MODULATION OF CONNEXIN43 EXPRESSION - EFFECTS ON CELLULAR COUPLING

Introduction: Gap junctions connect cardiac myocytes allowing propagation of action potentials, They contain intercellular channels formed by multiple different connexin proteins, The arrangement and type of gap junctions and the types, function, and interaction of connexin proteins determine intercellular resistance and can thereby influence conduction velocity and the potential for reentrant arrhythmias. Our goal was to develop genetically manipulable models to test the effects of altering expression of a major cardiac connexin (connexin43) on intercellular coupling and expression of other connexin proteins. Methods and Results: BHK cells that are poorly coupled and BWEM cells that are well coupled were stably transfected with plasmids containing connexin43 cDNA in antisense and sense orientations, RNA blots confirmed expression of the transfected transcripts, Immunoblots showed that connexin43 protein was reduced in the BHK antisense transfectants and increased in the BHK sense transfectants compared to the parental cells, It was not detectably changed in the BWEM antisense transfectant line compared to the BWEM parental cells, Transfection of connexin43 cDNA did not affect production of connexin45 mRNA and protein nor did transfection induce expression of other previously unexpressed connexin mRNAs, Cell coupling was assessed by intercellular diffusion of microinjected Lucifer yellow in confluent cell populations, Lucifer yellow passed to a mean of 3 +/- 3 neighboring parental BHK cells, to 8 +/- 8 neighbors in the sense connexin43 transfected BHK cells, and to only 2 +/- 2 neighbors in the antisense connexin43 transfected BHK cells (P < 0.05), In contrast, dye transfer did not differ significantly between the parental BWEM cells (mean transfer = 19 +/- 14 cells) and the BWEM connexin43 antisense transfectants (mean transfer = 15 +/- 12 cells) (P = 0.20). Conclusions: These data demonstrate that stable transfection,vith connexin43 cDNA constructs can result in detectable changes in connexin43 expression and cellular coupling without inducing compensatory changes in the cell's connexin phenotype and, therefore, may provide a basis for future attempts at specifically modulating connexin expression and intercellular resistance in cardiac tissues.