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

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.

The unstoppable connexin43 carboxyl-terminus: new roles in gap junction organization and wound healing.

Intercellular connectivity mediated by gap junctions (GJs) composed of connexin43 (Cx43) is critical to the function of excitable tissues such as the heart and brain. Disruptions to Cx43 GJ organization are thought to be a factor in cardiac arrhythmias and are also implicated in epilepsy. This article is based on a presentation to the 4th Larry and Horti Fairberg Workshop on Interactive and Integrative Cardiology and summarizes the work of Gourdie and his lab on Cx43 GJs in the heart. Background and perspective of recently published studies on the function of Cx43-interacting protein zonula occludens-(ZO)-1 in determining the organization of GJ plaques are provided. In addition how a peptide containing a PDZ-binding sequence of Cx43, developed as part of the work on cardiac GJ organization is also described, which has led to evidence for novel and unexpected roles for Cx43 in modulating healing following tissue injury.

Zonula occludens-1 alters connexin43 gap junction size and organization by influencing channel accretion.

Regulation of gap junction (GJ) organization is critical for proper function of excitable tissues such as heart and brain, yet mechanisms that govern the dynamic patterning of GJs remain poorly defined. Here, we show that zonula occludens (ZO)-1 localizes preferentially to the periphery of connexin43 (Cx43) GJ plaques. Blockade of the PDS95/dlg/ZO-1 (PDZ)-mediated interaction between ZO-1 and Cx43, by genetic tagging of Cx43 or by a membrane-permeable peptide inhibitor that contains the Cx43 PDZ-binding domain, led to a reduction of peripherally associated ZO-1 accompanied by a significant increase in plaque size. Biochemical data indicate that the size increase was due to unregulated accumulation of gap junctional channels from nonjunctional pools, rather than to increased protein expression or decreased turnover. Coexpression of native Cx43 fully rescued the aberrant tagged-connexin phenotype, but only if channels were composed predominately of untagged connexin. Confocal image analysis revealed that, subsequent to GJ nucleation, ZO-1 association with Cx43 GJs is independent of plaque size. We propose that ZO-1 controls the rate of Cx43 channel accretion at GJ peripheries, which, in conjunction with the rate of GJ turnover, regulates GJ size and distribution.

Quantitative analysis of ZO-1 colocalization with Cx43 gap junction plaques in cultures of rat neonatal cardiomyocytes.

The gap junction (GJ) is an aggregate of intercellular channels that facilitates cytoplasmic interchange of ions, second messengers, and other molecules of less than 1000 Da between cells. In excitable organs such as heart and brain, GJs configure extended intercellular pathways for stable and long-term propagation of action potential. In a previous study in adult rat heart, we have shown that the Drosophila disks-large related protein ZO-1 shows low to moderate colocalization at myocyte borders with the GJ protein Cx43. In the present study, we detail a protocol for characterizing the pattern and level of colocalization of ZO-1 with Cx43 in cultures of neonatal myocytes at the level of individual GJ plaques. The data indicate that ZO-1 shows on average a partial 26.6% overlap (SD = 11.3%) with Cx43 GJ plaques. There is a strong positive correlation between GJ plaque size and area of ZO-1 colocalization, indicating that the level of associated ZO-1 scales with the area of the GJ plaque. Qualitatively, the most prominent colocalization occurs at the plaque perimeter. These studies may provide insight into the presently unknown biological function of ZO-1 interaction with Cx43.

Knockout of the neural and heart expressed gene HF-1b results in apical deficits of ventricular structure and activation.

OBJECTIVE: Knockout of the neural and cardiac expressed transcription factor HF-1b causes electrophysiological abnormalities including fatal ventricular arrhythmias that occur with increasing frequency around the 4th week of postnatal life. This study addresses factors that may contribute to conduction disturbance in the ventricle of the HF-1b knockout mouse. Disruptions to gap junctional connexin40 (Cx40) have been reported in distal (i.e., apically located), but not proximal His-Purkinje conduction tissues of the HF-1b knockout mouse. This abnormality in myocardial Cx40 led us to address whether 4-week-old HF-1b knockout postnates display other disruptions to ventricular structure and function. METHODS: Western blotting and immunoconfocal quantification of Cx43 and coronary arteriole density and function were undertaken in the ventricle. Electrical activation was described by optical mapping. RESULTS: Western blotting and immunoconfocal microscopy indicated that overall levels of Cx43 (p<0.001) and percent of Cx43 localized in intercalated disks (p<0.001) were significantly decreased in the ventricular myocardium of knockouts relative to wildtype littermate controls. Analysis of the reduction in Cx43 level by basal and apical territories revealed that the decrease was most pronounced in the lower, apical half of the ventricle of knockouts relative to controls (p<0.001). Myocyte size also showed a significant decrease in the knockout, that was more marked within the apical half of the ventricle (p<0.05). Optical recordings of ventricular activation indicated apically localized sectors of slowed conduction in knockout ventricles not occurring in controls that could be correlated directly to tissues showing reduced Cx43. These discrete sectors of abnormal conduction in the knockout heart were resolved following point stimulation of the ventricular epicardium and thus were not explained by dysfunction of the His-Purkinje system. To further probe base-to-apex abnormalities in the HF-1b knockout ventricle, we analyzed coronary arterial structure and function. These analyses indicated that relative to controls, the apical ventricular territory of the HF-1b knockout had reductions in the density of small resistance vessels (p<0.01) and deficits in arterial function as assayed by bead perfusion (p<0.01). CONCLUSION: The HF-1b knockout ventricle displays abnormalities in Cx43 level, myocyte size, activation spread and coronary arterial structure and function. These abnormalities tend to be more pronounced in the apical territory of the ventricle and seem likely to be factors contributing to the pathological disturbance of cardiac conduction that characterizes the heart of the HF-1b knockout mouse.

Increased association of ZO-1 with connexin43 during remodeling of cardiac gap junctions.

The intercellular geometry of connexin43 (Cx43) gap junctional coupling is key to coordinated spread of electrical activation through the ventricle of the mammalian heart. A progressive redistribution of electrical and mechanical junctions into intercalated discs occurs during postnatal development. Breakdown of disc-localized pattern in the adult heart, to recapitulate immature distributions, is thought to be key to the genesis of conduction disturbance and arrhythmia. Recently, ZO-1 (a PDZ-MAGUK protein), has been suggested to have a role in generating coupling geometries between myocytes. We therefore investigated the codistribution of ZO-1 with Cx43 and N-cadherin in the adult rat ventricle using quantitative immunoconfocal and immunoelectron microscopy. These analyses indicated that, whereas ZO-1 and Cx43 codistribute within discs, only low to moderate point-by-point colocalization of Cx43 and ZO-1 is found within these domains compared with the relatively high level of colocalization between N-cadherin and ZO-1. By contrast, levels of association between Cx43 and ZO-1 increased rapidly and significantly (P<0.001) after partial or complete enzymatic dissociation of myocytes from intact ventricle--a treatment known to induce gap junction endocytosis. Coimmunoprecipitation using Cx43- and ZO-1-specific antibodies confirmed that significantly (P<0.03) increased ZO-1 is precipitated relative to Cx43 in freshly dissociated myocytes as compared with intact ventricle. On immunoblots, decreases in Cx43 relative mobility, consistent with increased phosphorylation, were observed following myocyte dissociation. The increased ZO-1-Cx43 association that occurs after remodeling of myocyte intercellular contacts indicates the possibility of unanticipated roles for ZO-1 in gap junction turnover during cardiac development and disease processes.