Vinculin Y822 phosphorylation regulates adhesion remodeling during cardiomyocyte maturation

Figures

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  Fig. 1.. VCL pY822 levels correlate with CM adhesion dynamics during heart development. (A) Western blots of heart lysates from E13.5, E17.5, P1, P7, P14 and 10-week-old WT (C57BL/6J) probed for VCL pY822 and total VCL. (B) Quantification of western blots in shown A. The graph shows the ratio of Y822 phosphorylated VCL (pVCL) to total VCL (n=4 hearts/time point). *P<0.05, ****P<0.0001 (one-way ANOVA with Dunnett's multiple comparison to the E13.5 stage). Error bars represent s.e.m. (C) Representative WT mouse heart sections stained for pY822 VCL (red), VCL (green), and nuclei (blue). Images are deconvolved maximum projections of two-slice z-stacks (0.175 μm step size). Individual pVCL and VCL channels are shown, along with the merged image. Images representative of three independent experiments. Scale bars: 20 µm.

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  Fig. 2.. Increased VCL pY822 in post-MI hearts requires ABL kinase. (A) Representative immunofluorescence images of sham-operated (Sham), day 7 (D7), and D14 post-MI border zone (BZ) heart sections from WT rats stained for VCL pY822 (pVCL, green), N-cadherin (Ncad, red), and nuclei (blue). Individual pVCL and Ncad channels are shown along with the merged image. Arrowheads indicate colocalization of pVCL and N-cadherin at the ICD. Images representative of three independent experiments. Scale bar: 50 µm. (B) Immunoblots of BZ lysates from Sham, D7, and D14 rat hearts probed for pY822 VCL (pVCL), total VCL, and GAPDH. (C) Immunoblots of WT and Abl CKO (Abl f/f; MHC-MerCreMer) mouse hearts with (+) or without (−) tamoxifen (Tam) treatment. Blots probed for ABL and GAPDH. (D) Scheme for Tam-induced depletion of ABL in adult mice before MI. Blots in B and C representative of two independent experiments. (E,F) Immunoblots (E) and quantitative analysis (F) of MI BZ lysates from WT and Abl CKO mice at day 7 post-MI (n=3 mice per condition). Left ventricular heart lysate from sham-operated animals was used as a control. *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA with Tukey's multiple…

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  Fig. 3.. Generation of Vcl Y822F mutant mice. (A) Schematic of VCL protein structure (top) indicating the location of the Y822F mutation and the Vcl gene locus (bottom) showing the exon 17 sequence targeted by the sgRNA. (B) Sanger sequencing of the nucleotide change confirmed the Vcl Y822F (YF) mutation in a heterozygous Vcl Y822 mutant mouse (YF/+). (C) PCR genotyping of WT (+/+), YF/+, and homozygous mutant (YF/YF) mice. The mutation destroyed an EcoRV site (GATATC>GATTTC) in exon 17 of the Vcl gene. The PCR product was digested with EcoRV, resulting in two WT bands or an undigested full-length band corresponding to the YF mutation. (D) Immunoblots of WT, YF/+, and YF/YF P4 heart lysates. (E) Quantification of total VCL protein levels in WT (n=4) and Vcl Y822F (n=5) P4 heart lysates. Error bars are s.e.m. (F) Representative immunofluorescence images of heart sections from WT and Vcl Y822F P1 mice stained for VCL pY822 (pVCL, red), α-actinin (ACTN2, green) and nuclei (blue). Individual pVCL and ACTN2 channels are shown along with the merged image. Images representative of three independent experiments. Scale bar: 25 µm. (G) Body weight of female and male WT and YF/YF mice at 20 and 40…

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  Fig. 4.. Increased cell–ECM adhesions in Vcl Y822F hearts. (A) Representative immunofluorescence images of P7 heart sections from WT and Vcl Y822F mice stained for α5 integrin (α5, red), fibronectin (FN, green) and nuclei (blue). Individual α5 and FN channels are shown along with the merged image. Images representative of three independent experiments. Scale bar: 50 µm. (B) Immunoblots of DOC-insoluble P7 heart lysates from WT and Vcl Y822F mice probed for FN, α5 and desmin. (C) Quantification of FN/desmin and α5/desmin ratios in WT and Vcl Y822F P7 heart lysates (n=6 mice of each genotype). **P<0.01; ***P<0.001 (two-tailed, unpaired Student's t-test). Error bars represent s.e.m.

