Abstract 1110: Relative Abundance of Connexin43 and Connexin45 in Native Cardiac Gap Junctions by Immunogold Electron Microscopy
Connexin45 (Cx45) and connexin43 (Cx43) form hybrid channels with reduced unitary conductance and dye permeability compared to Cx43 channels. Increased Cx45 in gap junction (GJ) channels in failing hearts may be responsible for alterations in conduction, abnormal propagation and arrhythmogenesis. In this study, we performed Cx43/Cx45 double label immunostaining on murine ventricular myocardium (n=9). Cx45 staining was present in some intercalated discs that also exhibited Cx43 staining. Similar results were seen with double label immunogold electron microscopy (EM) of murine ventricular tissue. To quantify the relative abundance of Cx43 and Cx45 in GJ plaques, we isolated GJ-enriched fractions using sucrose density centrifugation of mouse ventricular homogenates pooled from wild-type (WT) C57BL/6 mice (n = 4) and transgenic mice with cardiac selective overexpression of Cx45 (Cx45OE, n = 4), and measured the abundance of Cx43 and Cx45 in these plaques by immunogold EM. GJ plaques from WT mice consisted predominantly (87%) of Cx43 staining; only 13% of WT GJ plaques stained for both Cx43 and Cx45. No GJ plaques had Cx45 staining in the absence of Cx43 staining. A majority (55%) of GJ plaques from Cx45OE hearts, however, had both Cx45 and Cx43 staining; 45% of plaques from Cx45OE hearts contained Cx43 alone. The relative abundance of Cx43:Cx45 in GJ plaques containing both connexins was the same in WT (95:5) and Cx45OE (94:6) hearts. To demonstrate that Cx45 and Cx43 interact directly in the heart, we immunoprecipitated Cx43-associated proteins from murine ventricular lysates (1% Triton X-100, 0.1% SDS), which were then immunoblotted with a monospecific polyclonal anti-Cx45 antiserum. All of the Cx45 in these lysates was associated with Cx43 (n=3). Immunoblotting analysis of Cx45-associated proteins isolated from ventricular homogenates demonstrated that a fraction of immunoprecipitatable Cx43 was present in Cx45 immunoprecipitates (n=5). In conclusion, our data demonstrate that Cx45 associates with Cx43 in native cardiac GJ plaques, this association may be due to direct interactions between Cx43 and Cx45, and upregulation of Cx45 increases the number of GJ plaques containing Cx45 rather than increasing the number of Cx45 channels in each GJ plaque.