Abstract
Diabetic retinopathy (DR) is one of the most common causes of vision loss and blindness among the working-age population. High glucose (HG)-induced decrease in mitochondrial connexin 43 (mtCx43) level is known to promote mitochondrial fragmentation, cytochrome c release, and apoptosis in retinal endothelial cells associated with DR. In this study, we investigated whether counteracting HG-induced decrease in mtCx43 level would preserve mitochondrial integrity and prevent apoptosis. Rat retinal endothelial cells (RRECs) were grown in normal (N; 5 mM glucose) or HG (30 mM glucose) medium for 7 days. In parallel, cells grown in HG were transfected with Cx43 plasmid, or empty vector (EV), as control. Western blot (WB) analysis showed a significant decrease in mtCx43 level concomitant with increased cleaved caspase-3, Bax, cleaved PARP, and mitochondrial fragmentation in cells grown in HG condition compared to those grown in N medium. When cells grown in HG were transfected with Cx43 plasmid, mtCx43 level was significantly increased and resulted in reduced cleaved caspase-3, Bax, cleaved PARP and preservation of mitochondrial morphology with a significant decrease in the number of TUNEL-positive cells compared to those grown in HG alone. Findings from the study indicate a novel role for mtCx43 in regulating apoptosis and that maintenance of mtCx43 level could be useful in preventing HG-induced apoptosis by reducing mitochondrial fragmentation associated with retinal vascular cell loss in DR.
Highlights
Diabetic retinopathy is a long-term microvascular complication of diabetes
Rat retinal endothelial cells (RRECs) grown in High glucose (HG) medium and transfected with Cx43 plasmid showed a significant increase in cytosolic (132 ± 17% of HG; p < 0.05; Figure 1A) and mitochondrial connexin 43 (mtCx43) expression (181 ± 13% of HG; p < 0.05; Figure 1B) compared to those grown in HG medium
The current study indicates the beneficial effects of increasing mtCx43 expression which prevents apoptosis in RRECs grown in HG condition
Summary
Diabetic retinopathy is a long-term microvascular complication of diabetes. As the diabetic population keeps increasing, the prevalence of DR keeps surging and is estimated at 103 million globally [1]. We have observed that HG downregulates mtCx43 level concomitant with increased mitochondrial fragmentation and apoptosis in retinal endothelial cells [10]. The role of mtCx43 in regulating mitochondrial dynamics and apoptosis remains unclear, studies suggest that upregulating Cx43 in endothelial cells increases Cx43 level in the mitochondria [15,26,27], which could facilitate preservation of mtCx43 level, inhibition of cytochrome-c release, thereby preventing apoptosis [28]. Studies have shown that mtCx43 level impacts mitochondrial morphology and apoptosis in cardiomyocytes [8] and that mtCx43 overexpression reduces cytochrome c release and caspase-3 activation [29] It is unknown whether counteracting HG-induced decrease in mtCx43 level would facilitate preservation of mitochondrial morphology, prevent HG-induced mitochondrial fragmentation and thereby prevent apoptosis. Given the likely link between mtCx43 level and apoptosis, in this study, we have attempted to counteract HG-induced decrease in mtCx43 level by upregulating mtCx43 expression and investigate whether plasmid-mediated Cx43 overexpression is sufficient to maintain mtCx43 level and thereby prevent HG-induced mitochondrial fragmentation and apoptosis in RRECs
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