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.

Heterozygous mutations of the cardiac transcription factor Nkx2-5 cause atrioventricular conduction defects in humans by unknown mechanisms. We show in KO mice that the number of cells in the cardiac conduction system is directly related to Nkx2-5 gene dosage. Null mutant embryos appear to lack the primordium of the atrioventricular node. In Nkx2-5 haploinsufficiency, the conduction system has half the normal number of cells. In addition, an entire population of connexin40 -/connexin45 + cells is missing in the atrioventricular node of Nkx2-5 heterozygous KO mice. Specific functional defects associated with Nkx2-5 loss of function can be attributed to hypoplastic development of the relevant structures in the conduction system. Surprisingly, the cellular expression of connexin40, the major gap junction isoform of Purkinje fibers and a putative Nkx2-5 target, is unaffected, consistent with normal conduction times through the His-Purkinje system measured in vivo. Postnatal conduction defects in Nkx2-5 mutation may result at least in part from a defect in the genetic program that governs the recruitment or retention of embryonic cardiac myocytes in the conduction system. 1130The Nonstandard abbreviations used: AV nodal effective refractory period (AVERP); connexin40 (Cx40); embryonic day (E); interventricular septum (IVS); intracardiac electrogram (IEGM); stimulus (S).

Heterozygous mutations of the cardiac transcription factor Nkx2-5 cause atrioventricular conduction defects in humans by unknown mechanisms. We show in KO mice that the number of cells in the cardiac conduction system is directly related to Nkx2-5 gene dosage. Null mutant embryos appear to lack the primordium of the atrioventricular node. In Nkx2-5 haploinsufficiency, the conduction system has half the normal number of cells. In addition, an entire population of connexin40 -/connexin45 + cells is missing in the atrioventricular node of Nkx2-5 heterozygous KO mice. Specific functional defects associated with Nkx2-5 loss of function can be attributed to hypoplastic development of the relevant structures in the conduction system. Surprisingly, the cellular expression of connexin40, the major gap junction isoform of Purkinje fibers and a putative Nkx2-5 target, is unaffected, consistent with normal conduction times through the His-Purkinje system measured in vivo. Postnatal conduction defects in Nkx2-5 mutation may result at least in part from a defect in the genetic program that governs the recruitment or retention of embryonic cardiac myocytes in the conduction system. 1130The Nonstandard abbreviations used: AV nodal effective refractory period (AVERP); connexin40 (Cx40); embryonic day (E); interventricular septum (IVS); intracardiac electrogram (IEGM); stimulus (S).

Ventricular preexcitation, which characterizes Wolff-Parkinson-White syndrome, is caused by the presence of accessory pathways that can rapidly conduct electrical impulses from atria to ventricles, without the intrinsic delay characteristic of the atrioventricular (AV) node. Preexcitation is associated with an increased risk of tachyarrhythmia, palpitations, syncope, and sudden death. Although the pathology and electrophysiology of preexcitation syndromes are well characterized, the developmental mechanisms are poorly understood, and few animal models that faithfully recapitulate the human disorder have been described. Here we show that activation of Notch signaling in the developing myocardium of mice can produce fully penetrant accessory pathways and ventricular preexcitation. Conversely, inhibition of Notch signaling in the developing myocardium resulted in a hypoplastic AV node, with specific loss of slow-conducting cells expressing connexin-30.2 (Cx30.2) and a resulting loss of physiologic AV conduction delay. Taken together, our results suggest that Notch regulates the functional maturation of AV canal embryonic myocardium during the development of the specialized conduction system. Our results also show that ventricular preexcitation can arise from inappropriate patterning of the AV canal-derived myocardium.

Studies of cell lineage in the rat cerebral cortex have provided new insights into the mechanisms of neuronal and glial determination. They have shown that clonally related cells, marked with retrovirus injection at embryonic day 16 (E16), express the same glial or neuronal phenotype, suggesting that separate progenitors for each of these cell phenotypes exist in the ventricular zone at that stage of corticogenesis. However, it is not known if such committed progenitors are present in the ventricular zone before E16. Another important question concerns which neurochemical features are shared by clonally related cells of the adult cerebral cortex. In this study we have addressed the first question by injecting a retroviral vector expressing beta-galactosidase into the telencephalic ventricles of rat embryos at different stages (E14-E19). In order to classify clonally related neurons in the cerebral cortex of these rats, we have used postembedding immunohistochemistry for the amino acid neurotransmitters glutamate, aspartate, and GABA. Glutamate and GABA immunoreactivity marked nonoverlapping populations of cells that corresponded to the pyramidal and nonpyramidal neuron types of the rat cerebral cortex. Clonally related neurons, marked by retrovirus injection at any day between E14 and E19, homogeneously expressed one or other phenotype and accordingly displayed glutamate or GABA immunoreactivity. This finding indicates that committed progenitor cells for pyramidal and nonpyramidal neurons are present in the ventricular zone before E16. To investigate whether lineage dictates other features in clonally related neurons, we performed an immunohistochemical analysis for the calcium- binding proteins calbindin, parvalbumin, and calretinin in clusters of clonally related nonpyramidal neurons. The same calcium-binding protein was rarely found in members of the same cluster, suggesting that lineage does not control the expression of calcium-binding proteins in cortical nonpyramidal neurons. As a result of examining a large number of clonally related neurons from brains injected at different ages, we observed remarkable differences in number and laminar distribution of pyramidal and nonpyramidal neurons marked with retrovirus. Clusters of nonpyramidal neurons were usually composed of two or three cells, and resided in the cortical layers that were just being generated at the time of injection. Clusters of pyramidal neurons were larger and dispersed in several layers in the earlier injections; their size and laminar distribution were progressively reduced for later injections. These observations suggest the existence of different mechanisms that generate the pyramidal and nonpyramidal neurons of the cerebral cortex.