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. 2020 Sep 17;5(18):e140066.
doi: 10.1172/jci.insight.140066.

Cardiomyocyte adhesion and hyperadhesion differentially require ERK1/2 and plakoglobin

Maria Shoykhet  1 , Sebastian Trenz  1 , Ellen Kempf  1 , Tatjana Williams  2 , Brenda Gerull  2 , Camilla Schinner  1 , Sunil Yeruva  1 , Jens Waschke  1
Affiliations

Cardiomyocyte adhesion and hyperadhesion differentially require ERK1/2 and plakoglobin

Maria Shoykhet et al. JCI Insight. .

Abstract

Arrhythmogenic cardiomyopathy (AC) is a heart disease often caused by mutations in genes coding for desmosomal proteins, including desmoglein-2 (DSG2), plakoglobin (PG), and desmoplakin (DP). Therapy is based on symptoms and limiting arrhythmia, because the mechanisms by which desmosomal components control cardiomyocyte function are largely unknown. A new paradigm could be to stabilize desmosomal cardiomyocyte adhesion and hyperadhesion, which renders desmosomal adhesion independent from Ca2+. Here, we further characterized the mechanisms behind enhanced cardiomyocyte adhesion and hyperadhesion. Dissociation assays performed in HL-1 cells and murine ventricular cardiac slice cultures allowed us to define a set of signaling pathways regulating cardiomyocyte adhesion under basal and hyperadhesive conditions. Adrenergic signaling, activation of PKC, and inhibition of p38MAPK enhanced cardiomyocyte adhesion, referred to as positive adhesiotropy, and induced hyperadhesion. Activation of ERK1/2 paralleled positive adhesiotropy, whereas adrenergic signaling induced PG phosphorylation at S665 under both basal and hyperadhesive conditions. Adrenergic signaling and p38MAPK inhibition recruited DSG2 to cell junctions. In PG-deficient mice with an AC phenotype, only PKC activation and p38MAPK inhibition enhanced cardiomyocyte adhesion. Our results demonstrate that cardiomyocyte adhesion can be stabilized by different signaling mechanisms, which are in part offset in PG-deficient AC.

