Abstract 504: Co-Expression of Connexin40 or 45 Slows Connexin43-Dependent Conduction
Connexin (Cx) 43 is the major myocardial gap junction protein. In addition, Cx40 is expressed in atrial, and Cx45 in ventricular, myocardium. While it is accepted that gap junctions form homo- or heteromeric channels from these proteins, the functional effects of mixed channels on action potential (AP) propagation are largely unknown. We hypothesized that mixed expression of Cx40/Cx43 or Cx45/Cx43 would result in altered AP conduction velocity (CV) relative to Cx43 expression alone. Primary neonatal rat ventricular myocytes (NRVMs) were cultured in monolayers and AP CV assessed using optical mapping. Endogenous Cx43 expression was abundant as assessed by immunoblots. In contrast, Cx40 and 45 were not detectable. NRVMs were genetically modified using lentiviral vectors encoding Cx40, Cx43, Cx45, Cx43 siRNA or LacZ. On day 10 of culture, non-transduced and Cx43- or LacZ-transduced NRVMs displayed equal CVs (16 ± 2.0 cm/s), indicating a lack of effect of vector or Cx43 on CV. In contrast, transduction of NRVMs with Cx40 or Cx45 resulted in marked and significant slowing of CV to 8.4 ± 1.0 cm/s or 8.2 ± 0.7cm/s, respectively, using an equivalent vector dose. Co-expression of Cx40/Cx43 and Cx45/Cx43 was confirmed by immunoblots. To assess dependence of the latter finding on the presence of mixed gap junctions, endogenous NRVM Cx43 expression was knocked-down by siRNA and essentially replaced with exogenous Cx40 or Cx45. On day 12 of culture, NRVM CVs in non-transduced, non-silencing siRNA control, Cx43 siRNA knockdown and Cx43 siRNA knockdowns with either Cx40 or Cx45 overexpression were 11.0 ± 1.2, 11.5 ± 1.2, 8.7 ± 0.7, 12.6 ± 1.4 and 5.6 ± 0.5 cm/s, respectively. The latter three CVs were significantly different from control CVs. Immunoblots confirmed complete Cx43 knockdown and Cx40 or 45 replacement. In conclusion, in the presence of Cx43, expression of either Cx40 or Cx45 causes slowing of Cx43-dependent conduction. In the absence of Cx43, Cx40 and Cx45 dependent conduction occurs at a rate reflective of their unitary conductance.