Faculty Bibliography

OBJECTIVES: We sought to quantify the number and length of desmosomes, gap junctions, and adherens junctions in arrhythmogenic right ventricular cardiomyopathy (ARVC) and non-ARVC dogs, and to determine if ultrastructural changes existed. ANIMALS: Hearts from 8 Boxer dogs afflicted with histopathologically confirmed ARVC and 6 dogs without ARVC were studied. METHODS: Quantitative transmission electron microscopy (TEM) and Western blot semi-quantification of alpha-actinin were used to study the intercalated disc and sarcomere of the right and left ventricles. RESULTS: When ARVC dogs were compared to non-ARVC dogs reductions in the number of desmosomes (P = 0.04), adherens junctions (P = 0.04) and gap junctions (P = 0.02) were found. The number of gap junctions (P = 0.04) and adherens junctions (P = 0.04) also were reduced in the left ventricle, while the number of desmosomes was not (P = 0.88). A decrease in the length of desmosomal complexes within LV samples (P = 0.04) was found. These findings suggested disruption of proteins providing attachment of the cytoskeleton to the intercalated disc. Immunoblotting did not demonstrate a quantitative reduction in the amount of alpha-actinin in ARVC afflicted samples. All Boxers with ARVC demonstrated the presence of electron dense material originating from the Z band and extending into the sarcomere, apparently at the expense of the cytoskeletal structure. CONCLUSIONS: These results emphasize the importance of structural integrity of the intercalated disc in the pathogenesis of ARVC. In addition, observed abnormalities in sarcomeric structure suggest a novel link between ARVC and the actin-myosin contractile apparatus

Gap junctional channels are comprised of protein subunits called connexins. Connexin43 (Cx43) is abundantly expressed in astrocytes and is involved in metabolite supply of neurons and extracellular potassium ion homeostasis, thus modulating neuronal activity. In the hippocampus, Cx43 is also expressed in neural stem cells of the subgranular zone (SGZ) in the dentate gyrus (DG). Throughout adult life, the socalled radial glia- (RG-) like cells give rise to new neurons which reside in the granule cell layer of the DG. We have previously found that the astroglial connexins Cx43 and Cx30 are required for the proliferative activity of RG-like cells and adult neurogenesis. Connexins mediate not only intercellular exchange of small molecules but also adhesive interaction between cells via the cytoplasmic C-terminal tail which might be required for neurogenesis. To gain further mechanistic insight into connexin function related to adult neurogenesis, we employed Cx43 mutations: Mice expressing the point mutation Cx43G138R exhibit selective loss of intercellular coupling, but this mutant still contains the C terminus, so that adhesion exerted by Cx43 should be preserved. Cx43K258stop mice carry a C-terminal truncation of Cx43, which still shows intercellular coupling, at least in transfected Hela cells, but adhesive interactions should be deficient. Astroglia-specific Cx43G138R mice lacking Cx30 showed complete absence of interastrocytic coupling in the hippocampus, as expected. To our surprise, astroglia-specific Cx43K258Stop mice lacking Cx30 also exhibited uncoupling, contrary to the studies in cultured cells. With immunohistochemistry, we found that both the proliferative activity in the SGZ and the number of newborn neurons in the DG were significantly decreased in astroglia-specific Cx43G138R and Cx43K258stop mice. Our findings strongly favor a role of Cx43-mediated intercellular coupling between RG-like cells rather than Cx43-mediated adhesive interactions for adult neurogenesis in the hippocampus

Formins dynamically regulate actin microfilament assembly, influencing processes such as cell division, signal transduction, migration, and cell-cell contact formation. The actin cytoskeleton affects ion channel activity and formation of mechanical cell-cell junctions. As in vitro differentiation of embryonic stem cells (ESC) results in phenotypical immature cardiomyocytes, differences in formin expression might reflect alterations to the actin cytoskeleton of adult cardiomyocytes. Objective: To compare formin expression during heart development to the profile of in vitro differentiated cardiomyocytes. Methods: Samples: ESC and cardiac tissue from embryonic day 14.5, neonatal day 2 or 2 month old mice of the 129P2 strain (heart development); spontaneously contracting cardiomyocytes differentiated from 129P2 ESC (in vitro differentiation). Transcript analysis performed by qRT-PCR analysis (15 mouse formin genes, 2 stemness genes, 4 cardiomyocyte differentiation genes). Expression of candidate formin proteins was analyzed using commercially available Diap1 and Fhod1 antibodies. Results: qRT-PCR analysis revealed significant changes in formin expression for 9 of 15 formins during heart development. Four patterns of transcript changes were observed: formins expressed in ESC and decreasing during development (Diap1, Diap3); transcripts peeking during fetal heart development (Fhod1, Fmnl1, Grid2ip); transcripts altered during heart development compared to ESC and adult heart (Daam1, Daam2, Fmnl2, Fmnl3); transcript increasing during development (Fhod3). In vitro differentiated cardiomyocytes revealed significant increases in expression of embryonic formins (Grid2ip, Fhod1, Fmnl2) and formins otherwise not differentially expressed during heart development (Diap2, Fmn1, Fmn2, Inf2). Expression of Fhod1 and Diap1 protein was studied in ESC, neonatal cardiomyocytes and in vitro differentiated cardiomyocytes, verifying the observed changes in transcript expression. Conclusion: Formins are dynamically regulated during cardiac development. Modifying formin expression of in vitro differentiated cardiomyocytes could improve functional maturity and therefore their potential use in cell replacement therapy for cardiac repair

