ZER1 Restrains Pressure Overload‐Induced Cardiac Remodeling by Targeting DVL2 for Gly/N‐Degron‐Dependent Degradation

Figures

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  FIGURE 1. ZER1 is reduced in failing and hypertrophic hearts. (A) Schematic of the CRL2/Gly‐N‐degron module and comparison of CRL2/Gly‐N‐degron module scores between non‐failing (NF) and dilated cardiomyopathy (DCM) human heart samples from GSE116250. (B) Association between CRL2/Gly‐N‐degron module score and fetal gene program score based on NPPA, NPPB, and MYH7 expression in human samples from GSE116250. Pearson correlation with two‐sided p‐value. (C) Immunoblot analysis and quantification of ZER1 in human myocardial extracts from healthy donors and patients with heart failure (HF) (n = 4 per group). (D) Representative ZER1 immunohistochemistry in paraffin‐embedded human heart sections. Scale bar, 50 µm. (E) RT‐qPCR analysis of NPPA, NPPB, and MYH7 mRNA levels in human hearts (healthy, n = 4; HF, n = 4). Values were normalized to GAPDH. (F–H) Correlations between myocardial ZER1 protein levels and fetal gene expression (NPPA, NPPB, and MYH7) in human hearts. Pearson correlation with two‐sided p value. (I) Immunoblot analysis and quantification of ZER1 in mouse left ventricular lysates 4 weeks after sham or TAC (n = 3 per group). (J) Immunoblot analysis and quantification of ZER1 in adult…

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  FIGURE 2. Cardiac IP‐MS identifies DVL2 as an N‐terminus‐dependent ZER1 interactor. (A) Endogenous ZER1 immunoprecipitation followed by mass spectrometry (IP‐MS) using mouse left ventricular lysates. Volcano plot comparing ZER1 immunoprecipitation with IgG control. Significantly enriched proteins are shown in red. ELOC, Elongin C; CUL2, Cullin 2. (B, C) Reciprocal endogenous Co‐IP of ZER1 (B) and DVL2 (C) from WT mouse heart lysates. (D, E) Reciprocal endogenous Co‐IP of ZER1 (D) and DVL2 (E) in HL‐1 cardiomyocytes. (F) GST pull‐down showing direct interaction between GST‐ZER1 and His‐DVL2. (G) GST pull‐down showing interaction between bacterially expressed GST‐ZER1 and Flag‐DVL2 from transfected HEK293T cell lysates. (H, I) SPR analysis of ZER1‐DVL2 binding. GST‐ZER1 immobilized with His‐DVL2 as analyte (H), or His‐DVL2 immobilized with GST‐ZER1 as analyte (I). Dissociation constants (KD) are indicated. (J) Confocal microscopy showing co‐localization of endogenous ZER1 and DVL2 in neonatal mouse cardiomyocytes. Scale bar, 10 µm. (K) Co‐IP showing that mutation of DVL2 Ala1 and/or Gly2 to Asn (A1N, G2N, or A1N/G2N) abolishes ZER1‐DVL2 association in HEK293T cells. (L) Domain mapping of…

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  FIGURE 3. ZER1 drives proteasomal degradation of DVL2. (A) Immunoblot analysis of DVL2 following HA‐ZER1 overexpression in HEK293T cells. (B) Immunoblot analysis of endogenous DVL2 following ZER1 knockdown in HEK293T cells. (C, D) Cycloheximide (CHX) chase assays showing altered DVL2 stability with ZER1 overexpression (C) or ZER1 knockdown (D). (E–G) Effects of MG132 (10 µm) (E), MLN4924 (10 µm) (F), or phenylalanine (Phe, 20 µm) (G) on ZER1‐mediated DVL2 reduction in HEK293T cells. (H) Effects of glycine or alanine supplementation on DVL2 abundance and ZER1‐dependent DVL2 reduction. (I, J) Ubiquitination assays using ubiquitin lysine‐to‐arginine (KR) mutants (I) or lysine‐only (K‐only) mutants (J) to define linkage requirements for ZER1‐driven DVL2 polyubiquitination. Data are presented as mean ± SEM. Statistical analyses were performed using two‐way ANOVA followed by Holm‐Šídák's post hoc test. *p < 0.05 and **p < 0.01.

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  FIGURE 4. ZER1 restrains DVL2‐CaMKII‐HDAC4‐MEF2C signaling. (A–C) Immunoblot analysis of DVL2‐CaMKII‐HDAC4 signaling following HA‐ZER1 overexpression (A), ZER1 knockdown (B), or combined ZER1 and DVL2 knockdown (C) in HEK293T cells. (D) Immunoblot analysis of the ZER1‐DVL2‐CaMKII‐HDAC4 axis in WT and Zer1 KO mouse hearts after sham or TAC. (E–G) Immunohistochemistry and quantification of ZER1 and DVL2 in WT and Zer1 KO hearts after sham or TAC. Scale bar, 50 µm. (H, I) MEF2C‐dependent luciferase reporter assays in HL‐1 cells following HA‐ZER1 overexpression (H) or Zer1 silencing with or without Dvl2 knockdown (I). (J) Immunoblot analysis of DVL2‐CaMKII‐HDAC4 signaling in hiPSC‐derived cardiomyocytes following ZER1 knockdown, DVL2 knockdown, or combined knockdown. Data are presented as mean ± SEM. Statistical analyses were performed using one‐way or two‐way ANOVA followed by Holm–Šídák's post hoc test. ns, not significant; *p < 0.05, **p < 0.01, and ***p < 0.001.

