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ZO-1 Regulates Intercalated Disc Composition and Atrioventricular Node Conduction

Originally published Research. 2020;127:e28–e43



ZO-1 (Zona occludens 1), encoded by the tight junction protein 1 (TJP1) gene, is a regulator of paracellular permeability in epithelia and endothelia. ZO-1 interacts with the actin cytoskeleton, gap, and adherens junction proteins and localizes to intercalated discs in cardiomyocytes. However, the contribution of ZO-1 to cardiac physiology remains poorly defined.


We aim to determine the role of ZO-1 in cardiac function.

Methods and Results:

Inducible cardiomyocyte-specific Tjp1 deletion mice (Tjp1fl/fl; Myh6Cre/Esr1*) were generated by crossing the Tjp1 floxed mice and Myh6Cre/Esr1* transgenic mice. Tamoxifen-induced loss of ZO-1 led to atrioventricular (AV) block without changes in heart rate, as measured by ECG and ex vivo optical mapping. Mice with tamoxifen-induced conduction system-specific deletion of Tjp1 (Tjp1fl/fl; Hcn4CreERt2) developed AV block while tamoxifen-induced conduction system deletion of Tjp1 distal to the AV node (Tjp1fl/fl; Kcne1CreERt2) did not demonstrate conduction defects. Western blot and immunostaining analyses of AV nodes showed that ZO-1 loss decreased Cx (connexin) 40 expression and intercalated disc localization. Consistent with the mouse model study, immunohistochemical staining showed that ZO-1 is abundantly expressed in the human AV node and colocalizes with Cx40. Ventricular conduction was not altered despite decreased localization of ZO-1 and Cx43 at the ventricular intercalated disc and modestly decreased left ventricular ejection fraction, suggesting ZO-1 is differentially required for AV node and ventricular conduction.


ZO-1 is a key protein responsible for maintaining appropriate AV node conduction through maintaining gap junction protein localization.


Editorial, see p 298

Meet the First Author, see p 205

ZO-1 (Zona occludens 1), encoded by the tight junction protein 1 (TJP1) gene, is a cytoplasmic scaffolding protein critical to the function of the apical junctional complex.1–3 Its essential function as a regulator of paracellular permeability is well recognized.2,4 In addition to binding to transmembrane proteins that dictate paracellular permeability,5–7 ZO-1 can bind to the actin cytoskeleton and the adherens junction component, α-catenin.5,8–11 ZO-1 is widely expressed in multiple cell types; however, its function has mostly been studied in epithelial and endothelial cells.4,12,13

Although its function in the heart remains poorly defined,14 ZO-1 is known to be enriched at the myocardial intercalated disc,15–18 which contains multiple components, including gap junctions, adherens junctions, and desmosomes.17 ZO-1 has been shown to bind to multiple intercalated disc proteins, including CAR (coxsackie and adenovirus receptor19 and gap junction proteins Cx (connexin) 35, 36, 40, 43, and 4516,20–23, and vinculin.24 In addition, loss of ZO-1 binding partners Cx40, Cx43, CAR, α-catenin, or vinculin in cardiomyocytes all disrupt heart function23,25–29 and loss of CAR results in decreased ZO-1 localization at the intercalated disc.26,27 Taken together, these data suggest ZO-1 may be required for intercalated disc structure and function.

Given previous studies suggesting the importance of ZO-1 at the intercalated disc,17,30 we sought to understand how ZO-1 might affect cardiac physiology. To circumvent the embryonic lethality of constitutive total body ZO-1 knockout,31 we developed inducible heart-specific Tjp1 deletion mouse models that allow for interrogation of the role for ZO-1 in the adult myocardium and conduction system. We show that ZO-1 is critical to atrioventricular (AV) nodal conduction and only modestly contributes to ventricular myocardial function. These data shed insight into the role of ZO-1 and potentially other ZO-1 associated proteins in heart physiology.


Data Availability

All supporting data and materials presented within this article and in the Data Supplement are available from the corresponding authors by reasonable request. Please see the Major Resources Table in the Data Supplement.

Generation of Adult, Cardiac-Specific ZO-1 Deficient Mice

All protocols were approved by The University of Chicago Institutional Animal Care and Use Committee. Tjp1 floxed mice (Tjp1tm2c(KOMP)Wtsi, mouse genome informatics [MGI]:6272009) were generated by Jerrold Turner (The University of Chicago and Brigham and Woman’s Hospital) from Tjp1tm2a(KOMP)Wtsi (MGI:4451268, KOMP repository, University of California, Davis) mice and generously provided by Jerrold Turner.32 This was performed by crossing Tjp1tm2a(KOMP)Wtsi C57BL/6N mice with B6.Cg-Tg(ACTFLPe)9205Dym/J (MGI: 2448985) mice to delete the En2SA-LacZ-neo cassette between the Frt sites.32Tjp1 floxed mice were bred with mice expressing a tamoxifen-inducible Cre recombinase driven by the Myh6 (B6.FVB(129)-A1cfTg(Myh6-cre/Esr1*)1Jmk/J, MGI:3050453),33Hcn4 (Hcn4tm2.1(cre/ERT2)Sev, MGI:5529675),34 or Kcne1 (Tg(Kcne1-cre/ERT2)1Imos, MGI:5301931)35 promoter. To induce recombination, both male and female mice were injected with tamoxifen at 6 to 8 weeks of age as previously described.36Tjp1fl/fl littermates with no Cre were also dosed with tamoxifen and used as controls.

Gene Expression Analysis

Left ventricular tissue was harvested from anesthetized mice. RNA extraction, reverse transcription, and quantitative real-time polymerase chain reaction was performed as previously described.37 Mouse Tjp1 expression was measured by quantitative real-time polymerase chain reaction with exon 3 spanning primers specific to the deletion, F: 5′-TTTCAGAGTGGGGAAACCTCC-3′, R: 5′-CACTCTTCCTTAGCTGCTGAAC-3′. Transcript abundance was normalized to GAPDH.

