Hapln1 promotes dedifferentiation and proliferation of iPSC-derived cardiomyocytes by promoting versican-based GDF11 trapping

Figures

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  Fig. 1. Recombinant human hyaluronan and proteoglycan link protein 1 (rhHapln1) promotes the proliferation and dedifferentiation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in a dose-dependent manner. (A–D) After administration of various concentrations (0, 20, 100, and 500 ng/mL) of rhHapln1 for 72 h, proliferation markers of the hiPSC-derived cardiomyocytes were tested using immunofluorescence analysis. (A, C) Representative immunofluorescence (IF) images of the EdU incorporation assay and quantification of EdU-positive hiPSC-CMs (n = 3). (B, D) Representative IF images of Ki67 staining and quantification of Ki67-positive hiPSC-CMs (n = 3). (E–G) Real-time quantitative polymerase chain reaction (RT-qPCR) assays were used to test markers for cardiomyocyte maturity. (H) QPCR was used to assess dedifferentiation markers (RUNX1, DAB2, NKX2.5, and ACTA2) of hiPSC-CMs. (I, J) Immunoblots were performed to assess markers for hiPSC-CM dedifferentiation (NKX2.5, α-SMA, encoded by ACTA2 gene) as shown by the (I) representative blot images and (J) grayscale quantification. (K, L) 24 h after hypoxic treatment, cells were subjected to 6 h reoxygenation. Then, Cell…

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  Fig. 2. Versican binds with Nodal growth differentiation factor (NODAL) and growth differentiation factor 11 (GDF11). (A) Enzyme-linked immunosorbent assays (ELISAs) were performed to detect the concentration of versican in the culture medium of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) after 72 h recombinant human hyaluronan and proteoglycan link protein 1 (rhHapln1) treatment. (B) Proteins with validated/potential interactions with versican. (C) Gene Oncology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the genes encoding the proteins in panel B. (D) Binding of versican to selective chemokines. Anti-versican, anti-chondroitin sulfate (anti-CS) mAb CS56, or selective chemokines (5–100 ng/spot) were spotted onto nitrocellulose membranes. After blocking, biotinylated versican (3 μg/mL) was applied to the membrane. The binding of biotinylated versican was detected in immunoblotting. (E) Binding of NODAL or GDF11 to immobilized versican. Serial dilutions of NODAL or GDF11 (0–3 μg/mL) were added to the versican (300 ng/well)-coated wells and incubated for 2 h. After washing, the binding of chemokines to versican was measured by ELISAs. (F)…

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  Fig. 3. NODAL and GDF11 mRNA expression in fetal human hearts at the single-cell level. (A, C) Uniform manifold approximation and projection (UMAP) plot indicating the individual cells with GDF11 (A) or NODAL (C) expression in the human fetal heart (left) and the cell type distribution (right). (B, D) Boxplots comparing different types of fetal cardiac cells with (B) GDF11 or (D) NODAL expression. Images were generated with access provided by Single Cell Portal (https://singlecell.broadinstitute.org/single_cell).

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  Fig. 4. Recombinant human hyaluronan and proteoglycan link protein 1 (rhHapln1) promotes human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) proliferation via the transforming growth factor (TGF)-β/SMAD2/3 signaling. (A) Western blotting assays were conducted in the cytoplasmic and nucleus fractions to explore the effect of HAPLN1 on the SMAD2/3 phosphorylation in hiPSC-CMs (4 weeks, (4W)). Cells were treated with 20 or 100 ng/mL rhHapln1 for 72 h, with or without the presence of A-83-01 (1 μM). (B) Western blotting assays were conducted to explore the expression of the hiPSC-CM maturation markers (TNNI3 and MYL2) and dedifferentiation markers (NKX2.5 and α-SMA) in whole-cell lysate from hiPSC-CMs as treated in panel A. (C–F) The proliferation of hiPSC-CMs as treated in Fig. 4A was investigated using (C, D) EdU incorporation assays and (E, F) immunostaining of Ki67. (G) Western blotting assays were conducted to explore the expression of P21, Cyclin D1, Cyclin D2 in whole-cell lysate from hiPSC-CMs treated as in Fig. 4A. Two-way analysis of variance (ANOVA) with Bonferroni correction for post-hoc comparisons (D and F) were conducted. Data are presented as the mean ±…