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  FIGURE 5. Cardiomyocyte‐specific Zer1 deletion exacerbates pressure overload remodeling via DVL2. (A) Representative gross hearts and histological analyses from Zer1 fl/fl and Zer1‐cKO mice subjected to sham or TAC, with AAV9‐cTnT‐NC or AAV9‐cTnT‐shDvl2. H&E, MTT, and WGA staining are shown. Scale bars, 1 mm for H&E and 50 µm for MTT and WGA. (B) HW/BW ratios (n = 6 mice per group). (C) Quantification of cardiomyocyte CSA by WGA staining (n = 5 hearts per group; ten fields per heart). (D) Quantification of myocardial fibrosis (n = 5 hearts per group). (E, F) Echocardiographic evaluation of EF and FS (n = 6 mice per group). (G–I) RT‐qPCR analysis of Nppa, Nppb, and Myh7; values were normalized to Gapdh (n = 5 mice per group). (J) Immunoblot analyses of ZER1, DVL2, and CaMKII‐HDAC4 signaling in heart lysates from the indicated groups. Data are presented as mean ± SEM. Statistical analyses were performed using two‐way ANOVA followed by Holm–Šídák's post hoc test. ns, not significant; *p < 0.05, **p < 0.01, and ***p < 0.001.

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  FIGURE 6. ZER1 is required for Wwp1 knockdown‐mediated protection against pressure overload‐induced cardiac remodeling. (A) Schematic of the AAV9‐cTnT‐shWwp1 vector. (B) Experimental timeline for AAV9 delivery in WT and Zer1 KO mice subjected to TAC. (C) Representative gross hearts and histological analyses from WT and Zer1 KO mice subjected to sham or TAC, with AAV9‐cTnT‐NC or AAV9‐cTnT‐shWwp1. H&E, WGA, and Masson's trichrome staining are shown. Scale bars, 1 mm for H&E and 50 µm for MTT and WGA. (D) HW/BW ratios (n = 6 mice per group). (E) Quantification of cardiomyocyte cross‐sectional area by WGA staining (n = 5 hearts per group; ten fields per heart). (F) Quantification of myocardial fibrosis (n = 5 hearts per group). (G, H) Echocardiographic evaluation of EF and FS (n = 6 mice per group). Data are presented as mean ± SEM. Statistical analyses were performed using two‐way ANOVA followed by Holm–Šídák's post hoc test. ns, not significant; *p < 0.05, **p < 0.01 and ***p < 0.001.

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  FIGURE 7. Therapeutic cardiomyocyte‐specific ZER1 expression attenuates pressure overload remodeling. (A) AAV9‐cTnT‐Zer1 construct and experimental timeline for therapeutic ZER1 expression after TAC. (B) Representative gross hearts and histological analyses 4 weeks after sham or TAC. H&E, WGA, and Masson's trichrome staining are shown. Scale bars, 1 mm for H&E and 50 µm for WGA and Masson's trichrome. (C) HW/BW ratios (n = 7 mice per group). (D) Quantification of cardiomyocyte cross‐sectional area by WGA staining (n = 5 hearts per group; ten fields per heart). (E) Quantification of myocardial fibrosis (n = 5 hearts per group). (F, G) Echocardiographic evaluation of EF and FS (n = 7 mice per group). (H–J) RT‐qPCR analysis of Nppa, Nppb, and Myh7 mRNA levels; values were normalized to Gapdh (n = 5 mice per group). (K) Immunoblot analyses of ZER1, DVL2, and CaMKII/HDAC4 phosphorylation in hearts with the indicated treatments. Data are presented as mean ± SEM. Statistical analyses were performed using two‐way ANOVA followed by Holm–Šídák's post hoc test. *p < 0.05, **p < 0.01, and ***p < 0.001.

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  FIGURE 8. Working model of ZER1‐DVL2 regulation in pressure overload‐induced cardiac remodeling. Schematic model showing that pressure overload reduces cardiomyocyte ZER1, thereby weakening CRL2ZER1‐dependent DVL2 degradation and allowing DVL2 accumulation. Increased DVL2 activates the CaMKII‐HDAC4‐MEF2C axis and promotes fetal gene reactivation and pathological remodeling. Cardiomyocyte‐directed AAV9‐Zer1 restoration enhances K48‐linked DVL2 polyubiquitination and proteasomal degradation, thereby restraining this signaling axis and preserving cardiac structure and function.