Western Blot Analysis

Following heart harvest, left ventricle and AV node were identified and dissected under a dissecting microscope. These tissues were processed, and tissue homogenates were made as previously described.38 Bradford assay was used to determine the protein concentration of the homogenates and 25 µg of protein was loaded onto 5% (homemade) or 4% to 20% (Bio-rad) acrylamide gels for separation by SDS-PAGE. Immunoblotting and detection were performed as previously described.38 Mouse anti-ZO-1 antibody (Thermo Fisher, Waltham, MA, Catalog no. 339100) and Rabbit anti-GAPDH (Cell Signaling Technology, Catalog no. 5174) were used at 1:1000 dilution. CAR (Santa Cruz Biotechnology, Catalog no. sc-373791), Cx40 (Thermo Fisher Scientific, Catalog no. 37-8900), β-catenin (Cell Signaling Technology, Catalog no. 9562), and HCN-4 (potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4; Abcam, Catalog no. ab32675) were used at 1:300 dilution. Mouse, Rat, and Rabbit horseradish peroxidase conjugated secondary antibodies were used at 1:1000 dilution (Cell Signaling Technology, Catalog nos. 7076, 7077, and 7074, respectively). Blots were developed by enhanced chemiluminescence reagents (Promega, Madison, WI) and imaged using X-ray film or a ChemiDoc XRS system (Bio-rad).

ECG Recordings

ECGs were recorded as previously described.37,39,40 Briefly, mice were implanted with subcutaneous telemetry transmitters and recorded over 48 hours (ETA-F10; Data Science International, St Paul, MN).39 Arrhythmia analysis and measurement of ECG intervals were performed using Ponemah Physiology Platform software (Data Science International, St Paul, MN).

In Vivo Electrophysiology

In vivo electrophysiology was performed as previously described.37 A right internal jugular venous approach was used on anesthetized mice to make recordings from the atrium, His bundle, and ventricle via a Scisense 1.9 F octapolar catheter with 0.5 mm spacing (Transonic, Ithaca, NY) connected to ADI BioAmp and PowerLab apparatus (ADInstruments, Colorado Springs, CO). Data were recorded using LabChart Software (ADInstruments). Atrial and ventricular pacing protocols were driven by a custom Python-based pacing program. Effective refractory periods were measured using 8-beat S1 drive trains followed by a single extra-stimulus.


Echocardiography was performed on anesthetized mice as previously described.37 The heart was imaged with a Vevo 770 ultrasound (VisualSonics, Toronto, Canada) using a 30 MHz high-frequency transducer. B- and M-mode images were recorded in the parasternal long- and short-axis projections at the mid-papillary muscle level in both views. Left ventricular internal diameters, septal wall thickness, and posterior wall thickness were measured in at least 3 beats from each projection and averaged, and fractional shortening was calculated and used to determine left ventricular ejection fraction.

Ex Vivo Optical Mapping

Optical mapping was performed as previously described37 using an approved George Washington University animal protocol. Hearts were isolated and prepared as previously described.41 Briefly, a midsternal incision was performed on the mice, and hearts were excised and mounted on a Langendorff apparatus for retrograde perfusion and superfusion with warmed (37°C) and oxygenated (95% O2/5% CO2) Tyrode’s solution (in mmol/L: 128.2 NaCl, 4.7 KCl, 1.05 MgCl2, 1.3 CaCl2, 1.19 NaH2PO4, 20 NaHCO3, and 11.1 glucose). Each isolated heart was pinned at the apex to the bottom of the chamber to prevent stream-induced movement. After pinning the right atrial and left atrial appendages, the excitation-contraction uncoupler blebbistatin (10 μmol/L, Bio-techne, Minneapolis, MN) was used to eliminate motion artifacts from the optical signals.

Langendorff-perfused hearts were stained in line with perfusion with the voltage-sensitive dye di-4-ANEPPS (30 μL of a 2.6 mmol/L stock solution dissolved in dimethyl sulfoxide). Epicardial depolarization was measured by illuminating the tissue preparation with a 520 nm LED light. Emitted light was recorded through a 650 nm long-pass filter by a MiCAM Ultima-L CMOS camera (SciMedia) with high spatial (100×100 pixels, 230±20 μm/pixel) and temporal (1000–3000 frames/s) resolution. The acquired fluorescent signal was analyzed with custom-developed software.42

Immunofluorescence Staining

Five micrometer thick cryosections of mouse left ventricular tissue or 10 µm AV node containing tissue were fixed (1% paraformaldehyde), washed (1×phosphate-buffered saline), quenched (50 mmol/L NH4Cl), permeabilized (0.5% NP-40), and stained with primary antibody as previously described.43 Antibodies were used against the following intercalated disc proteins: ZO-1 (Thermo Fisher Scientific, Catalog no. 339100), ZO-2 (Thermo Fisher Scientific, Catalog no. 71-1400), Cx43 (Cell signaling technology, Catalog no. 3512), JUP (junctional plakoglobin; Abcam Catalog no. ab11799), Nav1.5 (Abcam, Catalog no. ab56240), β-catenin (Cell Signaling Technology, Catalog no. 9562), N-cadherin (Thermo Fisher Scientific, Catalog no. 333900), CAR (Santa Cruz Biotechnology, Catalog no. sc-373791), Cx45 (Thermo Fisher Scientific, Catalog no. PA5-77357), Cx40 (Thermo Fisher Scientific, Catalog no. 37-8900), and CD31 (Antio-Proteomie, Catalog no. mAP-0032). The sections were then stained with Alexa Fluor dye conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc, Catalog nos. 711-586-152 and 715-546-151), phalloidin (Thermo Fisher Scientific, Catalog no. A22287), and Hoechst 33342 (Thermo Fisher Scientific, Catalog no. 953557). ProLong Gold (Thermo Fisher Scientific, Catalog no. P36934) was used for mounting. Fluorescence images were taken using an IX81 Olympus microscope (Olympus, Waltham, MA) with 20× or 40× air or oil immersion objectives. Quantification was performed in MetaMorph 7 Image Analysis Software (Molecular Devices, San Jose, CA) with linescan of the fluorescence signal across the width of the intercalated disc. Control experiments using the appropriate secondary antibody alone were performed simultaneously with all immunofluorescence staining experiments to determine antibody specificity. Furthermore, for ZO-1, CAR, β-catenin, and JUP, gastrointestinal tissue was used as a positive control for the antibody. Blinded imaging and quantification were performed using identical exposures and matched imaging and processing conditions.


Immunohistochemical staining for ZO-1 was performed using anti-ZO-1 (Thermo Scientific, Catalog no. 339100, mouse monoclonal antibody, Clone: ZO-1-1A12) on formalin-fixed paraffin-embedded mouse heart sections. After deparaffinization and rehydration, tissue sections were treated with antigen retrieval solution (Agilent, Santa Clara, CA) in a steamer for 20 minutes. MOM blocking kit (Vector Laboratories, Burlingame, CA) was used. Anti-ZO-1 antibody (1:50) was applied on tissue sections for 1 hour incubation at room temperature in a humidity chamber. Following PBS wash, the antigen-antibody binding was detected with Envision+ system (Agilent) and DAB+ chromogen (Agilent). Tissue sections were counterstained with hematoxylin and mounted. Hematoxylin and eosin stain44 and trichrome stain (Millipore-Sigma, St Louis, MO) were performed using standard protocols. Slides were imaged using a Leica DM2000 Microscope (Leica Microsystems, Wetzlar, Germany) with a ProgRes C14plus color digital camera (Jenoptik, Jena, Germany). Control slides using the appropriate secondary antibody alone was used to confirm primary antibody specificity.

