N-Cadherin promotes cardiac regeneration by potentiating pro-mitotic β-Catenin signaling in cardiomyocytes

Abstract

Adult human hearts exhibit limited regenerative capacity. Post-injury cardiomyocyte (CM) loss can lead to myocardial dysfunction and failure. Although neonatal mammalian hearts can regenerate, the underlying molecular mechanisms remain elusive. Herein, comparative transcriptome analyses identify adherens junction protein N-Cadherin as a crucial regulator of CM proliferation/renewal. Its expression correlates positively with mitotic genes and shows an age-dependent reduction. N-Cadherin is upregulated in the neonatal mouse heart following injury, coinciding with increased CM mitotic activities. N-Cadherin knockdown reduces, whereas overexpression increases, the proliferation activity of neonatal mouse CMs and human induced pluripotent stem cell-derived CMs. Mechanistically, N-Cadherin binds and stabilizes pro-mitotic transcription regulator β-Catenin, driving CM self-renewal. Targeted N-Cadherin deletion in CMs impedes cardiac regeneration in neonatal mice, leading to excessive scarring. N-Cadherin overexpression, by contrast, promotes regeneration in adult mouse hearts following ischemic injury. N-Cadherin targeting presents a promising avenue for promoting cardiac regeneration and restoring function in injured adult human hearts.

Fig. 1. Identification of Cdh2 (N-Cadherin) as a novel mediator of cardiomyocyte (CM) proliferation/regeneration.

a Heatmap and cluster dendrograms of mRNAs expressed in isolated neonatal (P1 and P3) and adult CMs. b Gene ontology (GO) analysis of transcripts that were significantly upregulated in neonatal CMs revealed a significant enrichment in genes involved in mitosis, cell cycle, and DNA replication, consistent with the higher proliferative activity in neonatal CM. c RNA-Seq analysis revealed that genes involved in cell cycle progression, DNA synthesis, and proliferation (upper panel) and genes known to promote cardiac regeneration (lower panel) were expressed at a markedly higher level in neonatal (results from P1 and P3 neonatal CMs were pooled, n = 8), compared to adult (n = 7), CMs. A two-tailed t-test was utilized for comparison between groups. d Network heatmap and cluster dendrograms for the Weighted Gene Co-expression Network Analysis (WGCNA) on adult and neonatal CM RNA-Seq data. e Bubble plot and GO analysis of the genes in the blue co-expression module identified by WGCNA. f Cdh2 expression level showed a strong positive correlation with that of cell cycle-related genes in murine CMs. All experimental data are presented as means ± SEM. Exact p values are indicated in figures.

Fig. 2. Cardiac Cdh2 (N-Cadherin) level reduces dramatically beyond the regenerative age window and is upregulated in neonatal heart following injury.

a Cardiac Cdh2 expression level exhibited an age-dependent reduction from P1, P3 to adult CM (n = 3, 4, and 7, respectively). A one-way ANOVA test (p < 0.0001) was utilized followed by a Tukey post-test correction. b Immunoblotting showed that N-Cadherin and β-Catenin were significantly higher in neonatal P1 and sharply declined in P21 and adult mouse hearts (n = 10, 6, and 8, respectively). A one-way ANOVA test (p < 0.0001 in relative N-Cadherin and β-Catenin expression) was utilized followed by a Tukey post-test correction. c Schematic illustration of cardiac apical resection (AR) in P1 neonatal mice. CMs proliferation (by pH3 staining) and Cdh2 expression levels were assayed on P8 (day 7 post-AR) in apical (1), border (2 and 3) and remote zones (4 and 5). d Immunofluorescence (IF) staining showed a marked increase in pH3⁺ CMs (arrowheads) in post-resected, compared to sham-operated, neonatal mouse hearts, particularly in the apical and border zones. e Quantification of pH3⁺ CMs (n = 6 for sham^-operated; and n = 8 for AR neonatal mouse hearts). A two-tailed t-test was utilized for comparison between groups. f Quantitative RT-PCR (qRT-PCR) revealed a significant (one-way ANOVA, p = 0.0326) upregulation of Cdh2 in mouse hearts following AR injury, highest in the apical zone and reduced gradually toward the remote zone (n = 4 in each group). g IF staining showed a marked increase in N-Cadherin in apical and border region in post-resected, compared to sham-operated, neonatal mouse hearts. h Quantification of N-Cadherin intensity revealed a significant (one-way ANOVA, p = 0.0011) upregulation of N-Cadherin in mouse hearts following AR injury (n = 5 for sham-operated; and n = 6 for AR neonatal mouse hearts). These Cdh2 and N-Cadherin expression gradients were not observed in the sham-operated heart. All experimental data are presented as means ± SEM. Exact p values are indicated in figures.

