Heart failure is a major killer worldwide. Atrioventricular conduction block is common in heart failure; it is associated with worse outcomes and can lead to syncope and bradycardic death. We examine the effect of heart failure on anatomical and ion channel remodelling in the rabbit atrioventricular junction (AVJ). Heart failure was induced in New Zealand rabbits by disruption of the aortic valve and banding of the abdominal aorta resulting in volume and pressure overload. Laser micro-dissection and real-time polymerase chain reaction (RT-PCR) were employed to investigate the effects of heart failure on ion channel remodelling in four regions of the rabbit AVJ and in septal tissues. Investigation of the AVJ anatomy was performed using micro-computed tomography (micro-CT). Heart failure animals developed first degree heart block. Heart failure caused ventricular myocardial volume increase with a 35% elongation of the AVJ. There was downregulation of HCN1 and Cx43 mRNA transcripts across all regions and downregulation of Cav1.3 in the transitional tissue. Cx40 mRNA was significantly downregulated in the atrial septum and AVJ tissues but not in the ventricular septum. mRNA abundance for ANP, CLCN2 and Navβ1 was increased with heart failure; Nav1.1 was increased in the inferior nodal extension/compact node area. Heart failure in the rabbit leads to prolongation of the PR interval and this is accompanied by downregulation of HCN1, Cav1.3, Cx40 and Cx43 mRNAs and anatomical enlargement of the entire heart and AVJ.
Increased Connexin43 hemichannel opening is associated with inflammasome pathway activation and inflammation in a range of pathologies including ocular disorders, such as age-related macular degeneration (AMD) and diabetic retinopathy (DR). In this study, the effect on retinal function and morphology of clinically safe doses of orally delivered tonabersat, a small molecule connexin hemichannel blocker, was investigated in the light-damaged retina animal model of dry AMD and in a spontaneous rat model of DR. Clinical parameters (fundus imaging, optical coherence tomography (OCT), and electroretinography) and inflammatory markers (immunohistochemistry for Iba-1 microglial marker, astrocyte marker glial fibrillary acidic protein, and Connexin43 protein expression) were assessed. Tonabersat treatment reduced inflammation in the retina in parallel with preservation of retinal photoreceptor function when assessed up to 3 months post light damage in the dry AMD model. In the DR model, clinical signs, including the presence of aneurysms confirmed using Evans blue dye perfusion, were reduced after daily tonabersat treatment for 2 weeks. Inflammation was also reduced and retinal electrical function restored. Tonabersat regulates assembly of the inflammasome (NLRP3) through Connexin43 hemichannel block, with the potential to reduce inflammation, restore vascular integrity and improve anatomical along with some functional outcomes in retinal disease.
The cis benzopyran compound tonabersat (SB-220453) has previously been reported to inhibit connexin26 expression in the brain by attenuating the p38-mitogen-activated protein kinase pathway. We show here that tonabersat directly inhibits connexin43 hemichannel opening. Connexin43 hemichannels have been called "pathological pores" based upon their role in secondary lesion spread, edema, inflammation, and neuronal loss following central nervous system injuries, as well as in chronic inflammatory disease. Both connexin43 hemichannels and pannexin channels released adenosine triphosphate (ATP) during ischemia in an in vitro ischemia model, but only connexin43 hemichannels contributed to ATP release during reperfusion. Tonabersat inhibited connexin43 hemichannel-mediated ATP release during both ischemia and reperfusion phases, with direct channel block confirmed using electrophysiology. Tonabersat also reduced connexin43 gap junction coupling in vitro, but only at higher concentrations, with junctional plaques internalized and degraded via the lysosomal pathway. Systemic delivery of tonabersat in a rat bright-light retinal damage model (a model for dry age-related macular degeneration) resulted in significantly improved functional outcomes assessed using electroretinography. Tonabersat also prevented thinning of the retina, especially the outer nuclear layer and choroid, assessed using optical coherence tomography. We conclude that tonabersat, already given orally to over 1000 humans in clinical trials (as a potential treatment for, and prophylactic treatment of, migraine because it was thought to inhibit cortical spreading depression), is a connexin hemichannel inhibitor and may have the potential to be a novel treatment of central nervous system injury and chronic neuroinflammatory disease.
