ZO-1 determines adherens and gap junction localization at intercalated disks

Abstract

The disruption of the spatial order of electromechanical junctions at myocyte-intercalated disks (ICDs) is a poorly understood characteristic of many cardiac disease states. Here, in vitro and in vivo evidence is provided that zonula occludens-1 (ZO-1) regulates the organization of gap junctions (GJs) and adherens junctions (AJs) at ICDs. We investigated the contribution of ZO-1 to cell-cell junction localization by expressing a dominant-negative ZO-1 construct (DN-ZO-1) in rat ventricular myocytes (VMs). The expression of DN-ZO-1 in cultured neonatal VMs for 72 h reduced the interaction of ZO-1 and N-cadherin, as assayed by colocalization and coimmunoprecipitation, prompting cytoplasmic internalization of AJ and GJ proteins. DN-ZO-1 expression in adult VMs in vivo also reduced N-cadherin colocalization with ZO-1, a phenomenon not observed when the connexin-43 (Cx43)-ZO-1 interaction was disrupted using a mimetic of the ZO-1-binding ligand from Cx43. DN-ZO-1-infected VMs demonstrated large GJs at the ICD periphery and showed a loss of focal ZO-1 concentrations along plaque edges facing the disk interior. Additionally, there was breakdown of the characteristic ICD pattern of small interior and large peripheral GJs. Continuous DN-ZO-1 expression in VMs over postnatal development reduced ICD-associated Cx43 GJs and increased lateralized and cytoplasmic Cx43. We conclude that ZO-1 regulation of GJ localization is via an association with the N-cadherin multiprotein complex and that this is a key determinant of stable localization of both AJs and GJs at the ICD.

Fig. 1.

A: connexin-43 (Cx43; green) and zonula occludens-1 (ZO-1; red) colocalized in an adult rat intercalated disk oriented en face as reconstructed from a maximum projection of a confocal optical section series recorded on a Perkin-Elmer Spinning disk laser confocal microscope with a ×100 objective. Scale bar = 10 μm. B: same disk as in A showing areas of overlap between Cx43 and ZO-1 signal highlighted in a white colocalization mask. C: subregion of the colocalization mask image shown in B as indicated by highlight showing asymmetric polarization of colocalized ZO-1 (white) on the inside edges (as defined by dashed line) of large gap junctions (GJs) ringing the intercalated disk. D: quantification of the average percentage of colocalized area in the total disk ring, the inner ring edge, and outer ring edge of 11 intercalated disks obtained from 5 adult rats. Colocalized area was significantly greater in inner ring edge vs. outer ring edge (Mann-Whitney, P = 0.032).

Fig. 2.

A: domain organization of full-length ZO-1 and a ZO-1 truncation [dominant negative (DN)-ZO-1] construct incorporating the first 2 postsynaptic density 95/Drosophila disk large/ZO-1 (PDZ) domains of ZO-1 and a 10 amino acid myc sequence. N, NH₂ terminus; C, COOH terminus. B–F: confocal optical sections of neonatal myocytes cultured for 72 h or 24 h (D–F) following infection with adenovirus green fluorescent protein (Ad-GFP; B) or Ad-DN-ZO-1 (D–F) and immunolabeled for Cx43 (red) and myc (blue). *Position of high magnification. GFP localization occupies the green channel. Cx43 distribution in Ad-GFP-infected myocytes was localized primarily to the borders of myocytes (small arrowheads). Extensive Cx43 labeling in large particles was observed in the cytoplasm of DN-ZO-1-expressing cells (large arrowheads) and high magnification in B and C. G. Western blots for myc (top), Cx43 (middle), and GAPDH (bottom) in neonatal myocytes infected with Ad-GFP, Ad-DN-ZO-1, or no virus. H–I: quantification of Cx43 immunolabeling indicates significant increases in the size (H) and cytoplasmic distribution (I) of Cx43 aggregates following DN-ZO-1 expression compared with Ad-GFP-infected (GFP) and noninfected controls. SH3, SRC homology 3 domain; MAGUK, membrane-associated guanylate kinase. Scale bar = 20 μm. *P < 0.05.

Fig. 3.

