Transient induction of actin cytoskeletal remodeling associated with dedifferentiation, proliferation, and redifferentiation stimulates cardiac regeneration

Figures

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  Figure 1. Single-nucleus RNA sequencing analysis reveals that actin cytoskeletal remodeling contributes to the dedifferentiation, proliferation, and redifferentiation of cardiomyocytes after myocardial infarction. (A) Schematic diagram showing the experimental design for snRNA-seq analysis; sequencing was performed in isolated nuclei from hearts on postnatal Days 1 and 3 after surgery-induced myocardial infarction or sham. (B) Uniform manifold approximation projection (UMAP) visualization of different cardiomyocyte clusters. (C) Violin plots showing the expression of cardiomyocyte-specific genes (Tnnt2, Actc1, and Pcm1) and proliferating genes (Mki67, Anln, Top2a, Cenpe, and Pcna). (D) Fraction of cardiomyocyte populations in each snRNA-seq sample. (E) The cell cycle analysis in CM0, CM2, CM3, CM6, and CM8 cells. (F) Pseudotime analysis revealed a circular trajectory of CM0, CM2, CM3, CM6, and CM8 cells. (G) UMAP visualization of cardiomyocyte clusters in the circular trajectory revealed the regenerative processes of dedifferentiation, proliferation, and redifferentiation. (H) Pseudotime analysis revealed the sinusoidal expression of immature (Actc1, Myh7, and Tnni1), and mature (Cacna1c,…

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  Figure 2. Transient transfection of the 2D2P genes using a non-integrating lentivirus system induces transient actin remodeling and a transient increase in cardiomyocyte proliferation in vitro. (A) Heatmap showing the expression patterns of actin polymerization genes (Dmd, Ctnna3, Palld, Ank2, Fhod3, and Diaph3) and actin depolymerization genes (Cfl1, Pfn1, Tmsb10, and Tmsb4x) in CM0, CM2, CM3, CM6, and CM8 cells. (B) The ranking of maximum variation of expression (ΔExpression) in actin polymerization (Dmd, Ctnna3, Palld, Ank2, Fhod3, and Diaph3) and depolymerization (Cfl1, Pfn1, Tmsb10, and Tmsb4x) genes. (C) Staining of DAPI (blue), pH3 (red), and α-actinin (green) in neonatal mouse ventricular myocytes (NMVMs) from postnatal Day 7 (P7) mice (C1). Scale bar, 20 μm. Quantification of pH3+ cardiomyocytes in NMVMs from P7 mice treated with siRNA for 48 h. ∗P < 0.05 vs. Control. 6 repeats/∼600 cells in each group (C2). (D) Schematic diagram of the plasmid design for Tnnt2-2D2P polycistronic NIL. (E–H) Representative images of NMVMs from postnatal Day 7 mice treated with Control-NIL or Tnnt2-2D2P-NIL for 0, 4, or 8 days and immunostained for DAPI (blue), GFP (green), Ki67 (red), pH3 (red),…

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  Figure 3. Transient transfection of the 2D2P genes using Tnnt2-2D2P-NIL can improve cardiac function and reduce scar size after myocardial infarction by promoting cardiomyocyte proliferation in mice. (A) Schematic diagram of the experimental design for virus injection of Control-NIL or Tnnt2-2D2P-NIL in mice with myocardial infarction (MI). Echocardiographic analysis was performed at the age of 8 weeks as a baseline and 24 h, 7, 14, 21, and 28 days after MI. Intramyocardial injection of Tnnt2-2D2P-NIL or Control-NIL virus was performed 7 days after MI. EdU was intraperitoneally injected on 9, 11, and 13 days after MI. (B–E) Quantification of left ventricular ejection fraction (EF%, B), fraction shortening (FS%, C), left ventricular internal diastolic diameter (LVIDd, D), and left ventricular internal systolic diameter (LVIDs, E). n = 8–10. ∗P < 0.05 vs. Control-NIL. (F, G) Quantification of the change in EF% (ΔEF%, F) and FS% (ΔFS%, G) for each mouse between 7 and 28 days after MI. n = 8–10. ∗P < 0.05 vs. Control-NIL. (H) Serial sectioning was performed at 500 μm intervals from the ligation area to the apex. Representative images (H1) and quantification (H2) of infarct length by Masson's…

