Coordinated Developmental Remodeling of IGF/FGF–MAPK Signaling and Cytoskeletal Plasticity Coincides with the Loss of Cardiac Regenerative Capacity

Figures

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  Figure 2. Expression dynamics of IGF-1R, IGF-2R, InsR, PDGFRβ, FGFR1, and OSMR in the mouse heart. (A) Scheme of IGF-1, IGF-2, and insulin receptor complexes, approximate molecular weights (kDa), and ligand–receptor interactions. Arrow thickness indicates reported relative ligand-binding strength. α-SU and β-SU indicate receptor subunits. Panel adapted and modified from [35]. (B) Representative Western blots of IGF-1R, IGF-2R, and InsR from mouse hearts. Blots were probed with antibodies against the β-subunits of IGF-1R and InsR; therefore, both higher-molecular-weight precursor-associated bands (IGF-1R ~200 kDa; InsR ~220 kDa) and lower-molecular-weight β-subunit-associated bands (~95 kDa) were detected. IGF-2R migrates at ~220 kDa. Molecular weights (kDa) are indicated. (C) Quantification of IGF-1R-immunoreactive bands. Both detected bands decline significantly by P7 and remain low thereafter. (D) Quantification of InsR-immunoreactive bands. The ~95 kDa band shows a small non-significant increase at P7, whereas the ~220 kDa band increases significantly at P7 and then declines during maturation. (E) Quantification of IGF-2R (CI-M6PR) immunoreactive signal. IGF-2R shows the strongest…

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  Figure 1. Blotted membrane showing protein separation from postnatally developing hearts and schematic overview of receptor-mediated Ras/Raf/MEK/ERK signaling. (A) Representative Western blot membrane of whole-heart lysates from P3, P7, P14, P28, and adult mice. Data are presented as mean ± SEM (n = 4 hearts per group; in panel A: n = 2). Statistical analysis was performed using Student’s t-test and is shown in the results. For completeness, we have included the corresponding ANOVA results in the Supplementary Materials. RedAlert total protein staining is shown to document transfer quality and comparable protein loading across ages. A colored molecular weight marker (kDa) is shown at the right, and approximate protein localizations are indicated. (B) Schematic overview of growth factor receptor regulation of the canonical Ras → Raf → MEK → ERK cascade in cardiac cells. Ligand binding to membrane receptors promotes receptor phosphorylation and recruitment of Grb2/SOS, leading to conversion of RasGDP to RasGTP and subsequent activation of Raf isoforms, which phosphorylate MEK and propagate MAPK signaling. Proteins and phosphorylation events reported to promote pathway activation are shown in…

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  Figure 3. Postnatal reduction in B-Raf expression is accompanied by decreased MEK1/2 abundance and reduced regulatory phosphorylation in the mouse heart. (A) Representative Western blots for pan-Ras (recognizing H-, K-, and N-Ras), A-Raf, B-Raf, and C-Raf in mouse hearts at the indicated postnatal ages; quantification is shown in (B–E). (B) Ras signal declines significantly during maturation. (C) A-Raf shows a variable, non-significant increase at P14 followed by reduction in adults. (D) B-Raf is markedly downregulated during postnatal maturation, reaching ~12% of early postnatal levels by P28 and P56. (E) C-Raf shows a modest increase at P7 followed by reduction at later stages. (F) Representative Western blots for total MEK1, total MEK2, phospho-MEK1/2 (Ser217/221 for MEK1 and Ser221/225 for MEK2), phospho-MEK1 (Thr292), ERK1/2, and phospho-ERK1/2. (G) Total MEK1 declines significantly during maturation and reaches 32% in adult hearts. (H) Total MEK2 is significantly reduced by P28 and declines further to 8.3% in adults. (I) Phospho-MEK1/2 decreases markedly during maturation, reaching 8% in adults. (J) Phospho-MEK1 (Thr292) also declines sharply, reaching 5% in adults. Phosphorylation…