Use of Human Atrial Tissue

Human heart tissue was collected from organ donors (that were not used for heart transplantation but had no history of major cardiovascular diseases and had normal cardiac function) provided by Illinois Gift of Hope Organ & Tissue Donor Network (GOH). The studies were approved by the Human Study Committee of Rush University. The AV node containing region was dissected out and fixed with formalin. Immunofluorescence and IHC staining of AV node slides were performed using anti-ZO-1 (described above) and anti-Cx40 (Thermo Fisher Scientific, Catalog no. 37-8900) antibodies with the secondary antibodies described above. Immunofluorescence images were obtained using an Olympus IX81 Inverted Widefield Microscope (Olympus, Waltham, MA).

Transmission Electron Microscopy

AV node and surrounding tissues were dissected from mice 2 weeks post-tamoxifen treatment. Tissues were fixed, embedded, sectioned, and mounted onto carbon grids as previously described with the following differences.45 For primary fixation, 2% glutaraldehyde and 4% paraformaldehyde in 0.1 mol/L sodium cacodylate buffer were used. 1% osmium tetroxide was used for secondary fixation. Polymerization was done at 60°C for 48 hours in a vacuum oven. Sixty nanometer thickness sections were cut and mounted on carbon-coated copper grids. Brief staining in 100% uranyl acetate and lead citrate was performed. Semi-thin sections used for Toluidine blue staining were cut at 300 nm. Images were taken with a JEM-1400 Plus electron microscope (JEOL, Tokyo, Japan).


Data are reported as mean±SEM. For paired comparisons, Shapiro-Wilk tests were performed, in R studio (Boston, MA), to determine normality, followed by 2-tailed Student t tests, in Excel (Microsoft, Redmond, WA), unless otherwise noted. To confirm statistical significance derived from t tests, nonparametric Wilcoxon rank-sum (WRS) test was used, and significance is stated when sample sizes were low and normality could not be reliably determined. This is designated with WRS followed by the P value. For count-based quantification of heart block occurrence, 1-tailed Fisher exact test was performed by using R studio. In the cases that multiple parameters are obtained and compared in the same experiment, the number of tests are limited, and the experiments and analyses are performed sequentially rather than simultaneously. In addition, different types of assays were performed to support our conclusions. While we deem it appropriate to not perform multiple testing correction in the results shown here, we do report the confidence interval and acknowledge that the interpretation could potentially be considered a weakness of our study. Statistical significance is designated as *P<0.05, **P<0.01, and ***P<0.001 in the figures.


Generation of Adult Cardiac-Specific Tjp1 Knockout Mice

ZO-1 is widely expressed in multiple cell types and is a known regulator of paracellular permeability in epithelial and endothelial cells.1–4,12,13 Although ZO-1 localization at the intercalated disc is well-documented,16–18,46,47 the function of ZO-1 in the heart remains poorly defined. Since the complete loss of ZO-1 is embryonic lethal at day E11.5 due to vascular defects,31 we generated inducible tissue-specific knockout models to study cardiac-specific ZO-1 function. Tjp1 floxed mice were bred with cardiomyocyte-specific (Myh6 promoter) Cre/Esr1* transgenic mice (Figure 1A).32,33

Figure 1.

Figure 1. Inducible cardiomyocyte-specific tight junction protein 1 (Tjp1) deletion reduces ZO-1 (zona occludens 1) expression at intercalated discs.A, Breeding and treatment scheme for Tjp1 deletion (Tjp1fl/fl; Myh6Cre/Esr1*). B, Exon 3 specific Tjp1 mRNA levels in left ventricle (P=0.0041, Wilcoxon rank-sum [WRS]: P=0.016, n=5 Tjp1fl/fl, n=4 Tjp1fl/fl; Myh6Cre/Esr1* mice) and liver (P=0.15, n=3 Tjp1fl/fl, n=4 Tjp1fl/fl; Myh6Cre/Esr1* mice), as determined by quantitative real-time polymerase chain reaction (qRT-PCR). C, Western blot of left ventricular lysate (P=0.013, WRS: P=0.029, n=4 Tjp1fl/fl, n=3 Tjp1fl/fl; Myh6Cre/Esr1* mice). D, Representative images of immunofluorescent staining of ZO-1 in left ventricular tissue. CD31 was used to label endothelial cells. Arrows: intercalated disc, Arrow heads: endothelial cells. E, Representative images of immunofluorescent staining of ZO-1 in left ventricular tissue. JUP (Junction plakoglobin) was costained with ZO-1 to mark intercalated discs. Arrow: intercalated disc. F, Immunofluorescent staining along lines perpendicular to the intercalated disc are shown (P=0.019, WRS: P=0.050, n=3 mice per genotype, 18 intercalated discs per animal; Red: Tjp1fl/fl; Myh6Cre/Esr1*, Blue: Tjp1fl/fl, **P<0.01, *P<0.05). All studies were performed at 10 d post-tamoxifen injection (scale bar=10 µm). Two-tailed Student t test was used to calculate P values.

We first assessed Tjp1 deletion efficiency in the heart. Quantitative real-time polymerase chain reaction showed Tjp1fl/fl; Myh6Cre/Esr1* heart tissues have decreased Tjp1 exon3 containing mRNA (46±4% decrease, P=0.0041, WRS: P=0.016) relative to Tjp1fl/fl controls 2 weeks post-tamoxifen injection (Figure 1B). In contrast, liver expression of Tjp1 mRNA was unchanged between Tjp1fl/fl; Myh6Cre/Esr1* and Tjp1fl/fl mice (P=0.15). In the left ventricle, ZO-1 protein was reduced by 69±10% (P=0.013, WRS: P=0.029) by Western blot (Figure 1C). The partial loss of Tjp1 mRNA and ZO-1 protein in the left ventricle could be due to (1) incomplete Tjp1 locus recombination resulting in incomplete knockout or (2) the presence of noncardiomyocyte cells (such as endothelial cells and fibroblasts) not affected by cardiomyocyte-specific MerCreMer protein activity. To differentiate between these 2 possibilities, we next performed immunofluorescence staining for ZO-1 protein to determine its myocardial-specific expression. ZO-1 expression remained abundant in endothelial cells, as identified by CD31 (Figure 1D arrowheads). ZO-1 protein expression at the intercalated discs was significantly reduced (73±8% decrease, P=0.019, WRS: P=0.050; Figure 1D and 1E arrows, Figure 1F) as measured by peak intensity of immunofluorescence staining of ZO-1 at the intercalated disc. These findings support that incomplete Tjp1 knockout is due to a combination of both factors discussed above. There were no observed differences in cardiomyocyte actin organization or cell morphology.