Neonatal rat ventricular myocytes (NRVMs) infected with either Ad-DN-ZO-1 (A, C, D, and G) or Ad-GFP (B, E, F, and H) and immunolabeled for N-cadherin (blue) and ZO-1 (red). Boxed regions in A and B are shown at higher magnification in C–H. GFP localization occupies the green channel. C–F demonstrate single channel images of Cx43 and ZO-1 expression of boxed regions in A and B. Arrows indicate regions of cell borders that are magnified and shown in G and H without the green channel. Note that N-cadherin and ZO-1 colocalize frequently at myocyte-myocyte contacts in control (GFP) cultures, and the same 2 proteins are largely internalized in the cytoplasm of DN-ZO-1-expressing cultures. Scale Bar = 25 μm. I. Western Blot of noninfected (left), DN-ZO-1-infected, or α-carboxy terminal-1 (α-CT1)-treated NRVM lysates probes with anti ZO-1 or N-cadherin antibodies. J: NRVM lysates immunoprecipitated (IP) with anti-ZO-1, -N-cadherin (N-Cad), or nonimmunoprecipitated (input). Note: no N-cadherin detected in DN-ZO-1-infected cardiomyocyte lysates immunoprecipitated with ZO-1 antibodies compared with uninfected cells (control).

Fig. 4.

A: adult rat left ventricular myocardium harvested 48 h postinjection with DN-ZO-1. GFP-positive regions indicate areas of DN-ZO-1 expression. Scale bar = 10 μm. DAPI, 4,6-diamidino-2-phenylindole. B (top) and C (top): high magnification regions of A showing an intercalated disk from a GFP-negative (Ad-DN-ZO-1 noninfected; B) or GFP-positive (Ad-DN-ZO-1 infected; C) region with the ZO-1 N-cadherin colocalized pixels highlighted in white. B (bottom) and C (bottom): cytofluorogram plotting red ZO-1 signal vs. blue N-cadherin signal for the GFP-negative (B) and GFP-positive (C) region indicated above.

Fig. 5.

A–E: en face views of intercalated disks from 48 h Ad-DN-ZO-1-infected adult rat hearts from GFP-positive areas (A, B, and D) and GFP-negative areas (C and E). A: GFP labeling demonstrating Ad-DN-ZO-1-infected intercalated disk. Scale bar = 10 μm. B: same as A without green channel. Inset: Cx43-ZO-1 colocalization mask (white). C: noninfected disk from same heart shows normal disk morphology and Cx43-ZO-1 colocalization mask (white inset). D: Ad-DN-ZO-1-infected disks labeled for Cx43 (blue) and N-cadherin (red). E: control disk from DN-ZO-1-nonexpressing cell from same heart as D. F: total Cx43 ZO-1 colocalization (Coloc) did not significantly differ in fields containing GFP compared with areas absent GFP expression (n = 3; P > 0.05). G: quantification of inner vs. outer colocalization of Cx43 and ZO-1; n = 3 animals. H: quantification of Cx43 GJ size in the center vs. peripheral regions of adult disks; *P < 0.05.

Fig. 6.

A: a postnatal day 10 heart infected on postnatal day 1 with Ad-DN-ZO-1. B: green GFP fluorescence indicates that the ventricle of this heart has been extensively infected with virus. Adult 90-day myocytes infected in vivo with Ad-GFP (C) or Ad-DN-ZO-1 (D) were labeled for myc (shown in blue). Myocytes infected with Ad-DN-ZO-1 showed labeling for myc (D), indicating the expression of DN-ZO-1, whereas Ad-GFP-infected myocytes (E) did not. GFP localization occupies the green channel. Scale bar = 25 μm. F–K: single confocal optical sections of Cx43-immunolabeled rat cardiac myocytes infected with Ad-GFP (F, H, and J) or Ad-DN-ZO-1 (G, I, and K) and euthanized at 10 (A–B), 40 (C–D), or 90 (E–F) postnatal days. Scale bar =10 μm. Over the course of postnatal development, the Cx43 GJs exhibit a cytoplasmic distribution in DN-ZO-1-infected myocytes.

Fig. 7.

A–D: myocytes from hearts at 40 days of postnatal development were immunolabeled for Cx43 following infection in vivo with either Ad-DN-ZO-1 (A, B, and F) or Ad-GFP viruses at postnatal day 1 (C–E). Cx43 signal is shown in red and GFP fluorescence is in green. The Cx43 signal is displayed in black and white single channel images in B and D. Ad-GFP-infected cells maintained typical distributions of Cx43 at cell borders and at developing intercalated disks (D–F). Striking increases in lateralization and cytoplasmic labeling of large Cx43 particles were detected in Ad-DN-ZO-1-infected myocytes (A–D). N-cadherin staining of Ad-GFP (E) in Ad-DN-ZO-1 (F)-infected myocytes. *Adjacent uninfected cell with N-cadherin labeling at the intercalated disk. Quantification of immunolabeling in single optical sections from 40-day-old ventricles indicate significant increases in Cx43 GJ aggregate size (G), cytoplasmic internalization (H), and lateralization (I) following Ad-DN-ZO-1 infection compared with Ad-GFP-infected and noninfected controls. Scale bar =12 μm.