Limited Regeneration Potential with Minimal Epicardial Progenitor Conversions in the Neonatal Mouse Heart after Injury

Abstract

The regeneration capacity of neonatal mouse heart is controversial. In addition, whether epicardial cells provide a progenitor pool for de novo heart regeneration is incompletely defined. Following apical resection of the neonatal mouse heart, we observed limited regeneration potential. Fate-mapping of Tbx18^MerCreMer mice revealed that newly formed coronary vessels and a limited number of cardiomyocytes were derived from the T-box transcription factor 18 (Tbx18) lineage. However, further lineage tracing with SM-MHC^CreERT2 and Nfactc1^Cre mice revealed that the new smooth muscle and endothelial cells are in fact derivatives of pre-existing coronary vessels. Our data show that neonatal mouse heart can regenerate but that its potential is limited. Moreover, although epicardial cells are multipotent during embryogenesis, their contribution to heart repair through "stem" or "progenitor" cell conversion is minimal after birth. These observations suggest that early embryonic heart development and postnatal heart regeneration are distinct biological processes. Multipotency of epicardial cells is significantly decreased after birth.

Keywords: cell lineage tracing; epicardial cells; heart regeneration; neonatal mouse.

Figure 1.. Neonatal Heart Repair upon Ventricular Apex Resection

(A and B) Transverse sections of sham (A) and apex-resected hearts (B) at 3, 7, 14, 21, 30, and 60 dps. (C and D) High magnification of apex indicates amputation and clot at 3 dps (C1 and D1), and repair at 7–60 dps (C2–C6 and D2–D6). Trichrome staining of serial sections revealed that the apex was sealed with a large amount of fibrin between 7–30 dps (D2–D5), and fibrosis was replaced by the reconstituted wall by 60 dps (D6). Arrows indicate injury site. Scale bar, 1 mm. See also Figures S1 and S2.

Figure 2.. Limited Potential of Neonatal Heart Regeneration

(A–D) Transverse sections of representative hearts at 21 dps (A and B) and 60 dps (C and D), suggesting that the repairing process takes longer than 21 days. A significant portion of hearts (46.2%) cannot be fully regenerated at 60 dps (C4 and C5). Asterisks indicate injured regions and incomplete repair. (E) Quantification of repair degree at 21 dps and 60 dps determined by apex shape and fibrotic tissues. (F–H) Global view of hearts in sham (F) and surgery (G) groups. Ventricular morphology is quantitated by the ratio of maximal horizontal width (w) to maximal vertical height (h) (H). **p < 0.01 versus the sham control. Scale bar, 1 mm. See also Figures S1 and S2.

Figure 3.. Cell Proliferation in the Injured Hearts

(A) Diagram of pulse-chase EdU labeling of proliferating cells and the timeline for EdU injection and tissue collection. (B–I) Co-localization of EdU, cTNT, and nuclei (DAPI) in the apex (B1, D1, F1, and H1) and remote areas (B2, D2, F2, and H2) at 21–32 dps (B–E) and 32–43 dps (G–I) in the resected (B, C, F, and G) and sham (D, E, H, and I) groups. Asterisks indicate injury site. Arrows in (C1) and (G1) show EdU-positive cardiomyocytes, and arrows in (C2), (E), (G2), and (I) show EdU-positive non-cardiomyocytes (cTnT⁻). (J and K) Quantification of EdU-labeled proliferative cells (J) and cardiomyocytes (K) in sham and resected hearts at 32 dps and 43 dps. Quantitative analysis was performed on 5 fields at 100× magnification from 3 different hearts per group at each time point. *p<0.05, **p < 0.01 versus the control. Scale bar, 100 μm (white) and 25 μm (yellow). See also Figure S3.

Figure 4.. Accumulated Tbx18-Expressing Cells in the Injured Region

(A) DAPI staining at 3, 7, 14, 21, 30, and 60 dps. (B and C) Robust Tbx18-expressing cells (Tbx18^H2B-GFP-positive) in the injury site during repair (arrows) with high density at 3–21 dps (B1–B4 and C1–C4). (D and E) Immunostaining in the injured site at 21 dps (D) and 60 dps (E). Tbx18^H2B-GFP is not co-expressed with cTNT (D1 and E1) but is co-expressed with SM-MHC (D2 and E2), α-SMA (D3 and E3), and PDGFRβ (D4 and E4). Tbx18^H2B-GFP is not co-expressed with PECAM in the endothelial cells (D5 and E5). The top right corner images in (D) and (E) are high magnification of the areas outlined in each panel. Scale bar, 1mm (white) and 100 μm (yellow). See also Figures S4 and S5.

