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.