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. 2018 May 2;4(5):eaao5553.
doi: 10.1126/sciadv.aao5553. eCollection 2018 May.

The local microenvironment limits the regenerative potential of the mouse neonatal heart

Mario Notari  1 , Antoni Ventura-Rubio  1 , Sylvia J Bedford-Guaus  1   2 , Ignasi Jorba  3   4   5 , Lola Mulero  1 , Daniel Navajas  3   4   5 , Mercè Martí  1   2 , Ángel Raya  1   2   6
Affiliations

The local microenvironment limits the regenerative potential of the mouse neonatal heart

Mario Notari et al. Sci Adv. .

Abstract

Neonatal mice have been shown to regenerate their hearts during a transient window of time of approximately 1 week after birth. However, experimental evidence for this phenomenon is not undisputed, because several laboratories have been unable to detect neonatal heart regeneration. We first confirmed that 1-day-old neonatal mice are indeed able to mount a robust regenerative response after heart amputation. We then found that this regenerative ability sharply declines within 48 hours, with hearts of 2-day-old mice responding to amputation with fibrosis, rather than regeneration. By comparing the global transcriptomes of 1- and 2-day-old mouse hearts, we found that most differentially expressed transcripts encode extracellular matrix components and structural constituents of the cytoskeleton. These results suggest that the stiffness of the local microenvironment, rather than cardiac cell-autonomous mechanisms, crucially determines the ability or inability of the heart to regenerate. Testing this hypothesis by pharmacologically decreasing the stiffness of the extracellular matrix in 3-day-old mice, we found that decreased matrix stiffness rescued the ability of mice to regenerate heart tissue after apical resection. Together, our results identify an unexpectedly restricted time window of regenerative competence in the mouse neonatal heart and open new avenues for promoting cardiac regeneration by local modification of the extracellular matrix stiffness.