Loss of ZO-1 Disrupts Cardiac Conduction

To study the physiological role of ZO-1 in the heart, we first monitored mice longitudinally by ECG under anesthesia. Five days after tamoxifen treatment, Tjp1fl/fl; Myh6Cre/Esr1* mice had significantly prolonged PR interval (80±10% prolongation, P=4.8×10−4; Figure 2A and 2B), indicating first degree AV block. By 10-day post-tamoxifen, 4 of 11 mice (P=0.037, 1-tailed Fisher exact test was used because the fraction of mice with AV block can only increase from control) displayed complete dissociation of atrial and ventricular depolarization, indicating heart block with junctional escape rhythm, compared with none (0 of 12) in control mice (Figure 2C). Ambulatory telemetric ECG recordings were performed to assess RR and PR interval variation. At 1-week post-tamoxifen injection, Poincaré plots of the PR interval showed distinct separation between Tjp1fl/fl; Myh6Cre/Esr1* and Tjp1fl/fl control mice (Figure 2D) and average PR interval was significantly prolonged in Tjp1fl/fl; Myh6Cre/Esr1* mice (P=0.00021, WRS: P=0.050; Figure 2E). In contrast, dispersion of RR interval and average RR interval was not significantly different (P=0.98; Figure 2F and 2G). By 2 weeks post-tamoxifen injection, PR interval was not only prolonged but also highly variable consistent with third degree heart block and junctional escape rhythm (P=0.020, WRS: P=0.050). RR intervals remained similar (P=0.21; Figure 2H through 2K). Neither P wave (10.2±0.8 versus 12.3±0.7 ms, P=0.18) nor QRS complex durations (8.9±0.4 versus 10.4±0.5 ms, P=0.13) were altered between Tjp1fl/fl and Tjp1fl/fl; Myh6Cre/Esr1* mice. Additionally, tamoxifen-injected Myh6Cre/Esr1* control mice exhibited normal PR interval lengths (PR=32.7±14 ms). To confirm the specific location of the conduction defect, intracardiac electrophysiology studies were performed. Ten days after tamoxifen injection, the time interval from atrial to bundle of His depolarization was increased (A-H interval, P=0.0027, WRS: P=0.050, Figure 2L and 2M), but the interval from bundle of His depolarization to ventricular depolarization was unchanged (H-V interval, P=0.39, Figure 2L and 2N). The minimum cycle duration that is capable of maintaining 1:1 atrial to ventricular conduction time (Figure 2O; AV Wenckebach time) was prolonged (P=0.041, WRS: P=0.032). Together, A-H interval and AV Wenckebach prolongation are indicative of slowed AV nodal conduction. Intracardiac pacing studies showed normal atrial and ventricular refractory periods (P=0.50, P=0.25, not shown). These data suggest that removal of ZO-1 causes slowed AV nodal conduction but does not alter atrial myocardial conduction, ventricular myocardial conduction, or RR interval.

Figure 2.

Figure 2. Tjp1fl/fl; Myh6Cre/Esr1* mice have altered cardiac electrophysiology.A, Averaged telemetry signals 5 d after tamoxifen injection. B, Time course of PR interval following tamoxifen injection (P=0.50 [n=7 Tjp1fl/fl, n=8 Tjp1fl/fl; Myh6Cre/Esr1*] 0 d, P=4.8×10−4 [n=6 Tjp1fl/fl, n=9 Tjp1fl/fl; Myh6Cre/Esr1*] for 5 d, P=2.6×10−7 [n=9 Tjp1fl/fl, n=10 Tjp1fl/fl; Myh6Cre/Esr1*] for 10 d, P=3.8×10−8 [n=11 Tjp1fl/fl, n=10 Tjp1fl/fl; Myh6Cre/Esr1*] for 20 d, and P=3.1×10−8 [n=6 Tjp1fl/fl, n=10 Tjp1fl/fl; Myh6Cre/Esr1*] for 40 d or later). C, Sampling of 3 time points within a representative ECG trace in Tjp1fl/fl and Tjp1fl/fl; Myh6Cre/Esr1* mice (arrows: P waves). D and H, Representative poincaré plots, showing beat-to-beat PR interval variability, from Tjp1fl/fl; Myh6Cre/Esr1* and Tjp1fl/fl mice 5 or 10 d post-tamoxifen treatment. E and I, PR intervals 5 or 10 d post-tamoxifen treatment (P=2.1×10−4 for 5 d and P=0.020 for 10 d, Wilcoxon rank-sum [WRS]: P=0.050 for both, based on averages for each mouse. n=3 mice per genotype). F and J, Representative poincaré plots, showing beat-to-beat RR interval variability, from Tjp1fl/fl; Myh6Cre/Esr1* and Tjp1fl/fl mice 5 or 10 d post-tamoxifen treatment. G and K, RR intervals 5 or 10 d post-tamoxifen treatment (n=3 mice per genotype). L, Representative atrial electrogram and simultaneous surface ECG recordings. M, Atrial to His conduction time (P=0.0027, WRS: P=0.050, n=3 Tjp1fl/fl, n=3 Tjp1fl/fl; Myh6Cre/Esr1* mice). N, His to ventricular conduction time (P=0.39, n=3 Tjp1fl/fl, n=3 Tjp1fl/fl; Myh6Cre/Esr1* mice). O, Atrioventricular (AV) Wenckebach time (P=0.041, WRS: P=0.029, n=4 Tjp1fl/fl, n=5 Tjp1fl/fl; Myh6Cre/Esr1* mice; red: Tjp1fl/fl; Myh6Cre/Esr1*, blue: Tjp1fl/fl, ***P<0.001, **P<0.01, *P<0.05, ns indicates not significant). Two-tailed Student t test was used to calculate P values.