Fig. 3. Depletion of Cdh2/N-Cadherin reduces the proliferative capacity of neonatal CMs.

a EdU labeling experiments revealed that the proliferative capacity was significantly reduced in Cdh2 KD (n = 4), compared to Scramble control (n = 5), neonatal mouse CMs. A two-tailed t-test was utilized for comparison between groups. b qRT-PCR confirmed a ~70% reduction of Cdh2 in neonatal CMs treated with Cdh2-targeting shRNA (left panel, n = 17 for Scramble; and n = 14 for Cdh2 KD neonatal mouse CMs). The expression levels of cell-cycle related genes, including Ccnd1 (n = 12 for Scramble; and n = 10 for Cdh2 KD neonatal mouse CMs), Cdc16 (n = 18 for Scramble; and n = 15 for Cdh2 KD neonatal mouse CMs), and Cdk2 (n = 11 for Scramble; and n = 9 for Cdh2 KD neonatal mouse CMs) were markedly reduced in Cdh2 KD, compared to controls (right panel). Notably, Ctnnb1 (n = 7 in each group), which encodes β-Catenin, was not affected by Cdh2 KD. A two-tailed t-test was utilized for comparison between groups. c Immunoblotting revealed that the protein expression of N-Cadherin (n = 7 in each group), β-Catenin (n = 7 in each group), and cell-cycle protein Cyclin D1 (n = 7 for Scramble; and n = 6 for Cdh2 KD CMs) was dramatically reduced in Cdh2 KD, compared to Scramble control, CMs. A two-tailed t-test was utilized for comparison between groups. d Reduced proliferative capacity in Cdh2 KD neonatal CMs could be restored by Ctnnb1 OE (n = 4 for Scramble + Ctrl; n = 5 for Cdh2 KD + Ctrl; and n = 5 for Cdh2 KD + Ctnnb1 OE neonatal CMs). A one-way ANOVA test (p = 0.0057) was utilized followed by a Tukey post-test correction. All experimental data are presented as means ± SEM. Exact p values are indicated in figures.

Fig. 4. The pro-mitotic effects mediated by N-Cadherin in CMs depend on its interaction with β-Catenin.

a The protein half-life of β-catenin upon Cdh2/N-Cadherin knockdown in mouse HL-1 CMs was determined using a cycloheximide chase assay, which revealed an accelerated degradation of β-Catenin in Cdh2 KD, compared to Scramble control, CMs (n = 7 in each group at various time points). A two-way ANOVA test (Interaction p = 0.0001; Time points p < 0.0001; and Group p < 0.0001) was utilized followed by a Bonferroni’s multiple comparison test. b Co-immunoprecipitation (Co-IP) experiments using neonatal mouse cardiac lysates showed that N-Cadherin could be pulled down with β-Catenin, indicating their physical interaction (left panel). The Co-IP experiments were performed in three independent biological replicates. Schematic illustration of the functional domains of N-Cadherin protein. The β-Catenin binding motif (BBM) locates at the C-terminus of N-Cadherin. Cdh2: wild-type N-Cadherin; ΔCdh2: c-terminus-truncated N-Cadherin. c Immunoblotting revealed that β-Catenin was markedly increased in Cdh2-, but not ΔCdh2-, overexpressed mouse neonatal CMs (n = 5 for Ctrl; n = 5 for Cdh2 OE; and n = 4 for ΔCdh2 OE mouse neonatal CMs). Quantification of N-Cadherin and β-Catenin proteins was shown on the right. A one-way ANOVA test (p = 0.0306 in relative N-Cadherin expression; and p < 0.0001 in relative β-Catenin expression) was utilized followed by a Fisher’s LSD post-test correction. d Overexpression of WT, but not C-terminus-truncated (ΔCdh2), N-Cadherin led to increased neonatal CMs proliferation, suggesting that BBM is required for the promo-mitotic effects of N-Cadherin in CMs (n = 4 for Ctrl; n = 3 for Cdh2 OE; and n = 4 for ΔCdh2 OE mouse neonatal CMs). A one-way ANOVA test (p = 0.0059) was utilized followed by a Tukey post-test correction. All experimental data are presented as means ± SEM. Exact p values are indicated in figures.

