Alterations in Mitochondrial DNA in Corneal Fibroblast and Myofibroblast Post Injury.

Purpose: Mitochondria regulate cellular activity in a tissue-selective manner. The role of mitochondria in corneal fibrosis is elusive. This study investigated changes in mitochondrial DNA (mtDNA) and mitochondrial transcription factor A (TFAM) in human corneal stromal fibroblasts (CSFs) and corneal myofibroblast (CMFs) and effects on corneal fibrosis in vitro and ex vivo. Methods: Healthy donor human corneas were used to generate CSFs and ex vivo culture. CMF formation was induced by transforming growth factor beta-1 (TGFβ1) in vitro and human cornea by nitrogen mustard (NM) ex vivo. mtTFA/TFAM CRISPR/Cas9 KO plasmid, Lipofectamine CRISPRMAX, and TrueCut Cas9 Protein v2 were used for gene editing. Long-range PCR and quantitative reverse-transcription PCR (qRT-PCR) measured mtDNA transcription, mtDNA quantity, and ratios of mtDNA to nuclear DNA (nDNA). Immunofluorescence and immunoblotting quantified protein expression. The MitoSOX assay was used to analyze mitochondrial reactive oxygen species (mtROS). Results: Human CMFs showed significantly reduced mtDNA copies (P < 0.01) and mtDNA-to-nDNA ratios (P < 0.05) compared to CSFs. Significant time-dependent increases in mRNA levels of α-smooth muscle actin (αSMA) and nDNA-transcribed genes and decreases in TFAM and mtDNA-transcribed genes were noted during CSF transdifferentiation to CMFs (P < 0.05, P < 0.001, or P < 0.0001). Correspondingly, time-dependent decreases in TFAM and increases in Rieske iron-sulfur (Fe-S) and αSMA protein (P < 0.0001) and mtROS and ROS levels (P < 0.0001) were observed. TFAM silencing arrested fibrotic events and exhibited reduced αSMA and enhanced mtDNA (P < 0.001). The NM-induced fibrotic human cornea showed decreased TFAM and increased αSMA compared to naïve corneas (P < 0.01). Conclusions: We observed that mtDNA plays an important role in corneal fibroblast transdifferentiation to myofibroblast and that TFAM has the potential to modulate this process in an injured cornea. Additional studies are warranted.