To assess epicardial depolarization patterns, we performed optical mapping studies. Propagation of epicardial depolarization in isolated hearts from mice 20 days post-tamoxifen treatment was measured. The overall pattern of atrial and ventricular depolarization was not different between Tjp1fl/fl and Tjp1fl/fl; Myh6Cre/Esr1* hearts with only one focus of depolarization initiation occurring at or near the sinoatrial node in both genotypes (Figure 3A). There were no significant differences observed in atrial (P=0.86) and ventricular (P=0.84) activation times (Figure 3A and 3B). However, a 75±24% increase in AV conduction time was observed (P=0.0045, WRS: P=0.0043), consistent with electrophysiology studies (Figure 3 and Figure 2A, 2B, 2L, 2M, and 2O).

Figure 3.

Figure 3. Identification of prolonged atrioventricular (AV) conduction time by optical mapping.A, Representative activation maps of atria and ventricle in Tjp1fl/fl (left) and Tjp1fl/fl; Myh6Cre/Esr1* (right) mice. B, Atrial, ventricular, AV conduction times (P=0.0045, Wilcoxon rank-sum: P=0.0043, n=6 Tjp1fl/fl, n=5 Tjp1fl/fl; Myh6Cre/Esr1* mice). All studies were performed 20 d post-tamoxifen injection (red: Tjp1fl/fl; Myh6Cre/Esr1*, blue: Tjp1fl/fl, **P<0.01). Two-tailed Student t test was used to calculate P values.

Tjp1 Deletion Is Associated With Modestly Decreased Heart Function

Given the observed changes in conduction, we next asked whether loss of cardiomyocyte Tjp1 would cause decreased heart function. We measured left ventricular ejection fraction by echocardiography over the course of 10 weeks. Ejection fraction was unchanged in Tjp1fl/fl; Myh6Cre/Esr1* compared with Tjp1fl/fl controls in the first 10 days post-tamoxifen treatment (P=0.15). Beginning at 20 days post-tamoxifen treatment, average ejection fraction in Tjp1fl/fl; Myh6Cre/Esr1* mice was modestly decreased compared with Tjp1fl/fl controls (16±4%, P=5.3×10−5; Figure IA and IB in the Data Supplement). Forty days post-tamoxifen injection, the decreased ejection fraction in Tjp1fl/fl; Myh6Cre/Esr1* mice persisted without additional decrease in ejection fraction (Figure IB in the Data Supplement). A repeated measures mixed ANOVA was performed, and ejection fraction between Tjp1fl/fl and Tjp1fl/fl; Myh6Cre/Esr1* mice was significantly different (P=5.1×10−4 and partial eta squared=0.95). The heart rate was not significantly different at any time point (Figure IB in the Data Supplement). Left ventricular systolic and diastolic diameters are shown in Figure IIA through IIF in the Data Supplement. The small and persistent decrease in ejection fraction in Tjp1fl/fl; Myh6Cre/Esr1* mice at ≥20 days is due predominantly to increased internal diameter (P=0.0017, Figure IIC in the Data Supplement) during systole without significant reduction in diastolic diameter (P=0.090, Figure IID in the Data Supplement). The change in ejection fraction was not accompanied by gross or microscopic changes, including heart size (Figure IC in the Data Supplement), heart weight (Figure ID in the Data Supplement), or fibrosis as determined by trichrome staining (Figure IE in the Data Supplement) 12 weeks after tamoxifen injection. Thus, loss of ZO-1 causes only a small but significant change in cardiac function without evidence of gross or histological alterations.

ZO-1 Loss Disrupts AV Nodal Cx40 and CAR Localization and Perinodal Cx43 Localization

Due to AV nodal dysfunction following Tjp1 deletion, we assessed AV node morphology and ZO-1 expression in Tjp1fl/fl and Tjp1fl/fl; Myh6Cre/Esr1* mice. Using trichrome staining, we identified the AV node, which was not organizationally disrupted in Tjp1fl/fl; Myh6Cre/Esr1* mice (Figure 4A). In Tjp1fl/fl mice, immunohistochemical staining showed ZO-1 was highly expressed at the AV node. ZO-1 staining intensity was decreased by 49±10% (P=0.015, WRS: P=0.050, n=3 for each group) in AV nodes in Tjp1fl/fl; Myh6Cre/Esr1* mice (Figure 4B). Next, we assessed expression and localization of proteins important to AV nodal conduction. HCN-4 staining was high in the region identified as the AV node via trichrome stain (Figure 4C and 4D), further confirming its functional specialization. Cx40 and CAR stained the AV node strongly, whereas Cx43 was absent from the AV node but was strongly expressed in the adjacent myocardium (perinodal region; Figure 4E and 4F). Cx40 and CAR co-staining with the intercalated disc marker β-catenin showed that Cx40 and CAR are enriched at intercalated discs of AV nodes (Figure III in the Data Supplement). Cx40 staining intensity within the intercalated discs of the AV node was decreased by 70±8% in Tjp1fl/fl; Myh6Cre/Esr1* mice (P=0.050, WRS: P=0.050, n=3 animals per genotype; Figure 4G). Cx43 staining intensity at the intercalated discs of perinodal myocardium was decreased by 65±4% in Tjp1fl/fl; Myh6Cre/Esr1* mice (P=0.0018, WRS: P=0.050, n=3 animals per genotype; Figure 4H). CAR stained AV nodal cells in a punctate and membranous pattern at cell-to-cell junctions, and staining intensity was decreased by 39±6% in Tjp1fl/fl; Myh6Cre/Esr1* mice (P=0.011, WRS: P=0.050, n=3 animals per genotype; Figure 4I). Cx45, NaV1.5, β-catenin, and N-cadherin staining intensity were not changed in the AV node (Figure IV in the Data Supplement).

Figure 4.

