Electrical Propagation in Synthetic Ventricular Myocyte Strands From Germline Connexin43 Knockout Mice

Abstract

Abstract-To characterize the role of connexin43 (Cx43) as a determinant of cardiac propagation, we synthesized strands and pairs of ventricular myocytes from germline Cx43 Ϫ/Ϫ mice. The amount of Cx43, Cx45, and Cx40 in gap junctions was analyzed by immunohistochemistry and confocal microscopy. Intercellular electrical conductance, g j , was measured by the dual-voltage clamp technique (DVC), and electrical propagation was assessed by multisite optical mapping of transmembrane potential using a voltage-sensitive dye. Compared with wild-type (Cx43 ϩ/ϩ ) strands, immunoreactive signal for Cx43 was reduced by 46% in Cx43 ϩ/Ϫ strands and was absent in Cx43 Ϫ/Ϫ strands. Cx45 signal was reduced by 46% in Cx43 ϩ/Ϫ strands and to the limit of detection in Cx43 Ϫ/Ϫ strands, but total Cx45 protein levels measured in immunoblots of whole cell homogenates were equivalent in all genotypes. Cx40 was detected in Ϸ 2% of myocytes. Intercellular conductance, g j , was reduced by 32% in Cx43 ϩ/Ϫ cell pairs and by 96% in Cx43 Ϫ/Ϫ cell pairs. The symmetrical dependence of g j on transjunctional voltage and properties of single-channel recordings indicated that Cx45 was the only remaining connexin in Cx43Ϫ/Ϫ cells. Propagation in Cx43 Ϫ/Ϫ strands was very slow (2.1 cm/s versus 52 cm/s in Cx43 ϩ/ϩ ) and highly discontinuous, with simultaneous excitation within and long conduction delays (2 to 3 ms) between individual cells. Propagation was abolished by 1 mmol/L heptanol, indicating residual junctional coupling. In summary, knockout of Cx43 in ventricular myocytes leads to very slow conduction dependent on the presence of Cx45. Electrical field effect transmission does not contribute to propagation in synthetic strands. Key Words: Cx43 Ⅲ Cx45 Ⅲ very slow electrical propagation Ⅲ discontinuous propagation Ⅲ heptanol C onnexin (Cx) proteins form intercellular channels enabling the intercellular exchange of ions and small molecules. 1 In the heart, they facilitate rapid, coordinated electrical excitation, a prerequisite for normal rhythmic contraction. Three different connexins, Cx43, Cx40, and Cx45, are expressed in heart. 2,3 Cx43, the most abundant connexin, is found in ventricular and atrial myocardium. Cx40 is expressed in atrial tissue and the cardiac conduction system. Although Cx45 expression in working ventricular myocardium is modest compared with Cx43, Cx45 is vital for early embryogenesis and cardiac development. 4,5 Cx45 is also expressed in the sinoatrial and atrioventricular nodes. It colocalizes with Cx40 in the conduction system, 6 and with Cx43 in ventricular myocardium. 7 The three cardiac connexins form channels with unique properties. 8 Although multiple connexins are coexpressed in cardiac tissues, their interactions and contributions to electrical or metabolic function are not understood completely. Cx43 and Cx45 likely form heteromeric/heterotypic channels, 9 -12 which may fulfill distinct functions. Cardiac diseases that lead to arrhythmias are associated with gap junction remodeling. [13][14][15] Therefore, know...

Cell-to-cell coupling in engineered pairs of rat ventricular cardiomyocytes: relation between Cx43 immunofluorescence and intercellular electrical conductance

“…Immunofluorescence signals for Cx45 have been shown to be present in small amounts in rat, mouse, and human ventricles (3,18,33,35). The low level of Cx45 in ventricular myocardium was confirmed in our study, and interference from background fluorescence precluded a quantitative 3D analysis of this immunosignal.…”

“…In ventricular myocardium, Cx43 is the dominant protein, and Cx45 is present in small amounts. In certain species, Cx45 modifies average gap junction channel conductance (3). Cx43 and Cx45 are remodeled in pathophysiological states; in the case of cardiac failure, downregulation of Cx43 and upregulation of Cx45 are observed (1,12,35).…”

A possible mechanism of halocarbon-induced cardiac sensitization arrhythmias

“…Previously, the FPs have been shown to correlate well with the underlying cellular electrophysiology [8], and this arrangement allowed for evaluation of changes in the dominant pacemaker, a surrogate for ectopic activity, and of conduction velocity [9,10]. Mapping of two-dimensional cardiomyocytes cultures has proven an efficient methodology for exploring arrhythmic risk [9][10][11][12][13][14][15][16].…”

Modeling electrical activity of myocardial cells incorporating the effects of ephaptic coupling

“…In mice with heterozygous Cx43AE down-regulation, either a 23-44% decrease in propagation velocity (2)(3)(4) or no discernible decrease in propagation velocity (5)(6)(7)(8) was found. In Cx43 − ∕− mice with no expression of the protein, propagation was still found, although discontinuous and at much slower speeds (7). These experimental findings are in conflict with the classical understanding of how gap junctions determine propagation velocity.…”