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  Figure 4. Enhanced sarcomeric striation with stable Actn2, increased sarcomeric α-actin signal, and developmental expression of troponin-associated proteins in the myocardium. (A) Representative immunofluorescence images of left-ventricular myocardium from P3 and adult mice. Samples were co-stained for Actn2 and myomesin (green) and filamentous actin (phalloidin; red); nuclei were counterstained with DAPI (blue). Neonatal myocardium shows less sharply defined sarcomeric organization, whereas adult cardiomyocytes display more regular cross-striation consistent with mature sarcomere architecture. (B) Representative Western blots and quantification of Actn2 and sarcomeric α-actin across postnatal ages. Actn2 shows no significant change during maturation. Sarcomeric α-actin-associated signal increases progressively, reaching 140% in adulthood. (C) Western blots of TnC, TnI, and TnT. P3 samples serve as the reference baseline (100%). (D) Representative fluorescence images of P3 and adult mouse heart tissue stained for TnC, TnI, and TnT (green). F-actin is shown in red and nuclei in blue (DAPI). The scale bar represents 30 µm. Neonatal tissue shows less regular sarcomeric organization than adult…

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  Figure 5. Postnatal downregulation of dedifferentiation-associated markers and actin-regulating proteins during cardiac maturation. (A) Fluorescence microscopy images of mouse myocardium stained for non-muscle α-actinin-1 (Actn1; green). F-actin is visualized in red and nuclei in blue (DAPI). The scale bar represents 30 µm. P3 myocardium shows broad Actn1 distribution, whereas in adult myocardium signal is strongly reduced in the myocardial compartment and largely retained in vascular structures. (B) Western blots (a) and quantification (b–f) of Actn1 and actin-regulatory proteins. (b) Actn1 shows a transient rise at P7 followed by marked decline in adulthood. (c) Profilin-1 decreases significantly from P14 onward. (d) Cofilin-1 declines significantly during postnatal maturation. (e) Acta2 declines significantly during maturation. (f) Destrin levels also decrease significantly, reaching low adult levels. p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

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  Figure 6. Expression and phosphorylation-associated activation state of cardiac ERM proteins decline. (A) Western blots showing expression of ezrin, radixin, moesin, phospho-ERM (Thr567 in ezrin, Thr564 in radixin, Thr558 in moesin), and merlin during mouse postnatal development. (B) Ezrin decreases significantly from P7 onward and reaches 8% in adulthood. (C) Radixin declines more moderately and remains detectable in adult hearts. (D) Moesin decreases significantly from P14 onward and reaches low adult levels. (E) Phospho-ERM signal declines strongly during maturation. (F) Merlin decreases significantly during postnatal development. p < 0.01 (**), and p < 0.001 (***).

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  Figure 7. Increased cytochrome c and myoglobin expression and reduced Aurora B and phospho-histone H3 mark postnatal cardiac maturation. Western blot analysis and quantification of cytochrome c (A) and myoglobin (B) show increased expression during development. Cytochrome c rises significantly from P7 onward and remains elevated in adulthood. Myoglobin also increases progressively and reaches its highest levels in adults. Western blot and quantification of Aurora B (C) and phospho-histone H3 (D) show marked reduction in mitosis-associated signals during maturation. p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

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  Figure 8. Cardiomyocyte-centered schematic of postnatal cardiac maturation. This schematic summarizes the principal developmental transitions identified in this study within a cardiomyocyte-centered framework across postnatal maturation. Three distinct phases are delineated: (i) Proliferative phase (P1–P7): Characterized by high expression of cell-cycle-associated genes and elevated levels of insulin-like growth factor (IGF) receptors and fibroblast growth factor receptor 1 (FGFR1), consistent with active proliferative signaling. In parallel, components of the MAPK cascade, including B-Raf and MEK1/2, display increased expression and phosphorylation. The cytoskeleton is dominated by a highly dynamic non-sarcomeric actin network, with abundant actin-binding and remodeling proteins (e.g., ERM proteins, phosphorylated ERM, destrin/cofilin), while sarcomeres remain immature and poorly organized. Cellular metabolism is predominantly glycolytic. (ii) Intermediate phase (P7–P14): Marked by a progressive reduction in cell-cycle-associated gene expression and decreased abundance of IGF receptors, FGFR1, and MAPK pathway components, consistent with attenuation of proliferative signaling. This phase…