Catecholaminergic polymorphic ventricular tachycardia (CPVT) predisposes to ventricular arrhythmia due to altered Ca(2+) homeostasis and can arise from ryanodine receptor (RyR2) mutations including RyR2-P2328S. Previous reports established that homozygotic murine RyR2-P2328S (RyR2 (S/S)) hearts show an atrial arrhythmic phenotype associated with reduced action potential (AP) conduction velocity and sodium channel (Nav1.5) expression. We now relate ventricular arrhythmogenicity and slowed AP conduction in RyR2 (S/S) hearts to connexin-43 (Cx43) and Nav1.5 expression and Na(+) current (I Na). Stimulation protocols applying extrasystolic S2 stimulation following 8 Hz S1 pacing at progressively decremented S1S2 intervals confirmed an arrhythmic tendency despite unchanged ventricular effective refractory periods (VERPs) in Langendorff-perfused RyR2 (S/S) hearts. Dynamic pacing imposing S1 stimuli then demonstrated that progressive reductions of basic cycle lengths (BCLs) produced greater reductions in conduction velocity at equivalent BCLs and diastolic intervals in RyR2 (S/S) than WT, but comparable changes in AP durations (APD90) and their alternans. Western blot analyses demonstrated that Cx43 protein expression in whole ventricles was similar, but Nav1.5 expression in both whole tissue and membrane fractions were significantly reduced in RyR2 (S/S) compared to wild-type (WT). Loose patch-clamp studies similarly demonstrated reduced I Na in RyR2 (S/S) ventricles. We thus attribute arrhythmogenesis in RyR2 (S/S) ventricles resulting from arrhythmic substrate produced by reduced conduction velocity to downregulated Nav1.5 reducing I Na, despite normal determinants of repolarization and passive conduction. The measured changes were quantitatively compatible with earlier predictions of linear relationships between conduction velocity and the peak I Na of the AP but nonlinear relationships between peak I Na and maximum Na(+) permeability.
Stem cells with enhanced resistance to oxidative stress after in vitro expansion have been shown to have improved engraftment and regenerative capacities. Such cells can be generated by preconditioning them with exposure to an antioxidant. In this study we evaluated the effects of Tualang honey (TH), an antioxidant-containing honey, on human corneal epithelial progenitor (HCEP) cells in culture. Cytotoxicity, gene expression, migration, and cellular resistance to oxidative stress were evaluated. Immunofluorescence staining revealed that HCEP cells were holoclonal and expressed epithelial stem cell marker p63 without corneal cytokeratin 3. Cell viability remained unchanged after cells were cultured with 0.004, 0.04, and 0.4% TH in the medium, but it was significantly reduced when the concentration was increased to 3.33%. Cell migration, tested using scratch migration assay, was significantly enhanced when cells were cultured with TH at 0.04% and 0.4%. We also found that TH has hydrogen peroxide (H2O2) scavenging ability, although a trace level of H2O2 was detected in the honey in its native form. Preconditioning HCEP cells with 0.4% TH for 48 h showed better survival following H2O2-induced oxidative stress at 50 µM than untreated group, with a significantly lower number of dead cells (15.3 ± 0.4%) were observed compared to the untreated population (20.5 ± 0.9%, p<0.01). Both TH and ascorbic acid improved HCEP viability following induction of 100 µM H2O2, but the benefit was greater with TH treatment than with ascorbic acid. However, no significant advantage was demonstrated using 5-hydroxymethyl-2-furancarboxaldehyde, a compound that was found abundant in TH using GC/MS analysis. This suggests that the cellular anti-oxidative capacity in HCEP cells was augmented by native TH and was attributed to its antioxidant properties. In conclusion, TH possesses antioxidant properties and can improve cell migration and cellular resistance to oxidative stress in HCEP cells in vitro.