Expression of Multiple Connexins in Cultured Neonatal Rat Ventricular Myocytes

Abstract

Three gap junction proteins have been identified in mammalian cardiac myocytes: connexin43 (Cx43), connexin45 (Cx45), and connexin40 (Cx40). These proteins form channels with different electrophysiological properties and have different distributions in cardiac tissues with disparate conduction properties. We characterized the expression, phosphorylation, turnover, and subcellular distribution of these connexins in primary cultures of neonatal rat ventricular myocytes. Cx43, Cx45, and Cx40 mRNA were specifically detected in RNA blots. Immunofluorescent staining with antibodies specific for Cx43 and Cx45 revealed punctate labeling at appositional membranes, but no immunoreactive Cx40 was detected. Double-label immunofluorescence confocal microscopy of cultured myocytes revealed colocalization of Cx43 and Cx45. Cx43 and Cx45 were both identified by immunoprecipitation from [35S]methionine-labeled cultures, but anti-Cx40 antibodies did not precipitate any radiolabeled protein. Phosphorylated forms of both Cx45 and Cx43 were immunoprecipitated from cultures metabolically labeled with [32P]orthophosphate. Phosphoamino acid analysis demonstrated that Cx45 was modified on serine residues, and Cx43 was phosphorylated on serine and threonine residues. Pulse-chase labeling experiments demonstrated that the half-lives of Cx43 and Cx45 were 1.9 and 2.9 hours, respectively. Thus, both Cx43 and Cx45 turn over relatively rapidly, suggesting that myocardial gap junctions have the potential for dynamic remodeling. The results implicate multiple mechanisms of gap junction regulation that may differ for different connexins.

Autocrine Regulation of Myocyte Cx43 Expression by VEGF

“…It remains to be determined whether the marked increase in Cx43 expression observed in the present study was due to increased connexin synthesis, decreased connexin degradation, or a combination of these mechanisms. Based on previous studies demonstrating that Cx43 turns over rapidly (t 1/2Ϸ 1.5 hours) in cardiac myocytes 18,19 and that turnover of the contractile protein pool is suppressed in neonatal rat cardiac myocytes stretched by 1% to 5%, 20 we predict that at least part of the increase in Cx43 content observed in the present study was due to a diminished rate of degradation of Cx43.…”

Regulation of intercellular coupling in acute and chronic heart disease

“…Laird and co-workers (21) reported rapid turnover of Cx43 in cultured neonatal myocytes and this observation has been confirmed (22). Laing and co-workers (23) have characterized mechanisms of Cx43 degradation in primary cultures of neonatal rat ventricular myocytes using specific inhibitors of lysosomal and proteasomal pathways.…”

The effects of connexin phosphorylation on gap junctional communication

“…Agents that elevate intracellular cAMP have been shown to increase Cx43 phosphorylation and Cx43-mediated communication (Darrow, Laing, Lampe, Saffitz, & Beyer, 1995), the size and number of GJs (Atkinson et al, 1995), and the assembly of new GJs (Paulson et al, 2000). Such enhancement has been shown to result from increased synthesis (Mehta, Yamamoto, & Rose, 1992) and/or the increased trafficking of connexons to the plasma membrane (Burghardt, Barhoumi, Sewall, & Bowen, 1995;Holm, Mikhailov, Jillson, & Rose, 1999;Paulson et al, 2000).…”