Keywords: Arrhythmias; Cardiology; Cardiovascular disease; Cell migration/adhesion.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Cardiomyocyte cohesion regulation by different signaling pathways.
(A) Dissociation assays in HL-1 cardiomyocytes under basal and Ca2+-depleted conditions. *P ≤ 0.05, unpaired Student’s t test, n = 8. (B) Representative Western blots showing changes in signaling pathways between basal and Ca2+-depleted conditions. Fold changes in phosphorylation compared with –EGTA (basal conditions) are indicated. *P ≤ 0.05, unpaired Student’s t test, n = 6. (C) Dissociation assays in HL-1 cells, showing fold change of the number of fragments after treatment with F/R, Iso, PMA, SB20, or Aniso under basal (–EGTA) or Ca2+-depleted (+EGTA) conditions as compared with respective controls. DMSO serves as control for SB20. *P ≤ 0.05, 1-way ANOVA with Holm-Šidák correction, n = 6. (D) Dissociation assays in cardiac slice cultures obtained from WT mice in basal and Ca2+-depleted conditions. *P ≤ 0.05, unpaired Student’s t test, n = 8. (E) Dissociation assays in cardiac slice cultures obtained from WT mice, showing fold change of the number of dissociated cells after treating with F/R, Iso, SB20, PMA, or Aniso under basal and Ca2+-depleted conditions, normalized to the respective control slices. *P ≤ 0.05, 1-way ANOVA with Holm-Šidák correction, n = 6.
Figure 2
Figure 2. Phosphorylation of signaling proteins in basal and Ca2+-depleted conditions after treatments with mediators regulating cardiomyocyte cohesion.
(A) Representative Western blot of HL-1 cells treated with F/R, Iso, PMA, SB20, or Aniso under basal and Ca2+-depleted conditions. DMSO serves as control for SB20. (B–E) Quantification of changes in phosphorylation levels of (B) PG, (C) p38MAPK, (D) ERK1, and (E) ERK2, as compared with the respective controls upon treatment with mediators. *P ≤ 0.05, 1-way ANOVA with Bonferroni correction, n = 6–9. In C, Aniso was excluded from the statistical analysis, as the effect was so strong that all other effects were not significant anymore with 1-way ANOVA.
Figure 3
Figure 3. Inhibition of ERK1/2 alters cardiomyocyte cohesion in basal conditions and results in alteration of phosphorylation of PG, ERK1/2, and p38MAPK upon treatment with F/R and PMA.
(A) Dissociation assays in HL-1 cells treated with F/R, Iso, SB20, or PMA, with and without U0126 under basal and Ca2+-depleted conditions. DMSO serves as control for SB20. *P ≤ 0.05 as compared with the respective control, #P ≤ 0.05 as compared with the respective U0126-untreated condition, 1-way ANOVA with Holm-Šidák correction, n = 6. (B) Representative Western blot of HL-1 cells treated with F/R or PMA, with and without U0126 under basal and Ca2+-depleted conditions. n = 6. (C) Dissociation assays in HL-1 cells treated with F/R with and without U0126 or H89. *P ≤ 0.05 as compared with the vehicle control, #P ≤ 0.05 as compared with F/R, 1-way ANOVA with Holm-Šidák correction, n = 6. (D) Representative Western blot of HL-1 cells treated with F/R with and without U0126 or H89. n = 6.
Figure 4
Figure 4. ERK1/2 inhibition caused alteration of DSG2 localization.
Immunostainings of HL-1 cells treated with F/R, PMA, or SB20, with and without U0126, stained for N-CAD and DSG2 under basal conditions. DMSO serves as control for SB20 treatment. Arrows indicate increased localization of DSG2 in the membrane. Scale bar: 10 μm. n = 6.
Figure 5
Figure 5. Effect of Dsg2, N-Cad, Jup, or Dp knockdown on F/R-, Iso-, PMA-, or SB20-mediated increase in cardiomyocyte cohesion.
(A) Representative Western blots confirming decreased levels of DSG2, N-CAD, PG, and DP after siRNA-mediated knockdown. (B) Dissociation assay under basal conditions following siNT, siDsg2, siN-Cad, siJup, and siDp treatments. *P ≤ 0.05, 1-way ANOVA with Holm-Šidák correction, n = 6. (C) Dissociation assays showing fold changes in fragments as compared with the respective controls in HL-1 cells following siNT, siDsg2, siJup, or siDp treatments and administration of F/R, Iso, PMA, or SB20. DMSO serves as control for SB20. *P ≤ 0.05, 1-way ANOVAs with Holm-Šidák correction, n = 6. (D) Immunohistochemistry of ventricular cardiac slices obtained from WT and PG-deficient mice (Jup-KO) stained for PG protein. Scale bar: 50 μm. n = 6. (E) H&E and Picrosirius red stainings of cardiac slices obtained from WT and Jup-KO mice. Scale bar: 1 mm; 50 µm (high-magnification views). (F) Total collagen content of cardiac slices obtained from WT and Jup-KO mice. *P ≤ 0.05, Student’s t test with Welch correction, n = 6. (G) Dissociation assays in murine cardiac slice cultures comparing the number of dissociated cells in WT and Jup-KO cardiac slices. *P ≤ 0.05, unpaired Student’s t test, n = 6. (H) Dissociation assays in murine cardiac slices obtained from Jup-KO mice following F/R, Iso, PMA, SB20, and Aniso treatments. *P ≤ 0.05, 1-way ANOVA with Holm-Šidák correction, n = 6.
Figure 6
Figure 6. Schematic overview of signaling mechanisms involved in cohesion in HL-1 cardiomyocytes.
Adrenergic signaling (F/R and Iso), PKC activation (PMA), and p38MAPK inhibition (SB20) enhanced basal cardiomyocyte cohesion, referred to as positive adhesiotropy, in an ERK1/2-dependent manner. Positive adhesiotropy is possibly achieved through alterations in the interactions of the desmosomal proteins PG, PKP2, and DP (represented by dashed rectangle) that eventually lead to increased DSG2 translocation to the cell borders (represented by dashed arrow) and finally to positive adhesiotropy. On the other hand adrenergic signaling also acts via PKA-dependent PG phosphorylation at S665 and thereby increases basal cardiomyocyte cohesion. Under Ca2+-depleted conditions, adrenergic signaling, PKC activation, and p38MAPK inhibition lead to hyperadhesion independent of ERK1/2 (represented by dashed rectangle and straight arrow). Under the same conditions adrenergic signaling also induces PG phosphorylation, probably via PKA (shaded PKA), and thereby hyperadhesion (dashed arrow).
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