Figure 4. ZO-1 (zona occludens 1) loss disrupts atrioventricular (AV) node protein localization and abundance.A, Representative trichrome stain of mouse AV nodes. B, Representative immunohistochemical staining for ZO-1 in AV nodes (low and high magnification images of the same AV node). C and D, Representative trichrome (C) and HCN-4 (potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4) immunofluorescent (D) staining for AV node identification. E and F, Immunofluorescent staining of Cx40 (connexin 40) (E), Cx43 (E), and CAR (coxsackie and adenovirus receptor) (F) at low power (scale bar=100 µm). G–I, Representative high power images from boxed region of images in E and F (Cx40 in G, within AV node, Cx43 in H, in perinodal area, CAR in I, within AV node, scale bar=10 µm) and quantification of intercalated disc staining of Cx40 (G, within AV node), Cx43 (G, perinodal area), and CAR (I, within AV node). Quantification of immunofluorescent staining along lines perpendicular to the intercalated disc are shown. Peak intercalated disc intensity values were averaged per animal and compared (P=0.050 for Cx40, P=0.0018 for Cx43, and P=0.011 for CAR, Wilcoxon rank-sum [WRS]: P=0.050 for all, n=3 mice per genotype, 8 intercalated disc region measurements per mouse). AV nodes are circled by dashed lines. J, Western blot images of HCN-4 expression in AV node tissues compared with ventricular tissue in the same Tjp1fl/fl mice (n=3 mice). K, Representative Western blot images of AV node ZO-1, Cx40, CAR, and β-catenin and corresponding GAPDH loading control. L, Quantification of Western blot (P=0.0089, WRS: P=0.014 with a 95% CI of 100±24% [Tjp1fl/fl] and 50±8% [Tjp1fl/fl; Myh6Cre/Esr1*] for ZO-1, P=0.039, WRS: P=0.029 with a 95% CI of 100±42% [Tjp1fl/fl] and 41±28% [Tjp1fl/fl; Myh6Cre/Esr1*] for Cx40, n=4 mice per genotype; red: Tjp1fl/fl; Myh6Cre/Esr1*, blue: Tjp1fl/fl, ***P<0.001, **P<0.01, *P<0.05). All studies were performed 10 d post-tamoxifen injection. Peak intercalated disc intensity 2-tailed Student t test was used to calculate P values from immunofluorescence experiments and 1-tailed Student t test was used to test our hypothesis in the Western blots that ZO-1 and Cx40 were decreased in Tjp1fl/fl; Myh6Cre/Esr1*.

To determine whether overall Cx40 and CAR protein abundance are altered, we performed Western blot for ZO-1, Cx40, and CAR using homogenates of dissected AV nodes. AV nodal tissue dissection was confirmed by enrichment of HCN-4 in AV nodal tissue relative to ventricular tissue (Figure 4J). We observed that ZO-1 and Cx40 protein abundance was decreased by 50±6% (P=0.0089, WRS: P=0.014) and 59±11% (P=0.036, WRS: P=0.029), respectively, in dissected AV nodal regions of Tjp1fl/fl; Myh6Cre/Esr1* mice at 2 weeks of age (Figure 4K and 4L). CAR protein abundance also showed a downward trend, but it did not reach statistical significance (P=0.21, Figure 4K and 4L). Similar to immunofluorescence staining results, β-catenin protein abundance was not significantly different (P=0.11, Figure 4K and 4L). We further tested if similar changes could also occur in ventricular myocardium distant from the AV node. At the ventricular intercalated disc, Cx43 and CAR localization were significantly decreased (P=0.042 and P=0.0032, respectively, WRS: P=0.050 for both, n=3 animals per genotype), but β-catenin distribution was not significantly altered (P=0.21), as assessed by immunofluorescence staining (Figure V in the Data Supplement).

We next examined whether changes in AV node protein localization and total expression would yield discernable AV node ultrastructural changes. Transmission electron microscopy was performed on AV node containing sections, as determined by Toluidine blue staining (Figure VIA and VIB in the Data Supplement). No discernable differences in overall AV node architecture or in the appearance of the intercalated disc were noted between Tjp1fl/fl; Myh6Cre/Esr1* and control mice 2 weeks post-tamoxifen injection (Figure VIC and VID in the Data Supplement).

ZO-1 and Cx40 Colocalize in Human AV Nodes

Similar to our observation of high ZO-1 abundance in mouse AV nodes, we found ZO-1 is also highly expressed in AV nodes of humans (Figure 5A and 5B). It is also expressed at intercalated discs of atrial and ventricular cardiomyocytes (Figure 5A and 5B). Since Cx40 is a major AV node connexin,48,49 and Cx40 is decreased in Tjp1fl/fl; Myh6Cre/Esr1* mice, we stained for Cx40 in human AV node samples. ZO-1 colocalizes with Cx40 in both the AV node and atrial myocardial tissue but is not found in the ventricle (Figure 5C). Taken together, our human and mouse studies suggest abundant ZO-1 expression in the AV node.

Figure 5.

Figure 5. ZO-1 (zona occludens 1) colocalizes with Cx40 (connexin 40) in human atrioventricular (AV) nodes.A, Representative H&E stain of a normal human AV node (scale bar=1 mm). B, Immunohistochemical staining of ZO-1 in normal human atrium, AV node, and ventricle (scale bar=100 µm). C, Immunofluorescence staining of ZO-1 and Cx40 in human atrium, AV node, and ventricle (scale bar=10 µm). Arrows indicate intercalated discs.

Conduction System-Specific ZO-1 Loss Is Sufficient to Drive Heart Block

Upon determining that ZO-1 is abundantly expressed in mouse and human AV nodal tissues, we next tested the contribution of ZO-1 to cardiac conduction specifically by genetic dissection. We generated conduction system-specific (Tjp1fl/fl; Hcn4CreERt2) and AV bundle-His-Purkinje specific (Tjp1fl/fl; Kcne1CreERt2) Tjp1 deletion mouse models. Tjp1fl/fl; Hcn4CreERt2 mice developed increased PR interval (148±7% increase, P=2.7×10−7, WRS: P=0.029) and dissociation of atrial and ventricular depolarization 2 weeks after the start of tamoxifen treatment (Figure 6A through 6C). In contrast, none of the Tjp1fl/fl; Kcne1CreERt2 mice demonstrated heart block (Figure 6A through 6C). In line with this finding, the abundant ZO-1 expression found in the AV node was not observed in the AV bundle (Online Figure VII). Furthermore, despite the conduction system defects in the Tjp1fl/fl; Hcn4CreERt2 mice, there was no evidence of depressed myocardial function (P=0.51, Figure 7D), in contrast to our observations in Tjp1fl/fl; Myh6Cre/Esr1* mice (Figure IA and IB in the Data Supplement). These results show that ZO-1 expression in the conduction system proximal to the His bundle is critical to conduction. Furthermore, the data suggest myocardial dysfunction is independent of the conduction system defects in cardiac-specific Tjp1 deleted mice.

Figure 6.

