Downregulation of mitochondrial connexin 43 by high glucose triggers mitochondrial shape change and cytochrome C release in retinal endothelial cells.
OBJECTIVE
To determine connexin 43 (Cx43) localization in mitochondria and investigate the effects of high glucose (HG) on mitochondrial Cx43 (mtCx43) expression and whether altered mtCx43 channel activity is involved in promoting apoptosis in retinal endothelial cells.


METHODS
MtCx43 localization was determined using immunostaining, green fluorescent protein (GFP)-tagged Cx43 followed by confocal imaging, and Western blot analysis using protein isolated from mitochondria of rat retinal endothelial cells (RRECs). To assess HG effects on mtCx43 expression, RRECs were grown in normal (5 mM) or HG (30 mM) medium for 7 days, and mtCx43 protein level assessed by Western blot analysis. To determine if mtCx43 channel inhibition affected mitochondrial morphology, RRECs grown sparsely were left untreated or treated with β-glycerrhetinic acid (β-GA), an inhibitor of connexin channels, and imaged using confocal microscopy. Additionally, mitochondria isolated from RRECs were treated with β-GA, and cytochrome c release assessed by Western blot.


RESULTS
Cx43 localization on the mitochondria of RRECs was confirmed with immunofluorescence staining using Cx43 antibody and GFP-tagged Cx43 imaged in live cells. Western blot analysis indicated that Cx43 was located primarily on the inner mitochondrial membrane, and mtCx43 protein level was significantly reduced in RRECs grown in HG condition. Treatment of RRECs with β-GA significantly decreased mtCx43 phosphorylation, induced mitochondrial fragmentation, and isolated mitochondria treated with β-GA showed increased cytochrome c release.


CONCLUSIONS
HG-induced downregulation of mtCx43 protein resulting in decreased channel activity may promote mitochondrial morphology changes and cytochrome c release, suggesting a novel mechanism for hyperglycemia-induced apoptosis in diabetic retinopathy.
Department of Medicine and Ophthalmology, Boston University School of Medicine, Boston, MA 02118, USA.
Kyle
K
Trudeau
Tetsuya
T
Muto
Sayon
S
Roy
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002478:Cells, Cultured; D018031:Connexin 43 / Q000096:biosynthesis* / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism*; D003930:Diabetic Retinopathy / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D004195:Disease Models, Animal; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D005947:Glucose / Q000494:pharmacology*; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D012171:Retinal Vessels / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D013549:Sweetening Agents / Q000494:pharmacology
D018031:Connexin 43; D013549:Sweetening Agents; D045304:Cytochromes c; D005947:Glucose