Organ level Inhibition of Inter cellular communication by mutant connexin expression vector

点击翻译按钮获取中文摘要

Gap junctions are specialized cell-cell junctions that form intercellular channels and mediate the direct transfer of low molecular weight metabolites and ions. Intercellular communication via gap junction is believed to play important roles in the control of cell growth, differentiation and maintenance of homeostasis. Recently, it has been reported that several human hereditary diseases such as Charcot-Marie-Tooth disease, non-syndromic sensorineural deafness and skin diseases are caused by point mutations of gap junction protein (connexin) genes. However, the pathological processes of these diseases due to abnormalities in gap junctions are poorly understood.In the present study, we have established a method for visualization of cellular function in living cells, while identifying the localization of connexins in real-time. Expression vectors that contain fusion proteins of green fluorescent protein (GEP) and, either wild-type or a dominant-negative mutant connexin43 (Cx43) were cons … More tructed and transfected into primary neonatal rat cardiomyocytes and into communication-deficient HeLa cells. Intercellular communication was estimated by microinjection of gap junction-permeable fluorescent dye (Alexa 568,m.w. 730). Intracellular calcium dynamics in cardiomyocytes were monitored by a fluorescent calcium indicator (Fura Red) in combination with confocal scanning microscopy. Wild-type Cx43-GFP made functional gap junctions in otherwise communication-deficient Hela cells. In contrast, the mutated Cx43-GFP Inhibited dye coupling among primary neonatal rat cardiomyocytes in a dominant-negative manner. The mutated Cx43-GFP induced desynchronization of calcium transients among beating cardiomyocytes with a significantly higher frequency than in wild-type Cx43-GFP. These results indicate that dominant-negative Cx43 can induce inhibition of synchronous beating among cardiomyocytes through desynchronization of calcium transients, and suggest that inhibition of intercellular communication via gap junction might cause arrhythmia and contraction disturbance in the heart. Cx-GFP expression vectors provides a useful systems for studies of localization and function of gap junctions in vivo. Less