Cardiomyocyte-myofibroblast contact dynamism is modulated by connexin-43

Abstract

Healthy cardiomyocytes are electrically coupled at the intercalated discs by gap junctions. In infarcted hearts, adverse gap-junctional remodeling occurs in the border zone, where cardiomyocytes are chemically and electrically influenced by myofibroblasts. The physical movement of these contacts remains unquantified. Using scanning ion conductance microscopy, we show that intercellular contacts between cardiomyocytes and myofibroblasts are highly dynamic, mainly owing to the edge dynamics (lamellipodia) of the myofibroblasts. Decreasing the amount of functional connexin-43 (Cx43) at the membrane through Cx43 silencing, suppression of Cx43 trafficking, or hypoxia-induced Cx43 internalization attenuates heterocellular contact dynamism. However, we found decreased dynamism and stabilized membrane contacts when cellular coupling was strengthened using 4-phenylbutyrate (4PB). Fluorescent-dye transfer between cells showed that the extent of functional coupling between the 2 cell types correlated with contact dynamism. Intercellular calcein transfer from myofibroblasts to cardiomyocytes is reduced after myofibroblast-specific Cx43 down-regulation. Conversely, 4PB-treated myofibroblasts increased their functional coupling to cardiomyocytes. Consistent with lamellipodia-mediated contacts, latrunculin-B decreases dynamism, lowers physical communication between heterocellular pairs, and reduces Cx43 intensity in contact regions. Our data show that heterocellular cardiomyocyte-myofibroblast contacts exhibit high dynamism. Therefore, Cx43 is a potential target for prevention of aberrant cardiomyocyte coupling and myofibroblast proliferation in the infarct border zone.-Schultz, F., Swiatlowska, P., Alvarez-Laviada, A., Sanchez-Alonso, J. L., Song, Q., de Vries, A. A. F., Pijnappels, D. A., Ongstad, E., Braga, V. M. M., Entcheva, E., Gourdie, R. G., Miragoli, M., Gorelik, J. Cardiomyocyte-myofibroblast contact dynamism is modulated by connexin-43.

Keywords: Cx43; heart failure; heterocellular coupling; myocardial infarction.

Figure 1

SICM and the analysis of contact movement. A) Schematic of the SICM setup (left), cartoons representing the different cell configurations used here (top right), and the analysis using CellTrack (44) (bottom right). CellTrack (44) selects a set of random points (yellow squares) in the contact area that is selected (circled) and follows these throughout the scans to calculate the movement of the cell contact. B) Representative images of a CM–MFB contact, first scanned with SICM (left) and subsequently with the laser confocal (middle); an overlay showing the junction (SICM scan) and GFP-Cx43 in the junction (green) is shown on the right. C) Representative set of scans of the looped scans at representative time points. Each scan takes 4–6 min, generating between 10 and 20 images, although some may not be used as debris on the cell may interfere with the SICM scan. t, time; z, cell height. D) Representative image of CM–CM and CM–MFB Cx43 distribution at the cell–cell contacts. Red, β-catenin; green, Cx43; pink, vimentin; blue, DAPI. Cx43 was expressed at the CM–MFB interface. The white arrows indicate the presence of Cx43 at the cell–cell contacts. Scale bars, 10 μm.

Figure 2

Dynamism in control CM–CM, CM–MFB, and MFB–MFB contacts. A) Representative set of scans for each configuration. Cartoons indicate the configuration, as shown in Fig. 1. The red line is included for the contact between MFB–MFB for clarity. t, time; z, cell height. B) Movement speed of the contacts. All data are represented as means ± sem. *P ≤ 0.05, ***P ≤ 0.001.

Figure 3

Cx43 KD leads to decreased dynamism and reduced dye transfer. A) Representative set of scans for each configuration. Illustrations indicate the configuration, as shown in Fig. 1. t, time; z, cell height. B) Movement speed of the contacts after Cx43 KD using siRNA in only the myofibroblasts (in CM–MFB cultures) or in either cell type in the monocultures. C–E) Cx43 KD using Cx43-specific siRNA or Cx43–GFP shRNA in only the myofibroblasts leads to decreased dye transfer and therefore a reduced number of functional gap junctions. C) Analysis of the parachute assay. Cx43 siRNA, n = 52 images (for all parachute assays: the total number of images from 2 dishes per isolation for n = 3 isolations are indicated); control, n = 61 images. Cx43–GFP shRNA, n = 29 images; control, n = 29 images. D) Representative images of control and Cx43 siRNA. Calcein (green) is used as a cell-permeable dye, whereas CellTracker Orange (red) is a cell-impermeable dye. E) Representative images of control and Cx43–GFP shRNA. GFP (green) is used to identify myofibroblasts with Cx43 KD (only shown in the shRNA-treated images). Calcein Orange (orange) is used as a cell-permeable dye, whereas CellTracker FarRed (magenta) is a cell-impermeable dye. Scale bars, 100 μm. All data are represented as means ± sem. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 4