Connexin43 plays an important role in neuroprotection in experimental stroke models; reducing the expression of this gap junction protein in astrocytes enhances injury upon middle cerebral artery occlusion (MCAO). Because the C-terminal region of connexin43 isimportant for channel activity, we carried out MCAO stroke experiments in mice expressing a truncated form of connexin43 (Cx43DeltaCT mice). Brain sections were analyzed for infarct volume, astrogliosis, and inflammatory cell invasion 4 days after MCAO. Adult cortices and astrocyte cultures were examined for connexin43 (Cx43) expression by immunohistochemistry and Western blot. Cultured astrocytes were also examined for dye coupling, channel conductance, hemichannel activity, and Ca wave propagation. The Cx43DeltaCT mice exhibit enhanced cerebral injury after stroke. Astrogliosis was reduced and inflammatory cell invasion was increased inthe peri-infarct region in these mice compared with controls; Cx43 expression was also altered. Lastly, cultured astrocytes from Cx43DeltaCT mice were less coupled and displayed alterations in channel gating, hemichannel activity, and Ca wave properties. These results suggest that astrocytic Cx43 contributed to the regulation of cell death after stroke and support the view that the Cx43 C-terminal region is important in protection in cerebral ischemia

AIMS: Hearts of mice expressing K258stop in place of connexin43 (Cx43) protein were subjected to acute myocardial infarction in order to assess the importance of Cx43 regulation on infarct size and arrhythmia susceptibility. This mutation K258stop prevents chemical regulation of Cx43 channels, including by low intracellular pH. METHODS AND RESULTS: Langendorff-perfused hearts of mice harbouring one Cx43 knockout (KO) allele and one K258stop or Cx43 allele (K258stop/KO; Cx43/KO as control) were subjected to 1 h of ischaemia and 4 h of reperfusion by reversibly occluding the left anterior descending (LAD) coronary artery. Inducibility of ventricular tachyarrhythmias (VTs) was tested by applying an endocardial burst-pacing protocol during LAD occlusion. Separately, time course and the extent of acidification-induced closure of gap junction channels were tested by dual-voltage clamp. Infarct volume (as per cent of area at risk) was significantly larger in K258stop/KO hearts compared with Cx43/KO controls (42.2 +/- 3 vs. 30.4 +/- 1.7%, P = 0.004, n = 8 each). During LAD occlusion, K258stop/KO hearts had a higher incidence of pacing-induced VT and a higher frequency of occurrence of spontaneous premature ventricular beats. The occurrence of ventricular arrhythmias was also significantly larger in the K258stop/KO hearts during reperfusion. In separate experiments, we demonstrated reduced sensitivity to acidification-induced uncoupling in cell pairs obtained from K258stop/KO hearts. CONCLUSION: Loss of the regulatory domain of Cx43 leads to an increase in infarct size and increased susceptibility to arrhythmias following acute coronary occlusion

Transmigration of neutrophils through the microvascular endothelium is a cardinal event of acute inflammation. It has been suggested that gap junctions made of connexin43 (Cx43) may serve as a conducting pathway to spread inflammatory signals within the lung capillary network. To determine whether Cx43 contributes to neutrophil transmigration in vivo, the number of transmigrated neutrophils was monitored in lungs of Cx43 mouse models subjected to inflammation by intratracheal instillations of Pseudomonas aeruginosa lipopolysaccharide (LPS). Cx43 was detected in inflamed lungs independently of neutrophil recruitment, whereas Cx43 up-regulation was not detected in mice genetically protected from inflammation. Mice heterozygous for the Cx43 gene (gja1) showed a 56% (P < 0.01) reduction in airway neutrophil count. In contrast, increased (P < 0.05) neutrophil recruitment in response to LPS was observed in a mouse model expressing a mutant Cx43 with enhanced channel conductivity. In vitro adhesion assays showed that reduced conductivity of Cx43 channels with (43)Gap26, a Cx43 blocking peptide, decreased adhesion of neutrophils to endothelial cells. Finally, we found that instillation of (43)Gap26 in inflamed lungs reduced neutrophil transmigration by 65% (P < 0.05). These results indicate that inflammatory mediators up-regulate alveolar Cx43 to promote neutrophil recruitment to the airspace. Cx43 may therefore represent a pharmacological target in lung diseases characterized by excessive neutrophil recruitment to the airways