Figure 6. PR prolongation is due to ZO-1 (zona occludens 1) loss proximal to the bundle of His.A, Schematics of locations of ZO-1 deletion in Tjp1fl/fl; Myh6Cre/Esr1*, Tjp1fl/fl; Hcn4CreERt2, and Tjp1fl/fl; Kcne1CreERt2 mouse lines. B, PR intervals of Tjp1fl/fl; Hcn4CreERt2, and Tjp1fl/fl; Kcne1CreERt2 mice (P=2.7×10−7, Wilcoxon rank-sum: P=0.029, n=4 Tjp1fl/fl n=3 Tjp1fl/fl; Hcn4CreERt2). C, Representative surface ECG recordings in Tjp1fl/fl; Myh6Cre/Esr1*, Tjp1fl/fl; Hcn4CreERt2, and Tjp1fl/fl; Kcne1CreERt2 mice (n=5 mice per genotype). D, Ejection fraction in Tjp1fl/fl; Hcn4CreERt2 (P=0.51, n=3 Tjp1fl/fl n=4 Tjp1fl/fl; Hcn4CreERt2 and Tjp1fl/fl; Kcne1CreERt2 mice; P=0.1, n=4 mice per genotype). All studies were performed 10 d post-tamoxifen injection (***P<0.001, ns indicates not significant). Two-tailed Student t test was used to calculate P values.

Figure 7.

Figure 7. ZO-1 (zona occludens 1) maintains atrioventricular (AV) nodal Cx40 (connexin 40)-ZO-1-CAR (coxsackie and adenovirus receptor) complex to facilitate atrial to ventricular conduction.

Electrical conduction from the atrium to ventricle passes through the AV node (purple triangle). Top, Cx40-ZO-1-CAR protein complex exists at the cell-cell junctions within the AV node to maintain normal AV nodal conduction. Bottom, When ZO-1 expression is lost in AV nodal cells, Cx40 and CAR cannot concentrate at cell-cell junctions, decreasing cell-cell conductance, leading to impeded AV nodal conduction (AV node purple, ZO-1 blue, Cx40 green, Cx43 red, CAR magenta).


Healthy cardiac conduction and function relies on the coordinated electrical activity of distinct populations of cardiomyocytes. Electrical activity initiated at the sinoatrial node must propagate through atrial myocardium, the AV node, His-Purkinje system, and into ventricular myocardium. Disruption of cell-cell conduction results in cardiac arrhythmias and cardiomyopathies, a leading cause of morbidity and mortality worldwide.50–53 Intercalated disc proteins have been recognized as important regulators of cardiac conduction due to their function in maintaining electrical and physical coupling between cells. Here, we show inducible loss of the intercalated disc protein ZO-1 in cardiomyocytes of adult mice impedes AV node conduction and modestly affects ejection fraction.

Mice with induced cardiomyocyte-specific Tjp1 deletion (Tjp1fl/fl; Myh6Cre/Esr1* mice with tamoxifen injection) exhibit an AV nodal conduction defect with normal atrial and ventricular conduction (Figures 2 and 3). This interpretation is based on electrophysiological and optical mapping studies demonstrating increased PR interval, increased AH interval, increased AV Wenckebach time, and AV conduction time but unchanged HV interval, P wave, QRS complex duration, and atrial and ventricular activation time in Tjp1fl/fl; Myh6Cre/Esr1* mice (Figures 2 and 3). To assess (1) if the AV block is intrinsic to the conduction system or secondary to myocardial changes and (2) potential region-specific requirements of ZO-1 in the conduction system, we generated 2 additional inducible tissue-specific knockout models. Similar to induced cardiomyocyte-specific Tjp1 knockout (Tjp1fl/fl; Myh6Cre/Esr1*), tamoxifen-induced overall conduction system Tjp1 knockout (Tjp1fl/fl; Hcn4CreERt2) resulted in AV block without decreased ejection fraction (Figure 6), suggesting AV block is not secondary to myocardial changes. In contrast, tamoxifen-induced Tjp1 knockout distal to the AV node (Tjp1fl/fl; Kcne1CreERt2) did not manifest heart block. Taken together with our observation that ZO-1 is highly expressed in the AV node, but in the fast-conducting AV bundle, such high expression was not apparent (Figure VII in the Data Supplement), these results support that ZO-1 loss in the conduction system above the bundle of His is responsible for PR prolongation, and ZO-1 expression is critical to normal AV nodal conduction.

Given the observed AV node dysfunction following ZO-1 loss, we hypothesized that ZO-1 is important to the organization of proteins at the intercalated disc that facilitates cell-cell conduction. Immunofluorescence microscopy and Western blot studies showed decreased expression of Cx4049,54 and to a lesser extent, CAR,26,27 in the AV node in Tjp1fl/fl; Myh6Cre/Esr1 mice. This is in line with previous biochemical data demonstrating that connexins, CAR, and ZO-1 can reside within the same protein complex. Connexins contain a C-terminal PDZ (PSD95-DLG1-ZO-1) binding sequence that directly binds to the PDZ domains of ZO-1.16,22,55–57 CAR contains a PDZ binding C-terminal motif and can precipitate with ZO-1.19,26,58,59 Furthermore, genetic deletion of Gja5 (Cx40)49,54 and Cxadr (CAR)26,27 both slow AV nodal conduction. Together, these data suggest ZO-1 plays a key role in maintaining the connexin-ZO-1-CAR complex at the intercalated disc.

It is notable that although the PR interval was altered in cardiac and conduction system-specific Tjp1 deleted mice, there was no change in RR interval or heart rate. This observation of AV block with normal heart rate is in contrast to some models in which PR interval prolongation is associated with increased RR interval.60,61 Interestingly, PR prolongation with normal heart rate is also observed in Cxadr26,27 and Gja549 knockout mice. Our finding that Cx40 and CAR localization at the intercalated disc are affected by Tjp1 knockout along with biochemical interactions between ZO-1, CAR, and Cx40 (discussed above) and altered ZO-1 distribution in Cxadr knockout mice provide increasing evidence for functional interactions between these proteins. Together, these observations coalesce into a model in which ZO-1, CAR, and Cx40 physically associate to coregulate AV nodal conduction.

Although atrial and ventricular conduction speed were normal in Tjp1fl/fl; Myh6Cre/Esr1 mice, changes in ventricular intercalated disc organization and protein staining were still observed. By immunofluorescence microscopy, there was decreased Cx43 and CAR localization at the ventricular intercalated disc. These observations are consistent with previous findings that partial connexin loss is not sufficient to induce altered myocardial conduction,62–64 and Cxadr deletion in cardiomyocytes also does not significantly affect atrial and ventricular conduction, despite decreased connexin and ZO-1 localization at intercalated discs of ventricular cardiomyocytes.26,27 Our finding that Tjp1 knockout mice have prolonged AV nodal conduction times but normal ventricular conduction times suggests that AV nodal and ventricular cardiomyocyte conduction have different molecular requirements. Our observations support that AV nodal conduction may be particularly vulnerable to defects in the connexin-ZO-1-CAR complex (Figure 7).

