultures treated with 1,000 ng ml−1 are shown. pH3 quantification was performed automatically using imageXpress software (n = 174,850 cardiomyocytes from 35 images of control (Ctrl) and 37 images of rat-agrin-treated cultures and 32 images for all other samples (a); n = 38,542 cardiomyocytes from nine control samples and six samples in all other groups (b). c, Human iPSC–CM proliferation assay in the 3D patch culture system. n = 3 samples for one week, 4 samples for other treatments. Scale bars, 20 μm (a–c). d–f, Effects of human agrin on conduction velocity (d) and structural and functional maturation genes (e, f). n = 4 samples (d); n = 3 samples per group (e, f). mRNA expression ratios of MYL2/MYL7 and TNNI3/TNNI1 are shown in e. CV, conduction velocity. n = 4 samples (d); n = 3 samples per group (e, f). Data are presented as mean ± s.e.m. proliferation per field. *P<0.05, **P<0.01, ***P<0.001; statistical significance was calculated using a one-tailed t-test (d) or relative to untreated or 3-week control group using ANOVA followed by Dunnett’s post hoc test.

Article Snippet: The following primary antibodies were used: anti-rat recombinant Agrn (MAB550, R&D systems), anti-α-dystroglycan (sc-28534, Santa Cruz), anti-β-dystroglycan (Mandag2[7d11], DSHB), anti-syntrophin (sc-50460, Santa Cruz), anti-dystrophin (ab15277, Abcam), anti-Grb2 (sc-8034, Santa Cruz), anti-Myh6 (ab50967, Abcam), anti-actin (A2066, Sigma-Aldrich), anti-Actn1 (A2543, Sigma-Aldrich), anti-Des (ab8592, Abcam), anti-Yap (sc-15407, Santa Cruz) and anti-H3f3a (ab62642, Abcam).

Techniques: Immunofluorescence, Activity Assay, Software, Control, Proliferation Assay, Functional Assay, Expressing, One-tailed Test

Journal: Genes & Development

Article Title: Structural basis of agrin-LRP4-MuSK signaling

doi: 10.1101/gad.180885.111

Figure Lengend Snippet: Characterization of the agrin–LRP4 interaction. (A) Schematic domain organizations of rat agrin and LRP4. The boundaries of protein fragments used in this study are indicated. (NtA) N-terminal agrin domain; (FS) follistatin-like repeat; (LB) laminin-B-like domain; (S/T) serine/threonine glycosylation sites; (EG) EGF-like domain; (LG) laminin-G-like domain; (LDLa) LDL class A repeats; (YWTD) YWTD repeat-containing β propeller; (TM) transmembrane region. (B) Agrin LG3 (residues Leu 1759–Pro 1948) forms a stable complex with LRP4V396–A737 in solution, as demonstrated by size exclusion chromatography. (C) Analytic ultracentrifugation (AUC) studies of agrin LG3, agrin LG2/LG3 (residues Leu 1481–Pro 1948), and the agrin LG3–LRP4T353–A737 complex. Agrin LG3 and LG2/LG3 are both monomeric in solution, and the agrin–LRP4 complex dimerizes with an average Kd of ∼39 μM. (Bottom panel) Absorbance data (blue dots) fit to a single-species model (LG2/LG3 and LG3) or a monomer–dimer model (agrin LG3–LRP4 complex) (red line). (Top panel) Residuals from the fit.

Article Snippet: Rat neural agrin LG3 (residues Leu 1759–Pro 1948), containing the z8 (ELTNEIPA) sequence, was subcloned into a modified pAcGP67 vector (BD Biosciences) that harbors an N-terminal six-histidine (6His) tag and a HRV 3C protease cleavage site.

Techniques: Size-exclusion Chromatography

Journal: Genes & Development

Article Title: Structural basis of agrin-LRP4-MuSK signaling

doi: 10.1101/gad.180885.111

Figure Lengend Snippet: Binary complex of agrin LG3 (agrin) and LRP4V396–A737 (LRP4). (A) The association of agrin (green) and LRP4 (orange) is mostly mediated by the neuron-specific z8 alternative splicing sequence in agrin. This loop is unambiguously defined by excellent electron densities (2Fo − Fc map contoured at 1σ) (in cyan). (B) The structure of rat agrin (green) in the context of an agrin–LRP4 complex is superimposed on the structure of an apo form of chicken agrin that also has an eight-amino-acid insert at the B/z site (magenta; PDB code 1PZ8). The arrows indicate the boundaries of the disordered z8 loop of the apo chicken agrin. (C) Stereoview of the detailed interactions between agrin (green/cyan) and LRP4 (yellow). Key residues that are directly involved in complex interactions are shown as the ball-and-stick model. Hydrogen bonds are indicated by dotted lines. (D) Open-book view of the electrostatic potential of the z8-interacting surfaces (highlighted with squares). The negatively charged z8 loop is complementary to the positively charged surface in LRP4.

Article Snippet: Rat neural agrin LG3 (residues Leu 1759–Pro 1948), containing the z8 (ELTNEIPA) sequence, was subcloned into a modified pAcGP67 vector (BD Biosciences) that harbors an N-terminal six-histidine (6His) tag and a HRV 3C protease cleavage site.

Techniques: Sequencing

Journal: Genes & Development

Article Title: Structural basis of agrin-LRP4-MuSK signaling

doi: 10.1101/gad.180885.111

Figure Lengend Snippet: Agrin-induced tetramerization of agrin–LRP4 is critical for AChR clustering and MuSK phosphorylation. (A) The z8 interface plays a predominant role in assembly of the agrin–LRP4 complex. GST pull-down assays were performed between GST-agrin variants (containing a HA tag) and LRP4L23–A737 (containing a His tag). Agrin and LRP4 were detected by an anti-HA antibody and an anti-His antibody, respectively. (B) MuSK phosphorylation induced by agrin variants. Fully differentiated myotubes were treated with wild-type or mutant agrin at 100 nM. MuSK was immunoprecipitated with anti-MuSK polyclonal antibody, and phosphorylation was detected by immunoblotting with anti-phosphotyrosine antibody 4G10. The bar graph shows mean ± SD, n = 3; (*) P < 0.05 in comparison with the wild-type agrin. (C) Representative images of AChR clustering (red dots) on C2C12 myotubes induced by wild-type LG3, LG3-N1783A, or LG3-H1795L. (D) Quantitative analyses of AChR clustering assay for agrin variants. Data are mean ± SD based on three independent experiments.

Article Snippet: Rat neural agrin LG3 (residues Leu 1759–Pro 1948), containing the z8 (ELTNEIPA) sequence, was subcloned into a modified pAcGP67 vector (BD Biosciences) that harbors an N-terminal six-histidine (6His) tag and a HRV 3C protease cleavage site.

Techniques: Mutagenesis, Immunoprecipitation, Western Blot