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Figures

Fig. 1
Fig. 1. Regeneration of the heart in P1 mice following surgical resection of a small portion of the left ventricular apex.
(A) Survival of P1 mice that underwent apical amputation (AMP; n = 46) or not (SHAM; n = 31). Animal survival was determined immediately (0 h), after 3 hours (3 h), and the day after surgery (24 h). Means ± SEM. (B) Stereomicroscopic pictures of AMP and SHAM hearts at 21 dpa showed similar heart morphology with only minimal fibrosis evident in the external ventricular wall (black arrowheads). (C) Representative hematoxylin and eosin (H&E)– and trichrome-stained sections of the apex of resected hearts at various stages of regeneration. Cardiac tissue loss is evident at 3 and 7 dpa by the misalignment of tissue fibers (H&E inset) and by the presence of blue staining evidencing collagen deposition (trichrome stain inset). Notably, at 21 dpa, fiber misalignment and blue collagen deposits were evident only in tissue outside the ventricular wall. White arrowheads indicate acute inflammation, and black arrowheads indicate granulation tissue, whereas the dashed line marks the lesion border. Scale bars, 50 μm. Right: Quantification analysis of the scar area in P1 AMP versus SHAM at 21 dpa. (D) Immunofluorescence staining for troponin-I and DAPI at 3, 7, and 21 dpa. An asterisk indicates the healing area, whereas the dashed line marks the lesion border. Scale bars, 25 μm. (E) Number of proliferating CMs (DAPI+/Tnni3+/BrdU+ cells) per 0.1 mm2 counted in paraffin-embedded SHAM (n = 3) and AMP (n = 5) heart sections from BrdU multiple pulse-chase experiments at the indicated dpa. Data are means ± SEM. Only fields completely filled with cells were included in the analysis. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 2
Fig. 2. Scar and new myocardial tissue assessment following surgical amputation of the left ventricular apex in P2 mice revealed a lack of myocardial regeneration.
(A) Survival of P2 mice that underwent amputation (AMP; n = 91) or not (SHAM; n = 51) of a small portion of the left ventricular apex. Animal survival was determined immediately (0 h), after 3 hours (3 h), and the day after surgery (24 h). Data are means ± SEM. (B) Stereomicroscopic pictures of AMP and SHAM hearts at 21 dpa demonstrated the presence of extensive white patches on the external ventricular wall 21 days following surgery (black arrowheads). Scale bar, 1 mm. (C) Heart weight of P2 AMP (n = 3) and SHAM (n = 3) mice was determined at different time points following surgery. (D) Representative H&E- and trichrome-stained sections of P2 hearts at 21 dpa. Cardiac tissue loss is evident by the misalignment of tissue fibers (H&E inset) and by the presence of intense blue staining (trichrome inset). Dashed line marks the lesion border. Scale bar, 200 μm. Right: Quantification analysis of the scar area in P2 AMP versus SHAM at 21 dpa. ***P < 0.001. (E) Paraffin-embedded resected P2 hearts were sectioned and immunostained for troponin-I and DAPI. Representative images at various time points during the post-AMP process demonstrate an absence of newly formed myocardial tissue. An asterisk indicates the healing area, whereas the dashed line marks the lesion border. Scale bar, 25 μm. (F) Number of proliferating CMs (DAPI+/MF-20+/BrdU+ cells) per 0.1 mm2 counted in paraffin-embedded SHAM and AMP heart sections from BrdU multiple pulse-chase experiments at the indicated dpa. Data are means ± SEM; n = 3. Only fields completely filled with cells were included in the analysis. ns, not significant.
Fig. 3
Fig. 3. Differentially expressed genes in the ventricles of hearts from P1 and P2 mice.
(A) Heat map showing the most differentially expressed (up- or down-regulated) genes in individual hearts from P1 and P2 mice. Shaded color bar represents low (red) to high (green) expression level changes. (B) GO analysis of differentially expressed genes between P1 and P2 hearts showing that the most differentially expressed genes belong to structural constituents of myoskeleton and of the ECM. FDR, false discovery rate. (C) Left: Quantification of MF-20+/Ki67+ CMs at 1-day-old (P1) or 2-day-old (P2) newborn mice; n = 3. Right: Representative immunofluorescence pictures of P1 and P2 heart sections display positive signal for myosin heavy chain (MF-20) and Ki67. DAPI was used to visualize CM nuclei. Black arrowheads indicate double-positive CMs. (D) Quantification analyses (%) of the mononucleated (MONO-) or binucleated (BI-) CMs at different time points during cardiac regeneration carried on hearts of P1 and P2 mice.
Fig. 4
Fig. 4. P1 and P2 hearts show differences in the ECM expression and stiffness; BAPN treatment reduced tissue stiffness in P3 mice.
(A) Left: Protein levels of ECM components were detected in ventricular heart tissue of P1 and P2 mice by immunoblotting using specific antibodies against collagen II, collagen IV, elastin, and laminin. Junctional connexin 43 protein was used as a loading control. A representative blot from three independent experiments is shown for each panel. Notably, each panel was cropped to better illustrate the difference between P1 and P2. Full-length blots are presented in fig. S6A. Right: Densitometry analysis of three independent Western blot experiments. (B) Graph showing the overall stiffness as measured using AFM in decellularized sections of P1 and P2 neonatal mouse hearts; n = 3. (C) Immunofluorescence showing the distribution of collagens II and IV (top) and fibronectin and laminin (bottom) in heart sections of P3 mice after treatment with BAPN and in control littermates. Scale bars, 50 μm. (D) Graph showing the overall stiffness as measured using AFM in decellularized sections of control and BAPN-treated P3 neonatal mouse hearts; n = 3. *P < 0.05, **P < 0.01.
Fig. 5
Fig. 5. Decreasing ECM stiffness rescues heart regeneration capacity of P3 mice.
(A) Stereomicroscopic pictures of control or BAPN-treated P3 neonatal mouse hearts at 21 dpa showed similar heart morphology, but fibrotic patches (black arrowheads) were much more evident in the external ventricular wall of control hearts. (B) Representative trichrome-stained sections of control and BAPN-treated P3 neonatal mouse hearts at 21 dpa. Dashed lines mark the amputation plane. Scale bars, 200 μm. (C) Serial transverse sections of trichrome-stained hearts of two control and two BAPN-treated neonatal mice at 21 dpa. Collagen deposits are indicated by black arrowheads. (D) Quantification analysis of the scar area in control and BAPN-treated P3 neonatal mice at 21 dpa; n = 3. (E) Left: Representative immunofluorescence images of heart sections showing positive signal for myosin heavy chain (MF-20) and Ki67. DAPI was used to visualize CM nuclei. Black arrowheads indicate double-positive CMs. Scale bars, 25 μm. Right: Quantification of proliferating CMs (MF-20+/Ki67+ cells) in control and BAPN-treated P3 neonatal mouse hearts before (0 dpa) or at 21 dpa. Data are means ± SEM; n = 3. ***P < 0.001; ns, not significant.
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