Gap junction remodelling in human heart failure is associated with increased interaction of connexin43 with ZO-1

2.1. Human heart samples

The study was conducted on explanted hearts of patients with ICM (n = 5) or DCM (n = 5) undergoing orthotopic cardiac transplantation at the Royal Brompton and Harefield NHS Trust Hospitals (Table 1). Samples of human left ventricles (encompassing the full thickness of the free wall) were snap-frozen in liquid nitrogen. As controls, tissues from five normal hearts initially harvested for use as transplantation donor organs, but rejected for technical reasons, were used. The study was approved by the Brompton, Harefield and NHLI Research Ethics Committee (#04–101 and #01–194). The investigation conforms with the principles outlined in the Declaration of Helsinki.²⁴

2.2. Immunoconfocal and electron microscopy

Immunoconfocal microscopy was conducted on cryosections of tissue that was directly frozen, without prior aldehyde fixation.⁶ For immunoelectron microscopy, tissue was processed by low denaturation procedures and thin sections were immunogold labelled.^25,26 Primary antibodies used were anti-Cx43 (Chemicon International Ltd), anti-ZO-1 (Zymed, 40–2200), and anti-CD31 (Sigma). For details of antibody characterization and specificity, see Supplementary material.

2.3. Quantification of protein levels by western blotting

Western blotting and quantification were carried out as described previously⁸ using anti-ZO-1 (Zymed, 61–7300) or anti-Cx43 (Sigma) antibodies.

2.4. Quantification of mRNA levels by northern blotting

RNA extraction and northern blotting were carried out as previously described.²⁷ A cDNA encompassing nucleotides 3394–5548 of the human ZO-1 coding sequence was generated by PCR from human DNA, and cloned into pGEM^®-T Easy vector (Promega). Following release from the vector, the probe was used at a concentration of 2.5 ng ml⁻¹ after random primer labelling with ³²P dCTP. Northern blots were quantified following normalization against methylene blue stained 18S RNA.⁸

2.5. Quantitative analysis of immunolabelled Cx43 and ZO-1 by confocal microscopy

Quantification of ZO-1 and Cx43 immunolabelling was performed using a Leica TCS 4D confocal microscope and PC Image™ software (Foster Findlay Associates).⁶ Blind measurements were taken of the fluorescent label in 10 individual en face intercalated discs per section, selected by random scanning across the tissue. Serial optical sections (0.5 µm intervals) were acquired at ×63 magnification, with a setting of zoom4 and a 512 × 512-pixel image size, yielding a pixel size of 0.08 µm. Using PC Image™ software, individual complete discs were outlined by hand for separate quantification and thresholds for detection on a 255-point grey scale adjusted to reduce any background cell outlines. A pixel-by-pixel analysis was used to assess label for Cx43, ZO-1, and both Cx43 and ZO-1 (co-localized) label.

2.6. Extraction of non-junctional and junctional proteins

To enable separate analysis of Cx43 within gap junctions (the ‘junctional’ fraction) and that elsewhere in the cell (the ‘non-junctional’ fraction), Triton X-100 (TX-100) extraction was performed. The non-junctional fraction, comprising cytoplasmic Cx43 en route to the plasma membrane, Cx43 in the plasma membrane as undocked connexons, and Cx43 en route to degradation, is soluble in TX-100, whereas the Cx43 in the docked connexons of gap-junctional plaques is not. Samples of frozen left ventricle were pulverized,⁸ then homogenized by sonication in immunoprecipitation (IP) buffer (10 µl mg⁻¹ frozen tissue: 25 mM Tris, pH7.4; 150 mM NaCl; 1/250 mammalian tissue extract protease inhibitors [Sigma]; 1% TX-100). The samples were incubated on ice for 30 min, with vortexing every 5 min, and then centrifuged (15 000 rpm, 30 min, 4^oC). Non-junctional proteins in the supernatant were collected. Junctional proteins in the pellet were re-suspended in an equal volume (i.e. 10 µl mg⁻¹ tissue) of IP buffer containing 4 M urea. Samples were sonicated, incubated at room temperature for 30 min with vortexing, centrifuged (15 000 rpm, 30 min, 4°C), and junctional proteins collected in the supernatant. Urea (8 M) separates the component membranes of gap junctions,²⁸ thereby undocking connexons and making them soluble in TX-100 for processing. We have checked the validity of using 4 M urea to extract junctional connexins from non-purified gap-junctional membranes by solubilizing the final pellet with 20% SDS. Negligible connexin levels were detectable in this fraction by western blot.

