Cardiomyocyte cohesion is increased after ADAM17 inhibition

Figures

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  FIGURE 1. ADAM17 inhibition leads to positive adhesiotropy in Jup +/+ and Jup −/− mice. (A) Representative Western blot showing protein expression of pp38MAPK, p38MAPK, ADAM17 and PG in Jup +/+ and Jup −/− mice. NoStain Dye™ was used as loading control. (B–D): Quantification of protein expression assessed by Western blots shown in (A). (B) proADAM17 protein expression. (C) ADAM17 protein expression. (D) phosphorylation of p38MAPK. *p ≤ 0.05, unpaired Student’s t-test, N = 9 mice. (E) Dispase-based dissociation assay in murine cardiac slice cultures obtained from Jup +/+ and Jup −/− mice upon inhibition of ADAM17 by TAPI-1. *p ≤ 0.05, unpaired Student’s t-test, N = 7 for Jup +/+ and N = 6 for Jup −/− mice.

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  FIGURE 2. ADAM17 inhibition leads to positive adhesiotropy in HL-1 cardiomyocytes independent of EGFR. (A, B) Dispase-based dissociation assays in HL-1 cardiomyocytes upon inhibition of ADAM17 by TAPI-1, with representative pictures of the wells, (A) inhibition of ADAM17 for 90 min, N = 6 biological replicates. (B) inhibition of ADAM17 for 24 h, N = 5 biological replicates. *p ≤ 0.05, unpaired Student’s t-test. (C) Dispase-based dissociation assay in HL-1 cardiomyocytes after knockdown of Adam17 by siRNA. Knockdown efficiency was confirmed by Western blot. *p ≤ 0.05, unpaired Student’s t-test, N = 4 biological replicates. (D) Dispase-based dissociation assay in HL-1 cardiomyocytes after knockdown of Egfr by siRNA and treatment with TAPI-1 for 90 min. Knockdown efficiency was confirmed by Western blot, representative images are shown below the respective bar graphs. *p ≤ 0.05, unpaired Student’s t-test, N = 4 biological replicates.

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  FIGURE 3. ADAM17 inhibition leads to enhanced localization of DSG2 and N-CAD at the cell borders. (A) Maximum projections of immunostainings in HL-1 cardiomyocytes for N-CAD and DSG2 showing an increase of DSG2 (white arrows) at the cell borders after 90 min of TAPI-1 treatment. Z-scans spanning the whole cell volume, z-steps = 0.25 µm. Scale bar: 10 µm. (B) Quantification of colocalization of N-CAD and DSG2 in HL-1 cardiomyocytes, *p ≤ 0.05, unpaired Student’s t-test. Each data point represents one image, N = 8 biological replicates. (C) Dispase-based dissociation assay in HL-1 cardiomyocytes after knockdown of Dsg2 by siRNA and treatment with TAPI-1 for 90 min *p ≤ 0.05, unpaired Student’s t-test, N = 3 biological replicates. (D) Representative Western blot confirming Dsg2 knockdown with quantification. *p ≤ 0.05, paired Student’s t-test, N = 3 biological replicates (E) Immunostaining of DP in HL-1 cardiomyocytes after 90 min of TAPI-1 treatment using Phalloidin as membrane marker. Scale bar: 10 µm. (F) Quantification of colocalization of DP and Phalloidin. *p ≤ 0.05, unpaired Student’s t-test. Each data point represents one image, N = 5 biological replicates.

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  FIGURE 4. No change in desmosomal complex upon inhibition of ADAM17 by TAPI-1. (A) Representative Western blots for immunoprecipitation of DSG2 with quantification of co-immunoprecipitation (normalized to DSG2 pulldown) of (B) DP, (C) DES and (D) PG. IgG heavy chain (IgG hc) served as loading control for immunoprecipitated samples, *p ≤ 0.05, unpaired Student’s t-test, N = 4 biological replicates.

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  FIGURE 5. Desmosomal assembly is not enhanced upon inhibition of ADAM17. (A) Dispase-based dissociation assays in HL-1 cardiomyocytes after a Ca2+-switch followed by treatment with TAPI-1 for 90 min with representative pictures of the wells. N = 8, unpaired Student’s t-test, *p ≤ 0.05. (B) Maximum projections of immunostainings in HL-1 cardiomyocytes for N-CAD and DSG2 showing no changes in N-CAD and DSG2 localization after a Ca2+-switch followed by TAPI-1 treatment. Z-scans spanning the whole cell volume, z-steps = 0.25 µm. Scale bar: 10 µm. (C) Quantification of colocalization of N-CAD and DSG2 in HL-1 cardiomyocytes, *p ≤ 0.05, unpaired Student’s t-test. Each data point represents one image, N = 5 biological replicates.

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  FIGURE 6. Proposed mechanism of action. Inhibition of ADAM17 by TAPI-1 enhanced the localization of DP and DSG2 at the membrane. Since we observed no changes in desmosomal assembly or the desmosomal complex upon inhibition of ADAM17, we hypothesize that the increase in cardiomyocyte cohesion upon inhibition of ADAM17 could result from reduced DSG2 cleavage. In contrast, without ADAM17 inhibition, basal ADAM17 activity leads to DSG2 cleavage with subsequent desmosome disassembly.