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Background α Carboxyl terminus 1 (αCT1) is a 25-amino acid therapeutic peptide incorporating the zonula occludens-1 (ZO-1)-binding domain of connexin 43 (Cx43) that is currently in phase 3 clinical testing on chronic wounds. In mice, we reported that αCT1 reduced arrhythmias after cardiac injury, accompanied by increases in protein kinase Cε phosphorylation of Cx43 at serine 368. Herein, we characterize detailed molecular mode of action of αCT1 in mitigating cardiac ischemia-reperfusion injury. Methods and Results To study αCT1-mediated increases in phosphorylation of Cx43 at serine 368, we undertook mass spectrometry of protein kinase Cε phosphorylation assay reactants. This indicated potential interaction between negatively charged residues in the αCT1 Asp-Asp-Leu-Glu-Iso sequence and lysines (Lys345, Lys346) in an α-helical sequence (helix 2) within the Cx43-CT. In silico modeling provided further support for this interaction, indicating that αCT1 may interact with both Cx43 and ZO-1. Using surface plasmon resonance, thermal shift, and phosphorylation assays, we characterized a series of αCT1 variants, identifying peptides that interacted with either ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 or Cx43-CT, but with limited or no ability to bind both molecules. Only peptides competent to interact with Cx43-CT, but not ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 alone, prompted increased pS368 phosphorylation. Moreover, in an ex vivo mouse model of ischemia-reperfusion injury, preischemic infusion only with those peptides competent to bind Cx43 preserved ventricular function after ischemia-reperfusion. Interestingly, a short 9-amino acid variant of αCT1 (αCT11) demonstrated potent cardioprotective effects when infused either before or after ischemic injury. Conclusions Interaction of αCT1 with the Cx43, but not ZO-1, is correlated with cardioprotection. Pharmacophores targeting Cx43-CT could provide a translational approach to preserving heart function after ischemic injury.
Vancomycin, a commonly used antibiotic, can be nephrotoxic. Known risk factors such as age, creatinine clearance, vancomycin dose / dosing interval, and concurrent nephrotoxic medications fail to accurately predict nephrotoxicity. To identify potential genomic risk factors, we performed a genome-wide association study (GWAS) of serum creatinine levels while on vancomycin in 489 European American individuals and validated findings in three independent cohorts totaling 439 European American individuals. In primary analyses, the chromosome 6q22.31 locus was associated with increased serum creatinine levels while on vancomycin therapy (most significant variant rs2789047, risk allele A, β = -0.06, p = 1.1 x 10(-7)). SNPs in this region had consistent directions of effect in the validation cohorts, with a meta-p of 1.1 x 10(-7). Variation in this region on chromosome 6, which includes the genes TBC1D32/C6orf170 and GJA1 (encoding connexin43), may modulate risk of vancomycin-induced kidney injury.
BACKGROUND - Atrial fibrillation (AF) affects >30 million individuals worldwide and is associated with an increased risk of stroke, heart failure, and death. AF is highly heritable, yet the genetic basis for the arrhythmia remains incompletely understood.
METHODS AND RESULTS - To identify new AF-related genes, we used a multifaceted approach, combining large-scale genotyping in 2 ethnically distinct populations, cis-eQTL (expression quantitative trait loci) mapping, and functional validation. Four novel loci were identified in individuals of European descent near the genes NEURL (rs12415501; relative risk [RR]=1.18; 95% confidence interval [CI], 1.13-1.23; P=6.5×10(-16)), GJA1 (rs13216675; RR=1.10; 95% CI, 1.06-1.14; P=2.2×10(-8)), TBX5 (rs10507248; RR=1.12; 95% CI, 1.08-1.16; P=5.7×10(-11)), and CAND2 (rs4642101; RR=1.10; 95% CI, 1.06-1.14; P=9.8×10(-9)). In Japanese, novel loci were identified near NEURL (rs6584555; RR=1.32; 95% CI, 1.26-1.39; P=2.0×10(-25)) and CUX2 (rs6490029; RR=1.12; 95% CI, 1.08-1.16; P=3.9×10(-9)). The top single-nucleotide polymorphisms or their proxies were identified as cis-eQTLs for the genes CAND2 (P=2.6×10(-19)), GJA1 (P=2.66×10(-6)), and TBX5 (P=1.36×10(-5)). Knockdown of the zebrafish orthologs of NEURL and CAND2 resulted in prolongation of the atrial action potential duration (17% and 45%, respectively).
CONCLUSIONS - We have identified 5 novel loci for AF. Our results expand the diversity of genetic pathways implicated in AF and provide novel molecular targets for future biological and pharmacological investigation.
© 2014 American Heart Association, Inc.
Helicobacter pylori colonizes the human stomach and confers an increased risk for the development of peptic ulceration, noncardia gastric adenocarcinoma, and gastric lymphoma. A secreted H. pylori toxin, VacA, can cause multiple alterations in gastric epithelial cells, including cell death. In this study, we sought to identify host cell factors that are required for VacA-induced cell death. To do this, we analyzed gene trap and short hairpin RNA (shRNA) libraries in AZ-521 human gastric epithelial cells and selected for VacA-resistant clones. Among the VacA-resistant clones, we identified multiple gene trap library clones and an shRNA library clone with disrupted expression of connexin 43 (Cx43) (also known as gap junction protein alpha 1 [GJA1]). Further experiments with Cx43-specific shRNAs confirmed that a reduction in Cx43 expression results in resistance to VacA-induced cell death. Immunofluorescence microscopy experiments indicated that VacA did not colocalize with Cx43. We detected production of the Cx43 protein in AZ-521 cells but not in AGS, HeLa, or RK-13 cells, and correspondingly, AZ-521 cells were the most susceptible to VacA-induced cell death. When Cx43 was expressed in HeLa cells, the cells became more susceptible to VacA. These results indicate that Cx43 is a host cell constituent that contributes to VacA-induced cell death and that variation among cell types in susceptibility to VacA-induced cell death is attributable at least in part to cell type-specific differences in Cx43 production.