During brain development, young neurons closely associate with radial glial while migrating from the ventricular zone (VZ) to the cortical plate (CP) of the neocortex. It has been shown previously that gap junctions are needed for this migration to occur properly, but the precise mechanism responsible is still in question. Here, we used Cre recombinase, driven by the nestin promoter, to conditionally knock-out a floxed coding DNA of the connexin43 (Cx43) gene in mice. Radial glia in the VZ normally express connexin43. They undergo divisions that produce neurons and astrocytes and serve as migratory guides for the daughter cells that they produce. Based on histological analysis, we suggest that removing Cx43 from radial glia alters the normal lamination of the mouse neocortex. To monitor newborn neurons during development, we introduced a plasmid containing green fluorescent protein driven by a neuronal (Talpha1 tubulin) promoter into the embryonic neocortex using in utero electroporation. The transfected migrating neurons remain in the VZ/intermediate zone (IZ) of the Cx43 conditional knock-out (Cx43cKO) animals, whereas in Cx43(fl/fl) mice, neurons migrate through the IZ into the CP, indicating that deletion of Cx43 from nestin-positive cells disrupts neuronal migration. We were able to rescue migration of Cx43cKO neurons by electroporating a cytomegalovirus-Cx43 expression plasmid into the embryonic cortex. In contrast, a C-terminal truncated form of Cx43 failed to rescue neuronal migration. In addition, Cx43(K258stop) mice, in which Cx43 lacks the last 125 amino acid residues of the cytoplasmic C-terminal domain, gave results similar to those seen with the Cx43cKO mice. This study illustrates that deletion of the C-terminal domain of Cx43 alters neuronal migration in the neocortex

Introduction: Connexin43 (Cx43) gap junction channels close during myocardial infarction (MI). To assess the importance of Cx43 regulation on arrhythmia susceptibility, mice in which the coding region of Cx43 was replaced with a mutation lacking most of the carboxyl-terminal domain (K258stop) were subjected to MI. This mutation has been shown to prevent chemical regulation of Cx43 channels by low intracellular pH in vitro. Due to reduced viability of homozygous K258stop mice, studies were carried out in animals harboring one Cx43 knockout allele and one K258stop or Cx43 allele, respectively (i.e., K258stop/KO; Cx43/KO). Methods: Langendorff-perfused hearts (n=12 per group) were subjected to 1 hour of ischemia and 4 hours of reperfusion by reversibly occluding the left anterior descending (LAD) coronary artery. Hearts were monitored for spontaneous ventricular tachyarrhythmias (VT) and for inducibility of VT by endocardial burst pacing near the apex of the left ventricle (3 x 18 S1 stimuli; 80, 60, 40 and 20ms cycle length; 2.5 times threshold) 15, 30, 45 and 60 minutes after the onset of LAD occlusion. Results: 45 minutes after the onset of LAD occlusion, VT could be induced by at least one pacing frequency in 81.8% of K258stop/KO hearts. The number increased to 100% at 60 minutes. For Cx43/KO hearts only 41.7% and 75% of the hearts developed VT at 45 min and 60 min, respectively. The average number of VT events elicited (regardless of burst pacing frequency) was significantly higher in the K258stop/KO hearts (2.64 +/- 0.56 v. 0.83 +/- 0.39 at 45min; p: 0.014; 3.18 +/- 0.55 v.1.67 +/- 0.47 at 60min; p= 0.047). VT episodes were also of longer duration in the K258stop/KO group. During reperfusion, K258stop/KO hearts showed a higher incidence of spontaneous VT (85.7% v. 42.9% of hearts) and increased numbers of episodes (7.14 +/- 2.27 v. 1.57 +/- 0.95; p=0.043). Conclusions: Loss of the regulatory domain of Cx43 leads to an increased susceptibility to arrhythmias following acute coronary occlusion. Whether similar results would be obtained when Cx43 channels remain open but structurally intact remains to be determined

Haplodeficient mice expressing carboxyl-terminally truncated Cx43 (K258stop/KO), instead of the wild-type Cx43 isoform, reach adulthood and reveal no abnormalities in heart morphology. Here, we have analyzed the expression of K258stop protein and the morphology of gap junctions in adult hearts of these mice. Coimmunofluorescence analysis revealed reduced juxtaposition of K258stop with other junctional proteins at the intercalated disc. Immunoprecipitation studies documented changes in the interaction with previously described Cx43 binding proteins. Quantitative transmission electron and confocal microscopy confirmed the localization of K258stop gap junctions to the periphery of the intercalated disc and further revealed an increase in the size of K258stop gap junction plaques and a reduction in their number. Dual whole cell patch clamp analysis confirmed that K258stop gap junctions were functional, with single channel properties similar to those described in exogenous systems. We conclude that the carboxyl-terminal domain of Cx43 (Cx43CT) is involved in regulating the localization, number and size of Cx43 plaques in vivo. Conversely, protein interactions or posttranslational modifications taking place within the Cx43CT are not required for the assembly of functional gap junctions in the intercalated disc