Prior to IP, junctional proteins were desalted using Bio-Gel^® P-6 DG desalting gel (Bio-Rad) and IP buffer in Zeba spin columns (Pierce).

2.7. Cross-linking

Proteins were extracted in IP buffer containing PBS instead of Tris. Prior to the first centrifugation, an equal volume of 0.2 M ethanolamine, pH 8, containing 0.5 mM dimethyl-3-3′-dithiobispropionimidate (DTBP; Sigma) was added. Following incubation for 30 min at room temperature, an equal volume of 50 mM Tris, pH 7.4, was added to quench the reaction. Non-junctional and junctional protein extraction was then carried out as described above.

2.8. Co-immunoprecipitation

Protein samples were incubated with 2.5 µg of anti-ZO-1 (Zymed, 61–7300) or anti-Cx43 (Sigma) antibody overnight at 4°C with rotation, followed by 1 h at 4°C with 25 µl protein-A coated agarose beads (Pierce). Beads were washed with ice-cold IP buffer by centrifugation (2 min, 2500 rpm)/re-suspension. Bound proteins were desorbed by re-suspension in 20% SDS, containing 2.5% 2-mercaptoethanol. ZO-1/Cx43 interactions were quantified by western blotting using a polyclonal anti-Cx43 antibody (Sigma; favoured over the monoclonal anti-Cx43 (Chemicon) because of its higher sensitivity and ability to recognize all phosphorylated forms of Cx43), following normalization against Coomassie blue-stained actin. Control experiments, in which primary antibodies were omitted, excluded non-specific interactions.

2.9. Statistical analysis

All analysis was done using GraphPad Prism 4 (GraphPad Software Inc.). Data are expressed as mean ± SEM_. Statistical significance was evaluated with ANOVA. Statistical differences were judged significant at P ≤ 0.05.

3.1. Localization of ZO-1 in human ventricular cardiomyocytes

Immunoconfocal localization of ZO-1 in longitudinal sections of left ventricular tissue shows prominent signal which, at first sight, appears to outline individual myocytes, thereby suggesting the presence of ZO-1 throughout the entire myocyte plasma membrane (Figure 1A). However, careful comparison with the labelling patterns obtained for the endothelial marker, CD31, in both longitudinal and transverse planes, demonstrates that the apparent lateral labelling of the myocytes is, in fact, attributable to capillaries (Figures 1A–F). In longitudinal sections, CD31 highlights capillaries running alongside the myocytes (Figure 1B); transverse sections correspondingly show capillaries as small, prominently labelled circular profiles or streaks, with no signal of the myocytes that occupy the spaces between (Figure 1E). ZO-1 labelling of transverse sections showed similar small capillary profiles (Figure 1D), accounting for the apparent lateral label in longitudinal sections. There was no label circumscribing the myocyte bodies as would be expected if ZO-1 were present throughout the lateral plasma membrane. Periodic large discs were observed filled with positive ZO-1 signal occupying the entire myocyte cross-sectional area; these represented intercalated discs (Figure 1D). As confirmed by the merged images (Figures 1C and F), these observations indicate that, in human ventricular myocytes, ZO-1 is confined to the intercalated disc plasma membranes. This pattern of ZO-1 localization was not altered in the failing heart.

Immunogold labelling at the higher resolution afforded by electron microscopy confirmed and extended these conclusions. ZO-1 gold label was localized specifically to intercalated disc membranes; no label was apparent at the lateral plasma membrane or membranes of intracellular organelles (Figure 2). Within the intercalated disc, ZO-1 label was observed both in association with gap junctions, particularly at the junctional edge, and in intervening segments of disc membrane.

3.2. ZO-1 expression levels

To determine whether the expression levels of ZO-1 differed between control and failing ventricles, western and northern blot analyses were conducted. ZO-1 was detected by western blot as a prominent band migrating at ∼225 kDa (Figure 3A). Visual examination indicated increased levels of ZO-1 in both DCM and ICM vs. controls, which was confirmed by densitometric analysis following normalization against Coomassie blue-stained actin (P = 0.0025).