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  Fig. 5. Versican-based growth differentiation factor 11 (GDF11) trapping is required for the proliferation-promoting effects of hyaluronan and proteoglycan link protein 1 (Hapln1). (A) Western blotting assays were conducted in the cytoplasmic and nucleus fractions to explore the effect of GDF11 manipulation on HAPLN1-induced SMAD2/3 phosphorylation in hiPSC-CMs (4 weeks, (4W)). Cells were treated with 100 ng/mL Hapln1 for 48 h, with or without GDF11 (20 ng/mL) or GDF11-neutralizing antibody (anti-GDF11, 100 ng/mL). (B, C) Western blotting assays were conducted to explore the expression of (B) the human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) maturation markers (TNNI3 and MYL2), dedifferentiation markers (NKX2.5 and α-SMA) and (C) cell-cycle related proteins (P21, Cyclin D1, and Cyclin D2) in whole-cell lysate from hiPSC-CMs, as treated in Fig. 5A. (D) Western blotting assays were conducted of the cytoplasmic and nucleus fractions, to explore the effect of VCAN manipulation on HAPLN1-induced SMAD2/3 phosphorylation in hiPSC-CMs (4W). Cells with or without lentivirus-mediated VCAN knockdown were treated with 100 ng/mL HAPLN1 for 72 h. One VCAN knockdown group…

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  Fig. 6. Recombinant mouse hyaluronan and proteoglycan link protein 1 (rmHapln1) induced cardiac regeneration in adult mice. (A) A schematic diagram depicting left anterior descending (LAD) ligation in adult mice. (B, E) Heart section scar assessment following reconstitution buffer (control) or rmHapln1 treatment at indicated days after myocardial infarction (MI). Representative images are shown in Fig. 6B and were quantified in Fig. 6E (n = 6 mice per group for days 0, 4, 15, and 35). (C) Representative images and (D) quantitation of in vivo evaluation of cardiomyocyte cell-cycle re-entry in the peri-infarct region 21 days after myocardial infarction by Ki67 (top), AURKB (middle), and EdU (bottom). Quantitation was conducted based on five random fields of peri-infarct areas from 6 PBS and 6 rmHapln1 samples. (F, G) Serial echocardiographic measurements of the ejection fraction (EF%) and fractional shortening (FS) (n = 6 per group). Western blotting assays were conducted to assess (H) the phosphorylation of SMAD2/3 (H), versican, growth differentiation factor 11 (GDF11), and (I) NKX2.5 expression in the peri-infarct region at the indicated time points from tissues of two randomly selected…

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  Fig. 7. Recombinant mouse hyaluronan and proteoglycan link protein 1 (rmHapln1) also induces human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) proliferation. (A) Real-time quantitative polymerase chain reaction (RT-qPCR) and (B, C) western blotting were conducted to evaluate the expression of HAPLN1 at the mRNA and proteins levels in the heart tissues from embryonic (E14) and postnatal mice (P1, P7, and P35) (for qPCR, n = 5; for Western blotting, n = 3). The proliferative abilities of hiPSC-CMs (4weeks, (4W)) treated by rmHapln1 (100 ng/mL, 72 h) were investigated using (D, E) EdU incorporation assays and (F, G) immunostaining of Ki67. HiPSC-CMs (4W) were treated by conditioned medium from primary cardiomyocytes (CM) or fibroblasts (FB), with or without lentivirus-mediated HAPLN1 knockdown from P1 or P7 mice heart tissues for 72 h. Then, cell proliferation was investigated using (H) EdU incorporation assays and (I) immunostaining of Ki67. One-way analysis of variance (ANOVA) with Turkey post hoc tests was conducted in Figs. 7A and C. Two-way ANOVA with Bonferroni correction for post-hoc comparisons was conducted in Figs. 7H and I. Unpaired Welch's t-test was conducted…