Aim-Gap-junctional connexin43 (Cx43) has roles in multiple aspects of skin wound healingincluding scarring. The aim here was to study the effects of a cell-permeant peptide from the Cx43 carboxyl-terminus (CT) on scarring and regeneration following cutaneous injury. Materials & methods-The effects of Cx43 CT peptide were studied in mouse and pig models of cutaneous injury. The parameters assessed included neutrophil density, wound closure, granulation, regeneration and skin tensile properties. Results-Cx43CT-peptide prompted decreases in area of scar progenitor tissue and promoted restoration of dermal histoarchitecture and mechanical strength following wounding of skin. These changes in healing were preceded by peptide-induced reduction in inflammatory neutrophil infiltration and alterations in the organization of epidermal Cx43, including increased connexon aggregation. Conclusion-Cx43CT peptide promotes regenerative healing of cutaneous wounds and may have applications in tissues other than skin, including heart, cornea and spinal cord. KeywordsCx43; gap junctions; regenerative medicine; scar prevention; wound healing Correspondence to: Robert G Gourdie. Future perspectiveScarring and abrogated histocomplexity are normal sequalae of trauma, including that resulting from implanted materials such as tissueengineered scaffolds [55]. In ongoing work we are testing whether Cx43 CT peptides have therapeutic applications in implant biocompatibility, as well as in promoting regenerative healing of injured heart, cornea, retina and spinal cord. Ethical conduct of researchThe authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved. NIH Public Access Author ManuscriptRegen Med. Author manuscript; available in PMC 2010 January 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe gap junction (GJ) is an aggregate of intercellular channels that provides for direct cell-tocell interchange of metabolites, ions and other small molecules of approximately 1000 Da or less [1,2]. The individual channels of GJ aggregates in vertebrates are composed of integral membrane proteins encoded by the connexin family of genes, of which more than 20 members have been recognized [3]. It is well established that GJs, and the connexin subunits of GJs, function in various aspects of wound healing [4][5][6]. Reports for such functions have included roles for GJs and connexins in intercellular spread of injury signal, immune cell extravasation, inflammation, re-epithelization, wound closure and formation of the scar progenitor, granulation tissue. Intercellular communication is thought to be one aspect of GJ-based mechanisms regulating and affecting the response of tissues to injury [4,[6][7][8][9][10]. There is also mounting evidence th...

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Rationale Remodeling of connexin-43 (Cx43) gap junctions (GJs) is linked to ventricular arrhythmia. Objectives A peptide mimetic of the carboxyl-terminal (CT) of Cx43, incorporating a Post-synaptic-density/Disks-large/ZO-1 (PDZ)-binding domain, reduces Cx43/ZO-1 interaction and GJ size remodeling in vitro. Here, we determined: 1] Whether the Cx43-CT mimetic αCT1 altered GJ remodeling following left-ventricular (LV) injury in vivo, 2] If αCT1 affected arrhythmic propensity, and 3] The mechanism of αCT1 effects on arrhythmogenicity and GJ remodeling. Methods and Results A cryoinjury model generating a reproducible wound and injury border zone (IBZ) in the LV was used. Adherent methylcellulose patches formulated to locally release αCT1 (<48-hours) were placed on cryoinjuries. Relative to controls, Cx43/ZO-1 colocalization in the IBZ was reduced by αCT1 by 24-hours post-injury. Programmed electrical stimulation ex vivo and optical mapping of voltage-transients indicated that peptide-treated hearts showed reduced inducible-arrhythmias and increased ventricular depolarization rates 7-9 days post-injury. At 24-hours and 1-week post-injury, αCT1-treated hearts maintained Cx43 in intercalated disks (IDs) in the IBZ, whereas by 1-week post-injury controls demonstrated Cx43-remodeling from IDs to lateralized distributions. Over a post-injury time-course of 1-week, αCT1-treated IBZs showed increased Cx43 phosphorylation at serine368 (Cx43-pS368) relative to control tissues. In biochemical assays, αCT1 promoted phosphorylation of serine368 by PKC-ε in a dose-dependent manner that was modulated by, but did not require ZO-1 PDZ2. Conclusion αCT1 increases Cx43-pS368 in vitro in a PKC-ε-dependent manner and in the IBZ in vivo acutely following ventricular injury. αCT1-mediated increase in Cx43-pS368 phosphorylation may contribute to reductions in inducible-arrhythmia following injury.

A major limitation in studies of the injured heart is animal-to-animal variability in wound size resulting from commonly used techniques such as left anterior descending coronary artery ligation. This variability can make standard errors sufficiently large that mean separation between treatment and control groups can be difficult without replicating numbers (n) of animals in groups by excessive amounts. Here, we describe the materials and protocol necessary for delivering a standardized non-transmural cryoinjury to the left ventricle of an adult mouse heart that may in part obviate the issue of injury variance between animals. As reported previously, this cryoinjury model generates a necrotic wound to the ventricle of consistent size and shape that resolves into a scar of uniform size, shape, and organization. The cryo-model also provides an extended injury border zone that exhibits classic markers of remodeling found in surviving cardiac tissue at the edge of a myocardial infarction, including connexin43 (Cx43) lateralization. In a further extension of the method, we describe how we have adapted the model to deliver a cryoinjury to the apex of the heart of neonatal mice-a modification that may be useful for studies of myocardial regeneration in mammals.

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.N-cadherin; connexin-43; postsynaptic density 95/Drosophila disk large/zonula occludens-1; heart THE INTERCALATED DISK (ICD) is a specialized domain of electromechanical coupling between myocytes (6, 31). The disk incorporates three types of intercellular junctions: adherens junctions (AJs), desmosomes, and gap junctions (GJs). AJs and desmosomes provide for cadherin-mediated adhesion between myocytes and serve as a platform for cytoskeletal attachment. GJs are aggregates of cell-to-cell channels that mediate the exchange of small molecules (Ͻ1,000 Da) between cells and in the myocardium conduct the ion transients responsible for the intercellular propagation of the cardiac action potential. Based on early electron microscopic studies, the ICD was perceived more as a locus at which multiple junctional types were found rather than as an integrated unitary structure in its own right. However, the disk is now recognized as a functional unit specialized for electromechanical coupling in which AJs, GJs, and desmosomes, together with cytoskeletal proteins, are interlinked via a web of protein-protein interactions (31, 32).Over postnatal ventricular development, the remodeling of electromechanical junctions from lateral, side-by-side contacts between myocytes results in a preferential accumulation of AJs, desmosomes, and GJs at ICDs in the adult (2, 13, 16). The average size of GJs also increases during the postnatal growth of the ventricle (16,36). A distinct population ...