LncRNA Snhg1-driven self-reinforcing regulatory network promoted cardiac regeneration and repair after myocardial infarction

Figures

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  Figure 1. Snhg1 was highly expressed in fetal and MI hearts. (A) Quantification of Snhg1 expression in ventricles harvested from E16.5 to P56 mice by RT-qPCR (n = 5). (B) Detection of Snhg1 expression in human and mouse heart tissue by ISH (3-5 hearts). Brown dot clusters indicate Snhg1. (C) RT-qPCR analysis of Snhg1 in isolated neonatal cardiomyocytes(CMs), fibroblasts(FBs), endothelial cells (ECs) and smooth muscle cells (SMCs) (n = 5). (D-E) Co-staining of neonatal CMs, SMCs, FBs, and ECs to identify cells expressing Snhg1 by RNA-FISH in vitro (D) and in vivo (E) (n = 5). (F) RT-qPCR analysis of Snhg1 levels in neonatal and adult mouse hearts after MI (n = 6). (G) Detection of Snhg1 expression in adult and neonatal heart tissue after MI by ISH, brown dot cluster indicated Snhg1, n=5. (H) Co-staining with CM and FB markers to identify cells expressing Snhg1 in adult hearts after MI by RNA-FISH (n = 5). (I) RT-qPCR analysis of Snhg1 abundance in the cytoplasm and nucleus of neonatal CMs. (J) Detection of Snhg1expression in neonatal CMs by FISH.

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  Figure 2. Snhg1 overexpression promoted P7 CM proliferation. (A) EdU staining of P7 CMs transduced with Adv-NC or Adv-Snhg1, (647 CMs from 5 mice in the Adv-NC group, 723 CMs from 5 mice in the Adv-Snhg1group). CMs were immunostained for cardiac troponin T (cTnT) and nuclei were stained with DAPI. Arrows indicate positive CMs. (B) Immunostaining for pH3 of P7 CMs transduced with Adv-NC or Adv-Snhg1, (467 CMs from 5 mice in the Adv-NC group, 539 CMs from 5 mice in the Ad-Snhg1 group). (C-D) Immunostaining for Aurora B of P7 CMs transduced with Adv-NC or Adv-Snhg1, (536 CMs from 5 mice in the Adv-NC group, 652 CMs from 5 mice in the Adv-Snhg1 group). (E) Detection of cell cycle alterations in P7 CMs transfected with Adv-Snhg1 or Adv-NC by flow cytometry. (F) Time-lapse images of Snhg1-induced cytokinesis in P7 mouse CMs isolated from MYH6-mCherry transgenic mice (427 CMs from 5 mice in the Adv-Snhg1group). Panels are representative of images recorded during cell division (see Video 1). Arrows indicate CMs undergoing cell division. (G) Quantification of CM binucleation in P7 CMs transduced with Adv-Snhg1 or Adv-NC. 428 CMs from 5 mice in Adv-Snhg1 group at 24 h, 476 CMs from 5 mice in Adv-NC…

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  Figure 3. Snhg1 promoted CM proliferation in adult mice. (A) Schematic of the experiments in adult mouse hearts injected with AAV9. IF, immunofluorescence. (B) Representative in vivo bioluminescence and bright field images of adult mice captured on day 14 after injection of AAV9-Snhg1 virus. Arrow indicates the heart with GFP fluorescence. (C) ISH results confirming that Snhg1 was significantly increased in adult hearts injected with AAV9-NC or AAV9-Snhg1 (n = 5). Brown dot clusters indicate Snhg1. (D-E) EdU staining of CMs isolated from adult mouse hearts after transduction with AAV9-Snhg1 or AAV9-NC for 14 days (422 CMs from 5 mice in the AAV9-Snhg1 group, 359 CMs from 5 mice in theAAV9-NC group). (F) Immunostaining for pH3 of CMs isolated from adult mouse hearts 14 days after transduction with AAV9-Snhg1 and AAV9-NC (405 CMs from 5 mice in the AAV9-Snhg1 group, 367 CMs from 5 mice in the AAV9-NC group). (G) Representative bright field and cTnT immunostained images of CMs isolated from adult mouse hearts 14 days after transduction with AAV9-Snhg1 or AAV9-NC and quantification of the CM number (423 CMs from 5 mice in the AAV9-Snhg1 group, 378 CMs from 5 mice in the AAV9-NC group). (H-J)…

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  Figure 4. Snhg1 improved adult cardiac function post-MI. (A) Schematic of the MI experiments in adult mouse hearts injected with AAV9. Echo, echocardiography. IF, immunofluorescence. (B) Detection of Snhg1 expression in sham operated and infarcted adult mouse hearts by ISH analysis (n = 4). (C-D) Immunofluorescence for Ki67 and pH3 in adult mouse hearts 14 days after MI (n = 6). Arrows indicate positive CMs. (E-F) Immunofluorescence for TUNEL and IB4 in adult mouse hearts 14 days after MI (n = 6). Arrows indicate positive cells or vessels. (G) Immunohistochemistry for CD31 in adult mouse hearts 14 days after MI (n = 6). (H) Masson's trichrome-stained sections of adult mouse hearts 28 days after MI and quantification of infarct size (n = 6). (I) Echocardiography analysis of adult mouse hearts at 1, 14, and 28 days after MI (n = 10).

