THE EXPRESSION, PHOSPHORYLATION, AND LOCALIZATION OF CONNEXIN-43 AND GAP-JUNCTIONAL INTERCELLULAR COMMUNICATION DURING THE ESTABLISHMENT OFA SYNCHRONIZED CONTRACTION OF CULTURED NEONATAL RAT CARDIAC MYOCYTES

We analyzed the expression, phosphorylation, and localization of the m ajor cardiac gap-junction protein connexin 43 (Cx43) during the establ ishment of a synchronized contraction in confluent monolayers of prima ry cultured neonatal rat cardiac myocytes, combined with a functional assay of gap junctions by the microinjection-dye transfer method. Moni toring of the beating rate and synchronization by Fotonic Sensor showe d that at Day 1 of culture cardiac myocytes contracted spontaneously b ut irregularly, that the contractile rate increased with culture time, and that a synchronized contraction was gradually formed. At Day 7, t he confluent cells exhibited synchronous contraction with a relatively constant rate (125 +/- 20 beats/min). Cardiac myocytes expressed a la rge amount of Cx43 mRNA even at Day 1 and maintained the expression un til at least Day 7. Immunofluorescence of Cx43 showed that the localiz ation of Cx43-positive spots was mostly restricted to cell-cell contac ts between myocytes and that few Cx43-positive spots were present betw een myocytes and fibroblasts or between fibroblasts. The amount of Cx4 3 protein, the proportion of phosphorylated forms to the nonphosphoryl ated one, and the number and total area of Cx43-positive spots increas ed with culture time. Gap-junctional intercellular communication measu red by dye transfer assay was also increased with culture time and cor related well with the number and total area of Cx43-positive spots. Ou r systematic study suggests that a concerted action of the expression, phosphorylation, and localization of Cx43 and gap-junctional intercel lular communication plays a major role in the reestablishment of synch ronous beating of cultured neonatal rat cardiac myocytes. (C) 1994 Aca demic Press, Inc.