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  Fig. 5.. Loss of VCL pY822 disrupts VCL distribution and AJ composition. (A) Representative immunofluorescence images of P28 heart sections from WT and Vcl Y822F mice stained for VCL (red), β-catenin (β-Cat, green) and nuclei (blue). Individual VCL and β-Cat channels are shown, along with the merged image. White arrowheads mark ICDs, and double yellow arrowheads point to lateral membranes in the VCL image. The inset in the merged image is a high-magnification view of the boxed region, highlighting VCL recruitment to the ICD. Scale bar: 25 µm. (B) VCL ICD to lateral membrane fluorescence ratio in WT and Vcl Y822F P28 heart CMs. Data are presented as a SuperPlot, where individual measured CM membrane fluorescence ratios (data points) from one heart (biological replicate) are plotted as circles, and each heart is assigned a unique color (n=4 hearts per genotype). The mean ICD/lateral fluorescence ratio from each heart is overlaid on the individual data points as a larger, color-matched circle outlined in black. The mean±s.d. of the four hearts are shown as black bars. (C) β-catenin fluorescence intensity at CM ICDs was measured in four WT and four Vcl Y822F P28 heart sections. Data are…

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  Fig. 6.. Y822 phosphorylation regulates VCL recruitment to cell-cell contacts in cultured CMs. (A–D) Wild-type (A,B) or Vcl Y822F CMs (C,D) plated on fibronectin (FN) or collagen I (COL)-coated PDMS, fixed 72 h post-plating, and stained for F-actin, plakoglobin and VCL. (E) VCL contact to cytoplasmic fluorescence ratio from cells imaged in A–D was measured from three biological replicates. Data are presented as a Superplot, where each image is plotted as a circle (WT cells) or square (Y822F cells) and colored (shades of green for cells plates on FN; shades of blue for cells plated on COL I) to identify samples in each replicate. The mean fluorescence ratio of each replicate is overlaid on the individual data points as a larger, color-matched circle outlined in orange. The mean±s.d. of the three replicates are shown as black bars. **P<0.01; ***P<0.001 (one-way ANOVA with Tukey's multiple comparisons). Scale bars: 10 µm.

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  Fig. 7.. VCL Y822F dynamics are similar to VCL WT. (A,B) Images from FRAP experiments showing pre-bleach, post-bleach and recovery after 10 min in WT CMs expressing Emerald-tagged VCL WT or Vcl Y822F CMs expressing VCL Y822F at cell–substrate contacts (A) or cell–cell contacts (B). Yellow arrowheads mark the FRAP region. Scale bars: 5 µm. (C,D) Plots of mean±s.d. of FRAP recovery fraction over time for Emerald–VCL WT expressed in WT CMs (WT, dark gray circles mark the mean; light gray shaded area defines the s.d.) and Emerald–VCL Y822F expressed in Vcl Y822F CMs (green diamonds mark the mean; green shaded area defines the s.d.). Recovery plots are shown for cell–substrate (C) and cell–cell (D) contacts. The data were fit to a single exponential curve (WT, light gray line; Y822F, light green line). The number of experimental replicates and FRAP regions quantified were: cell substrate WT (3, 46), Y822F (3, 39); cell–cell WT (3,36), Y822F (4, 42). (E) Summary of the mobile fraction and recovery halftimes with 95% confidence interval.

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  Fig. 8.. Histological and cardiac function analysis of Vcl Y822F hearts. (A) Representative whole-mount images of hearts from Vcl +/+, Vcl Y822F/+, and Vcl Y822F/Y822F mice at 40 weeks (wks) of age. (B) Representative H&E-stained heart sections from 28-week-old mice. (C,D) Quantification of heart weight/body weight ratio (C) and heart weight/tibia length ratio (D) in Vcl +/+, Vcl Y822F/+, and Vcl Y822F/Y822F mice at 40 weeks of age. The number of mice studied is listed in the column. ns, not significant (one-way ANOVA with Tukey's multiple comparisons). (E–H) Serial echocardiographic analyses were performed on mice from 10 to 28 weeks (wks) of age. Comparison of (E) ejection fraction (EF) and (F) fraction shortening (FS) at 10 and 28 weeks of age, and (G) cardiac output (CO) and (H) stroke volume (SV) at 28 weeks of age is shown. The number of mice studied is listed in the column. *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA with Dunnett's multiple comparison versus WT group). Error bars represent s.e.m.