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  Figure 5. Loss of Snhg1 impaired neonatal cardiac regeneration. (A) Representative images and quantification of P1 CMs positive for EdU (321CMs from 5 mice in the si-Snhg1 group, 413 CMs from 5 mice in the si-NC group). Arrows indicate positive CMs. (B) Representative images and quantification of P1 CMs positive for pH3 (364 CMs from 5 mice in the si-Snhg1 group, 432 CMs from 5 mice in the si-NC group). Arrows indicate positive CMs. (C) Representative images and quantification of P1 CMs positive for Aurora B (356 CMs from 5 mice in the si-Snhg1 group, 437 CMs from 5 mice in the si-NC group). Arrows indicate positive CMs. (D) Detection of cell cycle alterations in P1 CMs by flow cytometry. (E) Schematic illustrating the procedure to construct myocardial cas9-tdTomato mouse and the delivery of Adv expressing sgRNA into heart of the myocardial cas9-tdTomato mouse. (F) ISH assays detecting Snhg1 expression in Cas9 mouse hearts 7 days after injection with Adv-sgRNA (Snhg1)-GFP or Adv-NC, n=3. (G-H) Immunofluorescence for Ki67 and pH3 in neonatal hearts 7 days after injection with Adv-sgRNA, n = 5. Arrows indicate positive CMs. (I) Schematic of the MI experiments in neonatal mouse hearts…

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  Figure 6. Snhg1 regulated CM proliferation via the PTEN/AKT/c-Myc pathway. (A) Volcano plot displaying the differentially expressed genes in P7 hearts. Red dots represent up-regulated expressed genes and blue dots represent downregulated expressed genes. (B) KEGG pathway enrichment scatter plot of the top 20 upregulated genes. x-axis indicates the enrichment factor, y-axis specifies the KEGG pathway. (C) Venn diagram showing the number of enriched KEGG pathway genes overlapping the cell cycle, PI3K-AKT, and Hippo pathways. (D) Representative gene modules of overlapping genes. (E) Silver-stained SDS-PAGE gel of proteins immunoprecipitated by an RNA pull-down assay of Snhg1 or its antisense RNA (AS-Snhg1). Arrow indicates the identified PTEN protein. (F) PTEN protein assayed by Western blotting. (G-H) RIP assay performed using an antibody against PTEN or negative IgG (n = 5). Purified RNA was used for RT-qPCR analysis, and enrichment of Snhg1 was normalized to the input. (I) PTEN protein levels measured by Western blotting in isolated CMs (n = 4). CHX was used to block protein synthesis. (J) Western blotting analysis of p-AKT, AKT, and c-Myc protein in isolated CMs with Snhg1 overexpression…

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  Figure 7. c-Myc upregulated Snhg1 expression by binding to its promoter region. (A) The two predicted c-Myc binding regions and sequences in the promoter region of Snhg1. (B) Silver-stained SDS-PAGE gel of proteins immunoprecipitated by a DNA pull-down assay of a biotinylated or unbiotinylated probe targeting Snhg1. Arrow indicates region of the gel excised for MS determination. c-Myc protein was assayed by Western blotting. (C) Image of an agarose gel for ChIP-qPCR using anti-c-Myc or anti-IgG antibodies to access binding between Snhg1 and c-Myc (n = 5). Purified RNA was used for RT-qPCR, and enrichment of Snhg1 was normalized to the input. (D) RT-qPCR analysis of Snhg1 levels in isolated P7 CMs of various groups (n = 5). (E-F) Immunofluorescence for pH3 of P7 CMs transfected with Adv-NC, Adv-Snhg1, si-c-Myc, or si-c-Myc + Adv-Snhg1 (327 CMs from 5 mice in the Adv-NC group, 439 CMs from 5 mice in the Adv-Snhg1group, 309 CMs from 5 mice in the si-c-Myc group, 352 CMs from 5 mice in the si-c-Myc + Adv-Snhg1 group). Arrows indicate positive CMs. (G) Western blotting analysis of c-Myc, PTEN, p-AKT, and AKT protein levels in isolated CMs overexpressing or depleted of c-Myc (n = 4). (H) RT-qPCR…

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  Figure 8. Mutant Snhg1 inhibited Snhg1-induced CM proliferation. (A) Construction of the overexpression vectors Adv-Snhg1-WT and Adv-Snhg1-MU. (B-C) RT-qRCR analysis of Snhg1 levels and Western blotting analysis of PTEN, PI3K, p-AKT, AKT, and c-Myc protein levels in isolated CMs (n = 4). (D) EdU staining of P7 CMs (427 CMs from 5 mice in the Adv-Snhg1-WT group, 387 CMs from 5 mice in the Adv-Snhg1-MU group). Arrows indicate positive CMs. (E) Aurora B immunofluorescence staining of P7 CMs (326 CMs from 5 mice in the Adv-NC group, 406 CMs from 5 mice in the Adv-Snhg1-WT group, 354 CMs from 5 mice in the Adv-Snhg1-MU group). Arrows indicate positive CMs. (F-H) Representative images from time-lapse videos of P7 CMs transduced with Adv-Snhg1-WT (F), Adv-Snhg1-MU (G), or Adv-NC (H). Representative images from time-lapse videos of P7 CMs transfected with Adv-Snhg1-WT (F), Adv-Snhg1-MU (G), or Adv-NC (H). CMs grown for 36 h immunostained for cTnT and stained with DAPI.