Figure 5.. Tbx18⁺ Cells Do Not Become Cardiomyocytes or Coronary Endothelial Cells after Birth

The neonatal mice were given a single subcutaneous dose of tamoxifen at P0, and the hearts were collected at postnatal day 30 for analysis. (A and B) Tbx18 progeny do not include cardiomyocytes (not co-localized with cTNT, A, or Nkx2.5, B). (C and D) Co-expression of Tbx18 progeny with SM-MHC (C4) and α-SMA (D4). (E) Tbx18 lineage is not co-expressed with PECAM in the coronary endothelial cells (E4). (A1), (B1), (C1), (D1), and (E1) are DAPI staining. GFP-positive cells in (A2), (B2), (C2), (D2), and (E2) are cells of Tbx18 lineage. (A3), (B3), (C3), (D3), and (E3) are antibody staining for cTNT, Nkx2.5, SM-MHC, α-SMA and PECAM, respectively. (A4), (B4), (C4), (D4), and (E4) are overlays of (A1–A3), (B1–B3), (C1–C3), (D1–D3), and (E1–E3), respectively. Scale bar, 100 μm. See also Figure S6.

Figure 6.. Tbx18 Lineage Analysis after Cardiac Injury

(A–K) Tbx18^MerCreMer;R26R^tdTomato (A–F and H–) and Tbx18^MerCreMer;R26R^lacZ (G) mice were injected with tamoxifen after apex resection. (A–F) Immunostaining was performed on Tbx18^MerCreMer;R26R^tdTomato hearts at 30 dps (A–D) and 60 dps (E and F). Arrows in (A), (B), (E), and (F) indicate tdTomato and cTNT double-positive cells in the apex at 30 dps (A and B) and 60 dps (E and F). Arrows in (C) and (D) indicate non-myocardial Tbx18 lineage (cTnT⁻) in the remote area (C) or in the apex of sham group (D). (B–D and F) Right-bottom corner images are high magnification of the areas indicated by arrows. (G) X-gal staining of Tbx18^MerCreMer;R26R^lacZ hearts revealed Tbx18-derived cardiomyocytes (arrows in G4–G6) in the apex at 60 dps. (G2), (G3), and (G4) are high magnification of the square areas in G1. (G5) and (G6) are high magnification of the square areas in (G4). Unnotched arrows (G2–G4) indicate X-gal negative cells. (H–K) Immunostaining on Tbx18^MerCreMer;R26R^tdTomato hearts at 21 dps (H) and 60 dps (I). tdTomato signals (arrows) are co-localized with SM-MHC in the apex at 21 dps and 60 dps (H and I). PECAM staining (unnotched arrows in J and K) is not co-localized with tdTomato (arrows) in the regenerative coronary at 21 dps and 60 dps. Right bottom corner images (H-K) are high magnification of areas indicated by notched arrows. Scale bar, 50 μm (white), 100 μm (black) and 20 μm (yellow). See also Figure S6.

Figure 7.. New Coronary SMCs and Endothelial Cells within the Ventricular Apex Are Derived from the Pre-existing Coronary Vessels

(A) Immunostaining of SM-MHC^CreERT2; R26R^mT/mG hearts at P0 with tamoxifen induction during gestation. (B–D) Hearts at 30 dps (B) and 60 dps (C and D) with apical resection at P1. GFP⁺ cells (A1, B1, C1, and D1) are cells of SM-MHC lineage. (A2), (B2), (C2), and (D2) are antibody staining for α-SMA. (A3), (B3), (C3), and (D3) are overlays of SM-MHC lineage (GFP⁺) with DAPI and α-SMA. α-SMA⁺ SMCs in the injured apex are from SM-MHC lineage (arrows in B3 and D3). Asterisk indicates injured area. (E and F) Regeneration of coronary endothelial after apical resection. Immunostaining of Nfatc1^Cre/+;R26R^tdTomato/+ hearts at 60 dps. (E) Nfatc1 progeny give rise to coronary endothelium (arrows in E4). (F) PECAM⁺ endothelial cells in the newly formed apex are from Nfatc1⁺ endocardial/endothelial lineage (arrows in F4). (E1) and (F1) are DAPI staining; (E2) and (F2) are PECAM staining; (E3) and (F3) are overlays of Nfatc1 lineage (tdTomato⁺) with DAPI. (E4) and (F4) are high-magnification images of the areas outlined in (E3) and (F3). They are overlays of PECAM staining and Nfatc1 lineage (tdTomato⁺). Scale bar, 100 μm.