RATIONALE - The Ca(2+) sensitivity of the myofilaments is increased in hypertrophic cardiomyopathy and other heart diseases and may contribute to a higher risk for sudden cardiac death. Ca(2+) sensitization increases susceptibility to reentrant ventricular tachycardia in animal models, but the underlying mechanism is unknown.
OBJECTIVE - To investigate how myofilament Ca(2+) sensitization creates reentrant arrhythmia susceptibility.
METHODS AND RESULTS - Using hypertrophic cardiomyopathy mouse models (troponinT-I79N) and a Ca(2+) sensitizing drug (EMD57033), here we identify focal energy deprivation as a direct consequence of myofilament Ca(2+) sensitization. To detect ATP depletion and thus energy deprivation, we measured accumulation of dephosphorylated Connexin 43 (Cx43) isoform P0 and AMP kinase activation by Western blotting and immunostaining. No differences were detected between groups at baseline, but regional accumulation of Connexin 43 isoform P0 occurred within minutes in all Ca(2+)-sensitized hearts, in vivo after isoproterenol challenge and in isolated hearts after rapid pacing. Lucifer yellow dye spread demonstrated reduced gap junctional coupling in areas with Connexin 43 isoform P0 accumulation. Optical mapping revealed that selectively the transverse conduction velocity was slowed and anisotropy increased. Myofilament Ca(2+) desensitization with blebbistatin prevented focal energy deprivation, transverse conduction velocity slowing, and the reentrant ventricular arrhythmias.
CONCLUSIONS - Myofilament Ca(2+) sensitization rapidly leads to focal energy deprivation and reduced intercellular coupling during conditions that raise arrhythmia susceptibility. This is a novel proarrhythmic mechanism that can increase arrhythmia susceptibility in structurally normal hearts within minutes and may, therefore, contribute to sudden cardiac death in diseases with increased myofilament Ca(2+) sensitivity.
The ryanodine receptors (RyRs) are a class of intracellular calcium release channels of which there are three isoforms. In striated muscle, isoform 1 and isoform 2 are mainly expressed in the terminal cisternae of sarcoplasmic reticulum of skeletal muscle and heart, respectively. Isoform 3 is widely distributed in tissues but in minuscule amounts. These channels release calcium ions from intracellular stores in excitation-contraction coupling for cell signaling. Here, we report the presence of skeletal muscle isoform 1 localized in the intercalated discs (IDs) of human and mouse hearts. By using RyR1 and connexin43 specific antibodies and dual immunofluorescent techniques, both were localized in the proximity of the IDs of human and mouse hearts. We confirmed that RyR1 is localized to the IDs by selective immunoprecipitation of RyR isoform 1 from a subcellular fraction containing IDs from human heart tissue. The functional significance of our observation remains to be elucidated as isoform 1 is involved in depolarization induced calcium release, unlike RyR isoforms 2 and 3 which appear to be involved in calcium induced calcium release.
Normal septation of the cardiac outflow tract requires migration of neural crest cells from the posterior rhombencephalon to the branchial arches and developing conotruncal endocardial cushions. Proper migration of these cells is mediated by a variety of molecular cues. Adhesion molecules, such as integrins, are involved in the interaction of neural crest cells with the extracellular matrix, while cadherins allow neural crest cells to interact with each other during their migration. Pax3 appears to be important for proliferation of neural crest precursors, and connexin-43-mediated gap junction communication influences the rate of migration. Endothelin and its receptors are required for normal postmigratory differentiation. Platelet-derived growth factor and retinoic acid have roles in neural crest migration and differentiation as well. Finally, the similarity between the cardiovascular malformations seen in the DiGeorge and 22q11 deletion syndromes and animal models of neural crest deficiency has led to the examination of the role of genes located near or within the DiGeorge critical region in neural crest migration.
Electrical synapses, or gap junctions, are widely distributed in the vertebrate retina and are thought to play critical roles in the transmission and coding of visual signals. To investigate the molecular basis of this form of neural communication in the retina, we have isolated, characterized, and functionally expressed a cDNA for a gap junction channel derived from the retina of the teleost fish Danio aquipinnatus (giant danio). The cDNA contained an open reading frame of 1146 nucleotides encoding a connexin with a predicted molecular mass of 43.3 kDa which shared extensive identity with Rattus norvegicus Cx43 (78%). This protein (DACX43) contained several consensus phosphorylation sequences in the c-terminal region, some of which are conserved among Cx43 orthologs. RNA blot hybridization revealed that DACX43 was expressed in the brain as well as in the retina. In addition, Southern analysis suggested that there are multiple copies of DACX43, or other closely related sequences, in the Danio aquipinnatus genome. When DACX43 was expressed by stable transfection in gap-junction-deficient mouse N2A neuroblastoma cells, functional gap junctions were formed as indicated by dual whole-cell recordings of electrical coupling. We conclude that DACX43 is a connexin43 ortholog, which is expressed in the retina of Danio aquipinnatus, and when translated is able to form functional gap junction channels.