Relative Contributions of Connexins 40 and 43 to Atrial Impulse Propagation in Synthetic Strands of Neonatal and Fetal Murine Cardiomyocytes

Abstract

Abstract-Atrial tissue expresses both connexin 40 (Cx40) and 43 (Cx43) proteins. To assess the relative roles of Cx40 and Cx43 in atrial electrical propagation, we synthesized cultured strands of atrial myocytes derived from mice with genetic deficiency in Cx40 or Cx43 expression and measured propagation velocity (PV) by high-resolution optical mapping of voltage-sensitive dye fluorescence. The amount of Cx40 and/or Cx43 in gap junctions was measured by immunohistochemistry and total or sarcolemmal Cx43 or Cx40 protein by immunoblotting. Progressive genetic reduction in Cx43 expression decreased PV from 34Ϯ6 cm/sec in Cx43 ϩ/ϩ to 30Ϯ8 cm/sec in Cx43 ϩ/Ϫ and 19Ϯ11 cm/sec in Cx43 Ϫ/Ϫ cultures. Concomitantly, the cell area occupied by Cx40 immunosignal in gap junctions decreased from 2.0Ϯ1.6% in Cx43 ϩ/ϩ to 1.7Ϯ0.5% in Cx43 ϩ/Ϫ and 1.0Ϯ0.2% in Cx43 Ϫ/Ϫ strands. In contrast, progressive genetic reduction in Cx40 expression increased PV from 30Ϯ2 cm/sec in Cx40 ϩ/ϩ to 40Ϯ7 cm/sec in Cx40 ϩ/Ϫ and 45Ϯ10 cm/sec in Cx40 Ϫ/Ϫ cultures. Concomitantly, the cell area occupied by Cx43 immunosignal in gap junctions increased from 1.2Ϯ0.9% in Cx40 ϩ/ϩ to 2.8Ϯ1.4% in Cx40 ϩ/Ϫ and 3.1Ϯ0.6% in Cx40 Ϫ/Ϫ cultures. In accordance with the immunostaining results, immunoblots of the Triton X-100 -insoluble fraction revealed an increase of Cx43 in gap junctions in extracts from Cx40-ablated atria, whereas total cellular Cx43 remained unchanged. Our results suggest that the relative abundance of Cx43 and Cx40 is an important determinant of atrial impulse propagation in neonatal hearts, whereby dominance of Cx40 decreases and dominance of Cx43 increases local propagation velocity. (Circ Res. 2006;99:1216-1224.)Key Words: atrial myocyte Ⅲ basic science Ⅲ cardiac gap junction connexins Ⅲ cardiovascular genomics Ⅲ cell culture Ⅲ conduction velocity Ⅲ connexin 40 Ⅲ connexin 43 Ⅲ mapping Ⅲ neonatal mouse cardiomyocytes Ⅲ optical mapping C onnexin (Cx) proteins enable the intercellular exchange of ions and small regulatory molecules and are important determinants of cardiac electrical propagation. 1 Three major connexins, Cx43, Cx40, and Cx45, are expressed in heart. 2,3 Cx43 is abundant in ventricular and atrial myocardium; Cx40 is expressed in atrial tissue and in the Purkinje system; Cx45 is present in the sinoatrial (SA) and atrioventricular (AV) nodes and colocalizes with Cx43 in ventricular myocardium. 4,5 Cx43 and Cx40 each form channels with relatively large pores, whereas Cx45 forms narrow channels (unitary channel conductances of 75, 130, and 30 pS, respectively). 6 Ventricular myocytes express abundant Cx43 and small amounts of Cx45. In contrast, atrial myocytes express large amounts of Cx43 and Cx40. In ventricular myocytes and transfected cells, Cx43 and Cx45 colocalize in gap junctions and may form heteromeric/heterotypic channels. 5,7-10 Heterotypic Cx43/Cx40 gap junction channels have been described in HeLa cell pairs, whereas the role of heteromeric Cx43/ Cx40 channels has not been fully clarified. 7,11 Changes in cell-to-cell...

Engineering Cardiac Cell JunctionsIn Vitroto Study the Intercalated Disc

“…In an earlier study, we engineered strands from murine atrial tissue using photomicrolithography to show that linear propagation velocity at a cellular level was markedly decreased with Cx43 ablation and signifi cantly increased versus WT with Cx40 ablation (Beauchamp et al, 2006). These fi ndings were in seeming confl ict with some of the aforementioned studies in whole mice, but in accordance with studies in human hearts.…”

“…This complexity might have several components: There could be interaction (1) between the atrial connexins at the level of expression at the ID, (2) at the level of gap junction channels with formation of heteromeric hemichannels (or connexons) and mixed gap junctions channels composed of all three atrial Cx subspecies, and (3) between connexins and expression of other functional complexes, such as ion channels. Thus, in our earlier work, we could show that the area occupied by Cx40 at the ID decreased with Cx43 ablation, whereas the Cx43 immunofl uorescence signal increased with Cx40 ablation (Beauchamp et al, 2006). These changes occurred with a constant cellular pool of Cx40 and Cx43 proteins, as demonstrated by whole cell Western blots, suggesting the possibility of interaction at the level of traffi cking.…”

“…This result points to an interaction between Cx40, Cx45, and Cx43 at the level of traffi cking. This is because whole cell levels of Cx43 and Cx40 proteins remained unchanged in the case of either Cx43 or Cx40 genetic ablation (Beauchamp et al, 2006). A defect of Cx40 and Cx43 traffi cking has been shown in experiments involving the co-expression of mutated Cx40 protein (rare somatic mutation found in patients with lone atrial fi brillation) with wild-type Cx43 (Gollob et al, 2006).…”

Reversible atrial gap junction remodeling during hypoxia/reoxygenation and ischemia: a possible arrhythmogenic substrate for atrial fibrillation

“…In this setting, alterations of cardiac connexins may be looked up as an important mechanism underlying the onset and/or the maintenance of the arrhythmia. Beauchamp et al (2006) demonstrated that connexins Cx40 and Cx43 play a key role in the propagation of atrial impulse. Some studies on human atrial samples of patients with chronic AF reported redistribution of Cx43 and Cx40 labeling towards the lateral borders of atrial myocytes and decreased levels of Cx43 per myocyte in both right atrial appendage and lateral Quantitative densitometric analysis of Cx43 protein at different time points (0, 24, 48 h of hypoxia).…”

“…While regulation of ventricular connexin has been investigated in several in vitro models, atrial connexin have been poorly investigated (Beauchamp et al 2006). In this study, we evaluated connexin expression and distribution in atrial cardiomyocytes (HL-1 cells) and the effects of hypoxia/reoxygenation, ischemia in association with normoxia and hypoxia, pro-inflammatory stimuli, neurotransmitters and angiotensin II (Ang II) on Cx43 and Cx40 in the same cells.…”