Fig. 5. The nuclear localization and activities of β-Catenin in the CMs are reduced with N-Cadherin depletion.

a IF staining revealed a strong co-localization between N-Cadherin and β-Catenin at the cell membrane in neonatal mouse CM (Scramble control, n = 4). The fluorescence intensity of β-Catenin in CM nuclei was dramatically reduced in Cdh2-depleted neonatal mouse CMs (n = 4 in each group). A two-tailed t-test was utilized for comparison between groups. b N-Cadherin and β-Catenin were highly co-localized at the cell membrane of neonatal mouse CM (Control, n = 4). The β-Catenin signal intensity in the nuclei was markedly increased in WT Cdh2-, but not ΔCdh2-, overexpressed neonatal mouse CMs, suggesting that N-Cadherin enhances nuclear retention of β-Catenin through BBM-mediated physical binding (n = 4 for Ctrl; n = 5 for Cdh2 OE; and n = 7 for ΔCdh2 OE mouse neonatal CMs). A one-way ANOVA test (p = 0.0096) was utilized followed by a Tukey post-test correction. c Immunoblots to detect β-Catenin in the cytoplasmic and nuclear fractions of HL-1 mouse CMs with and without Cdh2 KD. GAPDH and Lamin A/C were used as cytoplasmic and nuclear markers, respectively (n = 7 for Scramble; n = 6 for Cdh2 KD HL-1 mouse CMs). A two-tailed t-test was utilized for comparison between groups. d Luciferase reporter (TOPFLASH) assays showed that the transcriptional activity triggered by Wnt3a-induced β-Catenin activation was significantly suppressed in Cdh2 KD, compared with Scramble control, HL-1 mouse CMs, revealing the essential role of cardiac N-Cadherin in relation to the nuclear activity of β-Catenin (n = 5 in each group). A one-way ANOVA test (p = 0.0308) was utilized followed by a Fisher’s LSD post-test correction. All experimental data are presented as means ± SEM. Exact p values are indicated in figures.

Fig. 6. The pro-mitotic effect of N-Cadherin/β-Catenin cascade is conserved in human iPSC-derived CMs (hiPSC-CMs).

a Knocking down CDH2 in human iPSC-derived CMs (hiPSC-CMs) led to a significant reduction in their proliferative activity as evidenced by decreased EdU labeling (N = 3 in each group, n = 24 for Scramble; and n = 26 for CDH2 KD hiPSC-CMs). A two-tailed t-test was utilized for comparison between groups. b Ectopic expression of CTNNB1 in hiPSC-CMs rescued the suppressed proliferative activity resulting from CDH2 depletion (N = 3 in each group, n = 22 for Scramble + Ctrl; n = 38 for CDH2 KD + Ctrl; and n = 23 for CDH2 KD + CTNNB1 OE hiPSC-CMs). Overexpression of WT CDH2, but not C-terminus-truncated CDH2 (ΔCDH2), increased the proliferative activity (N = 3 in each group, n = 46 for Ctrl; n = 72 for CDH2 OE; and n = 65 for ΔCDH2 OE hiPSC-CMs) (c) and nuclear accumulation of β-Catenin (d) in hiPSC-CMs (N = 3 in each group, n = 487 for Ctrl; n = 998 for CDH2 OE; and n = 751 for ΔCDH2 OE hiPSC-CMs). A one-way ANOVA test (p = 0.0014 in b; p = 0.013 in c; and p < 0.0001 in d) was utilized followed by a Tukey post-test correction. N-Cadherin and β-Catenin were highly co-localized at the cell membrane of hiPSC-CMs (n = 4). All experimental data are presented as means ± SEM. Exact p values are indicated in figures.

Fig. 7. N-Cadherin is required for the regeneration of neonatal mouse heart following injury.