Western blot analyses of Cx43 protein levels accorded with previous studies showing down-regulation in the failing heart (P < 0.0001; Figure 3A and B). Plotting these data against ZO-1 protein levels revealed a significant negative correlation between Cx43 and ZO-1 (Figure 3C), i.e. low Cx43 levels correspond to high levels of ZO-1 (P = 0.0029; r² = 0.51).

ZO-1 mRNA was detected by northern blot as a single ∼8 kb band after 3–5 h exposure (Figure 4). Densitometric analysis following normalization against methylene blue stained 18S RNA showed no significant difference between transcript levels in control and failing hearts (P = 0.537).

3.4. Co-localization of ZO-1 and Cx43 at the intercalated disc

To assess the possibility of interaction between ZO-1 and Cx43, quantitative double-label immunoconfocal microscopy was carried out in serial confocal reconstructions of en face intercalated discs, and the extent of co-localization determined.

Cx43 labelling revealed the typical pattern comprising large gap junctions at the periphery of the disc enclosing smaller junctions in the interior zone (Figure 5A).^6,29,30 ZO-1 label was apparent across the entire face of the disc in both control and failing hearts. Double-labelled sections revealed substantial areas with Cx43 or ZO-1 signal only. However, a small proportion of co-localized signal was consistently apparent, indicating the existence of a pool of Cx43 situated where it could potentially interact with ZO-1.

Quantitative analysis demonstrated that Cx43 label per unit area of disc was reduced in failing ventricles compared with controls (P = 0.004; Figure 5B). In contrast, ZO-1 label was increased in failing ventricles compared with controls (P = 0.031; Figure 5B). The proportion of Cx43 signal co-localizing with ZO-1 was increased in failing ventricles compared with controls (P = 0.003; Figure 5C). The proportion of ZO-1 signal co-localizing with Cx43 did not differ between failing and control hearts owing to the overall increase in ZO-1 observed in the former (P = 0.155).

3.5. Co-immunoprecipitation of Cx43 and ZO-1

Increased co-localization of Cx43 with ZO-1 raises the possibility of, but does not strictly demonstrate, an increased Cx43/ZO-1 interaction in the failing heart. To determine the extent of any interaction, co-immunoprecipitation experiments were carried out.

Using anti-ZO-1 or anti-Cx43 antibodies, ZO-1 and Cx43 were co-immunoprecipitated from human ventricular samples (Figure 6). Owing to the limited availability of human samples, low levels of ZO-1/Cx43 interaction and high sensitivity of the anti-Cx43 antibody (Sigma) in western blot analysis, co-immunoprecipitation was quantified by western blotting for Cx43 following IP with anti-ZO-1 antibody (Figure 7A). Remnants of the IgG heavy chain from the antibody used for co-immunoprecipitation are visible, but are readily distinguishable from Cx43 signal (Figure 7A). Densitometric analyses of Cx43 brought down with ZO-1 following normalization against total actin (Figure 7B) indicated that in both non-junctional and junctional fractions, slightly more Cx43 interacts with ZO-1 in the failing ventricles compared with controls, although this difference between absolute levels did not attain statistical significance (P = 0.397 and P = 0.209, respectively; Figure 7B). However, as Cx43 is down-regulated in the failing left ventricle (Figure 3) and this occurs in both non-junctional and junctional fractions (data not shown; P = 0.053 and P = 0.023, respectively), Cx43 co-immunoprecipitation with ZO-1 is more meaningfully quantified relative to Cx43 levels in each sample (Figure 7C). This demonstrated that the proportion of Cx43 that interacts with ZO-1 is significantly increased in the failing ventricle compared with controls in both non-junctional and junctional fractions (P = 0.01 and P = 0.01, respectively).

Tissue was treated with the reversible cross-linking reagent DTBP to examine the Cx43/ZO-1 association in the in vivo setting, and to establish whether the interaction was altered during protein extraction. Densitometric analysis of western blots indicated that cross-linking did not alter the amount of Cx43 co-immunoprecipitated with ZO-1 in the non-junctional or junctional fractions from control (P = 0.759 and P = 0.246, respectively), DCM (P = 0.363 and P = 0.629, respectively), or ICM (P = 0.304 and P = 0.111, respectively) samples (data not shown).