Increased cellular coupling induced by 4PB. A) Representative images of control cocultures and cocultures with 4PB-treated myofibroblasts. Calcein (green) is used as a cell-permeable dye, whereas CellTracker Orange (red) is a cell-impermeable dye. Scale bars, 100 μm. B) Analysis of the parachute assay. 4PB treatment of cocultured cardiomyocytes and myofibroblasts (n = 29 images, n = 3 isolations) or myofibroblasts alone (n = 55 images, n = 3 isolations) leads to increased calcein transfer. C) Treatment of both cardiomyocytes and myofibroblasts, but not myofibroblasts alone, leads to reduced movement speed in the contact. D, E) Western blot analysis of Cx43 (n = 3–4 independent isolations). Quantification (D) and representative blots (E) are shown. AU, arbitrary units. All data are represented as means ± sem. *P ≤ 0.05, ***P ≤ 0.001.

Figure 5

Removing Cx43 by hypoxia and forcing Cx43 to the membrane by αCT1 peptide. A–D) Representative scans of control and treatments. z, cell height. E) Hypoxia reduces the dynamism between cells. F) αCT1 treatment leads to increased dynamism in control, but not in hypoxic, conditions. G) Treatment with the αCT1, but not the reverse, peptide leads to decreased dye transfer, showing that, although the plaque size increases, the number of functional gap junctions decreases. All data are represented as means ± sem. *P ≤ 0.05, ***P ≤ 0.001. For representative Western blots and quantification of Cx43 protein expression in cardiomyocytes and myofibroblasts treated with hypoxia or αCT1, see Fig. 7A, B.

Figure 6

Treatment with latrunculin-B abolishes contact dynamism and dye transfer. A, B) Representative SICM scans (A) and images of parachute assays (B) of control CM–MFB cultures and CM–MFB cultures treated with latrunculin-B from neonatal rats. z, cell height. C, D) Treatment with latrunculin-B leads to reduced dynamism (C) and a reduction in the number of functional contacts (D). B, D, Control, n = 35 images, n = 3 isolations; Latrunculin-B, n = 39 images, n = 3 isolations. For representative Western blots and quantification of Cx43 protein expression in cardiomyocytes and myofibroblasts treated with latrunculin-B, see Fig. 7A, B. E, F) Representative SICM scans (E) and images of parachute assays (F) of myofibroblasts cultures derived from adult rats with myocardial infarction that were treated with latrunculin-B or vehicle control (MFB–MFB). For experiments carried out using myofibroblasts from sham-treated adult rats, see Supplemental Fig. S4. G, H) Treatment with latrunculin-B leads to reduced dynamism (G) and a reduction in the number of functional contacts (H). F, H, Control, n = 24 images, n = 3 isolations; Latrunculin-B, n = 25 images, n = 3 isolations. CTO, CellTracker Orange; LatB, latrunculin-B. All data are represented as means ± sem. Scale bars, 100 μm. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 7

Quantification of Cx43 expression in control, hypoxia-, latrunculin-B-, and αCT1-treated samples. A) Cx43 and loading control [glyceraldehyde 3-phosphate dehydrogenase (GAPDH)] intensity bands in neonatal cardiomyocyte and myofibroblast cultures (n = 2–4 independent isolations). B) Quantification of Western blots shown in A. All data are represented as means ± sem; n = 2–4 independent isolations. C) Latrunculin-B treatment significantly decreases the Cx43 intensity in the Cx43 clusters (n = 15 images, n = 3 isolations). All data are represented as means ± sem. **P ≤ 0.01. D) Representative images of immunostaining of CM–MFB contacts. Red, α-SMA; green, Cx43; pink, vimentin; blue, DAPI. Cx43 was expressed at the CM–MFB interface. Scale bars, 10 μm. AU, arbitrary units; latrunculin-B, LatB.

Figure 8

Schematic of our current view on the role of Cx43 in CM–MFB dynamism. Max., maximum; min., minimum.