Mitochondria constantly fuse and divide to form a cellular network, and this dynamic fusion-fission controls their function and genome stability. Although both, mitofusin 1 and 2 (Mfn1 and 2) regulate outer mitochondrial membrane fusion, Mfn2 itself is sufficient to modulate mitochondrial metabolism. In diabetes, retinal mitochondria are swollen, respiration is impaired, Mfn2 is decreased and mtDNA is damaged. Diabetes also facilitates epigenetic modifications, altering gene expressions without changing their DNA sequences. This study aims to investigate the role of DNA methylation of Mfn2 in the development of diabetic retinopathy.
Methods: Human retinal endothelial cells, incubated in normal (5mmols/L) or high glucose (20mmols/L) for four days, in the presence/absence of DNA methyltransferase (Dnmt) inhibitors (5-Azacytidine or Dnmt1-siRNA), were analyzed for methylated cytosine (5mC) at Mfn2 promoter by immunocapturing method. Mitochondrial integrity was evaluated by membrane permeability, coenzyme Q: cytochrome-c reductase (complex-III) activity, and transcripts of mtDNA-encoded cytochrome b (Cytb). Similar measurements were made in the retinal microvessels from streptozotocin-induced diabetic mice receiving 5-Aza (2.5mg/kg, i.p.) or Dnmt1-siRNA (2µg/2µl, intravitreal).
Results: High glucose increased 5mC levels at Mfn2 promoter by over 2 fold, and decreased Mfn2 expression. Dnmt inhibition ameliorated glucose-induced increase in 5mC at Mfn2 and mitochondrial membrane permeability, and decrease in Mfn2 expression, complex III activity and Cytb transcripts. Similarly, in mouse retinal microvessels, inhibition of Dnmt prevented diabetes-induced increase in 5mC at Mfn2 promoter and decrease in Mfn2 expression.
Conclusions: In diabetes, epigenetic modifications of Mfn2 promoter impair mitochondrial functional and genomic stability. Thus, targeting DNA methylation could help maintain mitochondrial homeostasis, and halt/retard the development of diabetic retinopathy.
R. Kowluru: None. M. Mishra: None.
