PKCɛ mediates serine phosphorylation of connexin43 induced by lysophosphatidylcholine in neonatal rat cardiomyocytes

Abstract

Lysophosphatidylcholine (LPC) is a potent pro-arrhythmic derivative of the membrane phosphotidylcholine, which is accumulated in heart tissues during cardiac ischemia. However, the cellular mechanism underlying LPC-induced cardiomyocyte damage remains to be elucidated. This study focuses on the effects of LPC on cardiomyocyte gap junction. At 30μM, LPC decreased the spontaneous contraction rates of cardiomyocytes, and caused arrhythmic contraction without affecting cell viability. Connexin43 (Cx43) was seen as large plaques at cell junctions in control cells, whereas upon LPC treatment, the intensity of Cx43 staining was decreased in a concentration-sensitive manner and Cx43 staining appeared as tiny dots at cell junctions with a corresponding increase in cytoplasmic punctate staining. This distributional change of Cx43 was accompanied by an impairment of the gap junction intercellular communication (GJIC). Further, LPC treatment induced protein kinase C (PKC) activation, and PKC-dependent Cx43 phosphorylation at serine (Ser) 368. Pre-treatment with a specific PKCɛ inhibitor, eV1-2, prevented the LPC-induced Cx43 phosphorylation at Ser368 and the loss of Cx43 from gap junctions, both of which may disturb GJIC functions. Furthermore, siRNA knockdown of PKCɛ in H9c2 cells prevented LPC-induced serine phosphorylation of Cx43, confirming the role of PKCɛ in Cx43 serine phosphorylation. Double labeling immunofluorescence showed that LPC increased the colocalization of Cx43 with ubiquitin, and pretreatment with MG132 effectively prevented LPC-induced gap junction disassembly. LPC increased the ubiquitination of Cx43, which was blocked by eV1-2 pretreatment, suggesting that LPC accelerated the intracellular degradation of Cx43 via the ubiquitin-proteasomal pathway. It can be concluded that LPC destroyed the structure and function of gap junctions via PKCɛ-mediated serine phosphorylation of Cx43. PKCɛ inhibitors might therefore be effective in prevention of LPC-related diseases.

Keywords: Cardiomyocytes; Connexin43; Cx43; GJIC; Gap junction; LPC; Lysophosphatidylcholine; PKC; PKCɛ; Serine 368 phosphorylation; ZO-1; connexin43; gap junction intercellular communication; lysophosphatidylcholine; protein kinase C; zonula occludens-1.