MODULATION OF CONNEXIN43 EXPRESSION - EFFECTS ON CELLULAR COUPLING

Introduction: Gap junctions connect cardiac myocytes allowing propagat ion of action potentials, They contain intercellular channels formed b y multiple different connexin proteins, The arrangement and type of ga p junctions and the types, function, and interaction of connexin prote ins determine intercellular resistance and can thereby influence condu ction velocity and the potential for reentrant arrhythmias. Our goal w as to develop genetically manipulable models to test the effects of al tering expression of a major cardiac connexin (connexin43) on intercel lular coupling and expression of other connexin proteins. Methods and Results: BHK cells that are poorly coupled and BWEM cells that are wel l coupled were stably transfected with plasmids containing connexin43 cDNA in antisense and sense orientations, RNA blots confirmed expressi on of the transfected transcripts, Immunoblots showed that connexin43 protein was reduced in the BHK antisense transfectants and increased i n the BHK sense transfectants compared to the parental cells, It was n ot detectably changed in the BWEM antisense transfectant line compared to the BWEM parental cells, Transfection of connexin43 cDNA did not a ffect production of connexin45 mRNA and protein nor did transfection i nduce expression of other previously unexpressed connexin mRNAs, Cell coupling was assessed by intercellular diffusion of microinjected Luci fer yellow in confluent cell populations, Lucifer yellow passed to a m ean of 3 +/- 3 neighboring parental BHK cells, to 8 +/- 8 neighbors in the sense connexin43 transfected BHK cells, and to only 2 +/- 2 neigh bors in the antisense connexin43 transfected BHK cells (P < 0.05), In contrast, dye transfer did not differ significantly between the parent al BWEM cells (mean transfer = 19 +/- 14 cells) and the BWEM connexin4 3 antisense transfectants (mean transfer = 15 +/- 12 cells) (P = 0.20) . Conclusions: These data demonstrate that stable transfection,vith co nnexin43 cDNA constructs can result in detectable changes in connexin4 3 expression and cellular coupling without inducing compensatory chang es in the cell's connexin phenotype and, therefore, may provide a basi s for future attempts at specifically modulating connexin expression a nd intercellular resistance in cardiac tissues.