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  Figure 4. Single-nucleus RNA sequencing analysis reveals regenerative effect of Tnnt2-2D2P-NIL treatment. (A) Schematic diagram showing the experimental design for snRNA-seq analysis; sequencing was performed on post-MI hearts 7 days after virus injection. (B) Uniform manifold approximation projection (UMAP) visualization of different cell clusters. (C) UMAP visualization grouped by different samples. (D) Violin plots showing the expression of cell-specific genes, including cardiomyocytes (Tnnt2, Actc1, and Pcm1), fibroblasts (Postn, Fbn1, and Col5a1), endothelial cells (Pecam1, Flt1, and Tie1), smooth muscle cells (Pdgfrb, Notch3, and Cspg4), epicardium cells (Wt1, Upk3b, and Muc16), and blood cells (Mrc1, Fyb, and Cd86). (E) UMAP visualization of different cardiomyocyte clusters. (F) Violin plots showing the expression of cardiomyocyte-specific genes (Tnnt2, Actc1, and Pcm1) and proliferating genes (Mki67, Anln, Top2a, and Cenpe). (G) UMAP visualization grouped by different cardiomyocyte clusters and samples (G1); Fraction of cardiomyocyte populations in each snRNA-seq sample (G2). (H) Feature plots showing the expression of proliferating genes (Mki67, Anln, Top2a, and Cenpe) grouped by…

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  Figure 5. Transient transfection of the 2D2P genes using Tnnt2-2D2P-NIL induces transient actin remodeling of cardiomyocytes in mice after myocardial infarction. (A, B) Representative images (A1, B1) of mouse hearts at the infarct border and remote zones stained with wheat germ agglutinin (WGA, green), DAPI (blue), and CTnI (red). Scale bar, 20 μm. Quantification of cardiomyocyte size (A2, B2) and ΔR (A3, B3) of Control-NIL or Tnnt2-2D2P-NIL treated hearts in infarct border and remote zones at 14 or 21 days after myocardial infarction (MI). 150–300 cells from 5 to 7 mice for cardiomyocyte size quantification. ∼120 cells from 5 to 7 mice for ΔR quantification. ∗P < 0.05 vs. Control-NIL. (C) Representative images (C1) and histograms (C2) showing the distribution of cardiomyocyte orientation angles in Control-NIL or Tnnt2-2D2P-NIL treated hearts at 14 or 21 days after MI. Scale bar, 20 μm. 330–370 cells from 5 to 7 mice. ∗P < 0.05 vs. 14D-Control-NIL; #P < 0.05 vs. 14D-Tnnt2-2D2P-NIL. (D, E) Representative images (D1, E1) and quantification (D2, E2) of the percentage of cardiomyocytes with disorganized F-actin in Control-NIL or Tnnt2-2D2P-NIL treated hearts in infarct border and remote zones…

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  Figure 6. Tnnt2-2D2P-NIL treatment for 4 months results in a maintained improvement of cardiac function without obvious ventricular dilatation, thickened septum, or fatal arrhythmia. (A) Schematic diagram of the experimental design for the injection of Control-NIL or Tnnt2-2D2P-NIL viruses in mice. Echocardiographic analysis was performed at the age of 8 weeks as a baseline and 24 h, 1, 4, 8, 12, and 16 weeks after myocardial infarction (MI). Intramyocardial injection of the Tnnt2-2D2P-NIL or Control-NIL virus was performed 7 days after MI. (B–E) Quantification of left ventricular ejection fraction (EF%, B), fraction shortening (FS%, C), left ventricular internal diastolic diameter (LVIDd, D), and left ventricular internal systolic diameter (LVIDs, E). n = 6–10. ∗P < 0.05 vs. Control-NIL. (F, G) Quantification of the change in EF% (ΔEF%, F) and FS% (ΔFS%, G) for each mouse between 1 and 16 weeks after MI. n = 6–8. ∗P < 0.05 vs. Control-NIL. (H) Quantification of lung weight/body weight ratio in Control-NIL- or Tnnt2-2D2P-NIL-treated mice at 16 weeks after MI. n = 6–8. ∗P < 0.05 vs. Control-NIL. (I) Serial sectioning was performed at 500 μm intervals from the ligation area to the apex.…

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  Figure 7. Actin cytoskeletal remodeling contributes to the dedifferentiation, proliferation, and redifferentiation of cardiomyocytes. Transient transfection of the 2D2P genes using Tnnt2-2D2P-NIL can promote cardiomyocyte expansion and regenerative repair after myocardial infarction.