Dominant-negative connexin43-EGFP inhibits calcium-transient synchronization of primary neonatal rat cardiomyocytes.
Recent studies using mice with genetically engineered gap junction protein connexin (Cx) genes have provided evidence that reduced gap-junctional coupling in ventricular cardiomyocytes predisposes to ventricular arrhythmia. However, the pathological processes of arrhythmogenesis due to abnormalities in gap junctions are poorly understood. We have postulated a hypothesis that dysfunction of gap junctions at the single-cell level may affect synchronization of calcium transients among cardiomyocytes. To examine this hypothesis, we developed a novel system in which gap-junctional intercellular communication in primary neonatal rat cardiomyocytes was inhibited by a mutated (Delta130-137) Cx43 fused with enhanced green fluorescent protein (Cx43-EGFP), and calcium transients were imaged in real time while the mutated Cx43-EGFP-expressing cardiomyocytes were identified. The mutated Cx43-EGFP inhibited dye coupling not only in the liver epithelial cell line IAR 20 but also in primary neonatal rat cardiomyocytes in a dominant-negative manner, whereas wild-type Cx43-EGFP made functional gap junctions in otherwise communication-deficient HeLa cells. The mutated Cx43-EGFP induced desynchronization of calcium transients among cardiomyocytes with significantly higher frequency than wild-type Cx43-EGFP. These results suggest that dysfunction of gap-junctional intercellular communication at the single-cell level could hamper synchronous beating among cardiomyocytes as a result of desynchronization of calcium transients.
Oyamada
Yumiko
Y
Department of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine, Kyoto, 602-8566, Japan.
Zhou
Wuxiong
W
Oyamada
Hideto
H
Takamatsu
Tetsuro
T
Oyamada
Masahito
M
D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism*; D002450:Cell Communication; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D017629:Gap Junctions / Q000378:metabolism; D015870:Gene Expression; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000378:metabolism*; D009154:Mutation; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection
D018031:Connexin 43; D008164:Luminescent Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D002118:Calcium