a Experimental design to determine the requirement of N-Cadherin for the regeneration of neonatal mouse heart following injury. b Comparing to αMHC-Cre^ERT2 control animals (n = 6), Cdh2 transcript was reduced by ~50% in cardiac-specific Cdh2-haploinsufficient animals (n = 8). The mRNA level of β-Catenin, Ctnnb1, was unchanged in the mouse hearts following Cdh2 depletion. A two-tailed t-test was utilized for comparison between groups. c β-Catenin protein levels were markedly downregulated in αMHC-Cdh2^fl/+ (n = 8), compared with control (n = 6), LV. There was no discernible change in the levels of total and phosphor-(pY216) GSK3β in αMHC-Cdh2^fl/+ compared with control, LV, revealing that the activity of β-Catenin destruction complex is not involved in N-Cadherin-dependent β-Catenin regulation. A two-tailed t-test was utilized for comparison between groups. d Masson’s trichrome staining of neonatal αMHC-Cdh2^fl/+ (n = 8) and αMHC-Cdh2^+/+ (n = 6) mouse cardiac sections 21 days following AR. While control animals achieved complete regeneration, αMHC-Cdh2^fl/+ mice failed to regenerate the injured heart, leaving a significantly larger scar in the cardiac apex. Quantification of scar/fibrotic area is shown in the right. A two-tailed t-test was utilized for comparison between groups. e The proportion of EdU⁺ CMs was dramatically reduced in αMHC-Cdh2^fl/+ (n = 8), compared to control (n = 6), mouse heart following AR injury. Arrowheads: de novo CMs, hollow arrowheads: proliferating non-CMs. A two-tailed t-test was utilized for comparison between groups. All experimental data are presented as means ± SEM. Exact p values are indicated in figures.

Fig. 8. Replenishing Cdh2/N-Cadherin in the adult mouse heart drives CM self-renewal and ameliorate MI-induced cardiac dysfunction.

a Experimental design to determine if replenishing Cdh2 in the adult heart could promote CM proliferation and mitigate post-MI cardiac dysfunction. b AAV9-mediated cardiac-specific overexpression of Cdh2/N-Cadherin led to a marked upregulation of β-Catenin protein, but not mRNA, in mouse LV (RNA-Cdh2: n = 16 for cTnT-CFP; and n = 15 for cTnT-Cdh2, RNA-Ctnnb1: n = 16 in each group; Protein: n = 15 for cTnT-CFP; and n = 14 for cTnT-Cdh2 adult heart). A two-tailed t-test was utilized for comparison between groups. c Echocardiographic analysis revealed a smaller infract region, reduced left ventricle end-diastolic dimension (LVIDd, n = 8 for cTnT-CFP; and n = 11 for cTnT-Cdh2 adult heart) and increased ejection fraction (EF, n = 10 for cTnT-CFP; and n = 9 for cTnT-Cdh2 adult heart) in animals receiving cTnT-Cdh2, compared to control (cTnT-CFP), AAV9 vectors. ΔEF indicates the change of EF following MI injury comparing to pre-MI baseline. A two-tailed t-test was utilized for LVIDd analysis. A two-way ANOVA test (Interaction p = 0.6614; Time points p < 0.0001; and Group p = 0.009) was utilized followed by a Fisher’s LSD test for ΔEF analysis. d Cardiac sections were collected below the ligation level with 400 µm intervals, followed by Masson’s Trichrome staining. Replenishing Cdh2 led to a marked reduction in the scar size following MI (n = 8 for cTnT-CFP; and n = 11 for cTnT-Cdh2 adult heart). A two-tailed t-test was utilized for comparison between groups. (e) The number of proliferative CMs (EdU⁺/PCM1⁺) was significantly increased in animals transduced with cTnT-Cdh2, compared to control, AAV9 (n = 7 for cTnT-CFP; and n = 6 for cTnT-Cdh2 adult heart). Arrowhead: de novo CMs, hollow arrowhead: proliferating non-CMs. A two-tailed t-test was utilized for comparison between groups. All experimental data are presented as means ± SEM. Exact p values are indicated in figures.

Fig. 9. Replenishing Cdh2/N-Cadherin in the adult mouse heart drives CM self-renewal and division.

a The αMHC-Cre^ERT2;MADM reporter mouse line was utilized to quantify CMs undergoing self-renewal/division by counting single color-labeled (red or green) CMs. cTnT-Cdh2 AAV9 transduction led to a significantly increased number of newly divided CMs (arrowheads) than those treated with control AAV9 (n = 6 in each group). A two-tailed t-test was utilized for comparison between groups. b The αMHC-Cre^ERT2;mTmG reporter mice were utilized to determine the origin of de novo CMs following injury. After tamoxifen induction 2 weeks before MI surgery, CMs were labeled with GFP, whereas non-CMs were labeled in red. Arrowheads indicate CMs derived from non-CM origins, which were labeled with membrane-targeted tdTomato. The ratio of tdTomato⁺/GFP⁺ CMs was comparable in cTnT-Cdh2- and cTnT-CFP AAV9-treated groups (n = 7 for cTnT-CFP; and n = 6 for cTnT-Cdh2 adult heart). A two-tailed t-test was utilized for comparison between groups. All experimental data are presented as means ± SEM. Exact p values are indicated in figures.