抄録
We studied the expression and localization of the major cardiac gap junction protein connexin 43 (Cx43) during the establishment of a synchronized contraction in confluently cultured neonatal rat cardiac myocytes, combined with a functional assay of gap junctional intercellular ccaniunication (GJIC) by using the microinjection-dye transfer method. We analyzed, by Fotonic Sensor, effects of heptanol on synchronized contraction of primary neonatal rat cardiac myocytes cultured at confluent density. We also examined the effect of heptanol on the changes in GJIC, on the expression and localization of Cx43, and on intracellular Ca2+ fluctuations by confocal laser scanning microscopy of myocytes loaded with the fluorescent Ca2+ indicator fluo 3.
Monitoring of the beating rate and synchronization by Fotonic Sensor showed that the contractile rate increased with culture time, and that a synchronized contraction was gradually formed. GJIC was also increased with culture time and correlated well with the total area of Cx43-positive spots and the amount of Cx43 protein. At day 6 of culture, numerous myocytes exhibited spontaneous, synchronous contractions, excellent dye coupling and synchronized intracellular Ca2+ fluctuations. At the concentrations of 2.0 and 2.5 mmol/L, heptanol reversely inhibited synchronized contraction, WIC and synchronization of intracellular Ca2+ fluctuations in the myocytes without preventing contraction and changes of intracellular Ca2+ in individual cells. Brief exposure (5-20 min) to heptanol (2.0 mmol/L) did not cause detectable changes in the expression and localization of Cx43, despite strong inhibition of WIC. These results suggest that GJIC plays an important role in synchronous intracellular Ca2+ fluctuations, which facilitate synchronized contraction of cardiac myocytes.
