335 - Modulation of dynamic mitochondria by epigenetics in diabetic retinopathy

Abstract

Introduction Mitochondria are highly dynamic, and they continually fuse and divide. Fusion mixes the contents of two mitochondria, and dilutes injured mitochondrial proteins and DNA. Outer membrane fusion is mediated by fusion proteins, mitofusins (Mfn), and Mfn2 itself is sufficient to modulate mitochondrial metabolism. In the pathogenesis of diabetic retinopathy, mitochondrial structure and DNA are damaged, oxygen consumption is decreased and Mfn2 levels are decreased, and damaged mitochondria accelerates capillary cell apoptosis. Gene expression is also regulated by epigenetic modifications, and these modifications are implicated in the development of diabetic retinopathy. Aim of this study is to investigate the role of epigenetic modifications in the regulation of Mfn2 in diabetic retinopathy. Methods In human retinal endothelial cells, incubated in normal (5mM) or high glucose (20mM) for four days, in the presence/absence of DNA methyltransferase (Dnmt) inhibitors (5-Azacytidine or Dnmt1-siRNA), methylated cytosine (5mC) levels at Mfn2 promoter were quantified by immunecapturing method. Mitochondrial stability was evaluated by measuring membrane permeability, complex-III activity and transcription of mtDNA (cytochrome b, Cytb), and cell apoptosis was quantified by ELISA method using monoclonal antibodies against DNA and histones. Key parameters were evaluated in the retinal microvessels from streptozotocin-induced diabetic mice receiving intravitreal administration of Dnmt1-siRNA. Results Cells exposed to high glucose had increased 5mC levels at Mfn2 promoter. Dnmt inhibition ameliorated increase in 5mC levels, decrease in Mfn2 expression, and protected mitochondrial stability. In the same cells, glucose-induced increase in apoptosis was also ameliorated. Similarly, Dnmt1-siRNA prevented diabetes-induced increase in 5mC at Mfn2 promoter and decrease in its expression. Conclusions DNA methylation of Mfn2 promoter in diabetes, impairs mitochondrial stability, leading to accelerated apoptosis. Thus, targeting Mfn2 DNA methylation could help maintain mitochondrial homeostasis, and halt/retard the development of diabetic retinopathy.