Abstract
Purpose:
To evaluate whether downregulation of Cx43 expression and gap junction intercellular communication (GJIC) promotes apoptosis in rat retinal Müller cells.
Methods:
Retinal Müller cells (rMC-1) were grown in normal medium (N; 5 mM glucose) or HG medium (30mM) for 7days. In parallel, cells grown in HG were transfected with Cx43 siRNAs. To avoid “off target” effects of siRNA, three Cx43 siRNAs targeted to different locations of the Cx43 transcript were independently transfected. To assess the effect of Cx43 downregulation on gap junction intercellular communication (GJIC), cells from the four groups were subjected to scrape load dye transfer assay. In parallel, the number of apoptotic cells was determined using TUNEL assay.
Results:
Cx43 protein expression was significantly decreased in rMC-1 cells grown in HG (67±19%, p<0.01, n=4) and in cells transfected with each of the three Cx43 siRNAs (72±13%; 79±9%; 73±8%, p<0.05, n=5) compared to cells grown in normal medium or cells transfected with scrambled siRNA. Cells grown in HG or cells transfected with Cx43 siRNA(s) exhibited significant reduction in GJIC compared to the normal untransfected cells or cells transfected with scrambled siRNA. The number of apoptotic cells was significantly increased concomitantly with downregulation in Cx43, GJIC activity.
Conclusions:
HG-induced apoptosis is mediated, at least in part, through Cx43 downregulation and reduced GJIC activity in rMC-1 cells. Impairment of cell-cell communication between Müller cells negatively impacts cell function and survival and contribute to HG-induced apoptotic cell death, in diabetic retinopathy.
Keywords: 499 diabetic retinopathy •
498 diabetes •
603 Muller cells