We demonstrate that ZO-1 loss in myocardial tissue is associated with modestly decreased ejection fraction. In contrast, neither of the conduction specific knockout models demonstrated this phenotype (Figure 6D). This suggests that loss of ZO-1 can independently affect conduction and ejection fraction, and decreased functional output is due directly to loss of ZO-1 in the myocardium and not secondary to conduction defects. We did not observe a progressive loss of ejection fraction and sudden death of the Tjp1fl/fl; Myh6Cre/Esr1* mice, which could be explained by the presence of compensatory mechanisms that may be important for preserving myocardial function following loss of ZO-1. For example, ZO-2, a closely related protein to ZO-1, may play a role in such compensatory mechanisms. ZO-1 and ZO-2 both work to regulate epithelial function, including tight junction function and apical membrane organization.65–67 Although we could readily detect ZO-2 expression in cardiac endothelial cells, we failed to detect ZO-2 at the intercalated disc 10 days following ZO-1 knockout (Figure VII in the Data Supplement), long-term studies are needed to understand whether ZO-2 along with other protein expression and localization are altered and limit ZO-1 induced myocardial changes.

Recently, TJP1 variations have been associated with arrhythmogenic cardiomyopathy in patients,68 but AV nodal dysfunction was not observed in the 4 probands studied by De Bortoli et al.68 We do not currently know why such a difference exists, and further investigation will be required to determine the potential role of ZO-1 on AV nodal function in humans with arrhythmogenic cardiomyopathy. Another difference between our ZO-1 knockout mouse model and the patients of De Bortoli et al68 is the absence of fibro-fatty replacement of myocardial tissue up to 12 weeks after Tjp1 deletion in the mice. This difference may be due to the adult onset, cardiomyocyte-specific nature of the ZO-1 loss in our mouse model compared to patients carrying pathogenic TJP1 variants, who have altered ZO-1 function in all cell types, throughout their life. Since ZO-1 may influence many aspects of cardiac (eg, coronary vasculature) as well as other organ system development and physiological function, the clinical symptoms may be influenced by all of these cell types and organs. Long-term studies will be required to further evaluate whether histological features of arrhythmogenic cardiomyopathy can manifest in mice following knockout of Tjp1.

ZO-1 has been implicated in other forms of cardiomyopathy without Tjp1 mutations. For example, expression of a pathogenic mutant of TMEM-43 (transmembrane protein 43) associated with arrhythmogenic cardiomyopathy causes loss of ZO-1 at the intercalated disc-like cell-cell junctions and decreases cell-cell conduction velocity in HL-1 cells.54 Furthermore, our analysis of published Gene Expression Omnibus datasets suggest TJP1 expression is altered in hearts of cardiomyopathy patients (GDS2205 and GDS1362) and isoproterenol treated mice (GDS3684). Together, these findings suggest ZO-1 expression or localization change can influence cardiomyopathy pathogenesis.

In conclusion, we demonstrate ZO-1 is necessary for maintaining gap junction protein abundance and localization, and loss of ZO-1 results in AV node dysfunction, and when deleted from myocytes throughout the heart, results in modest decrease in cardiac function. Understanding how ZO-1 function is altered in patients with cardiomyopathy and other heart diseases will be important and may provide novel therapeutic approaches.

Nonstandard Abbreviations and Acronyms


coxsackie and adenovirus receptor




potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4


junctional plakoglobin


mouse genome informatics




tight junction protein 1


transmembrane protein 43


zona occludens 1


We thank Terri Li, Can Gong, Xin Jiang, and Mark Lingen from The University of Chicago Human Tissue Research Center Core, Yimei Chen, Anthony Chang, and James Brainer for assistance with electron microscopy, and Rostislav Bychkov and Richard Telljohann for assistance with mouse atrioventricular (AV) node identification. Tjp1 floxed (Tjp1tm2c(KOMP)Wtsi) mice were generated and generously provided by Jerrold R. Turner with the support of the National Institutes of Health (NIH; R01 DK061931 and R01 DK068271). We thank Sylvia M. Evans for the Hcn4CreERt2 mice.

Supplemental materials

Online Figures I–VIII

Major Resources Table

Uncut Gel Blots


*L.S. and C.R.W. contributed equally to this article.

For Sources of Funding and Disclosures, see page e41.

The Data Supplement is available with this article at

Correspondence to Christopher Weber, Pathology, University of Chicago, 5841 S Maryland Ave, Chicago, IL 60637, Email
Le Shen, Pathology and Surgery, University of Chicago, 5841 S Maryland Ave, Chicago, IL 60637, Email


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Novelty and Significance

What Is Known?

  • The tight junction protein, ZO-1 (zonula occludens 1) maintains and regulates barrier function in epithelia and endothelia.

  • ZO-1 is found in the heart at cardiomyocyte junctions, called intercalated discs.

  • ZO-1 interacts with proteins that regulate cell-to-cell communications, called Cxs (connexins), as well as other cell-cell junction components.

What New Information Does This Article Contribute?

  • Heart block is a common patient condition whereby the electrical signals slow or are blocked as they flow from atria to ventricle.

  • Loss of ZO-1 in cardiomyocytes of adult mice results in heart block.

  • Atrioventricular (AV) node conduction, the electrical connection between the atria and ventricle, as opposed to conduction in either chamber, is most sensitive to ZO-1 loss.

  • Expression and localization of Cx40 (connexin 40), which is important for AV nodal conduction, are disrupted in ZO-1 deleted AV nodes.

Intercalated discs, found at the ends of cardiomyocytes, provide mechanical and electrical coupling between individual cells. However, our understanding of the molecular determinants of intercalated disc structure and function remains limited. In this study, we investigated the role of ZO-1, a cytoskeletal protein that can link transmembrane proteins and cytoskeletal components to stabilize multiprotein complexes, in the intercalated disc. We found that loss of ZO-1 in cardiomyocytes disrupts Cx localization at intercalated discs and results in PR prolongation and dissociation of atrial and ventricular depolarization, indicating heart block. Furthermore, we found ZO-1 is highly expressed in the AV node compared with surrounding atrial and ventricular tissue and loss of ZO-1 leads to decreased localization of Cx40 and CAR (coxsackie and adenovirus receptor) at AV node intercalated discs. This work shows ZO-1 is important for regulating AV conduction and given previous findings from Cx40 and CAR knockout mice, we propose ZO-1, Cx40, and CAR form a functional complex within intercalated discs to regulate AV conduction. These findings suggest a need to understand the structural-functional relationships of the Cx-ZO-1-CAR protein complex at the intercalated disc, which may lead to novel ways to relieve heart blocks in patients.


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