# Myogenic Differentiation (MyoD) Gene Expression in Cornea and Role in Corneal Myofibroblast Dedifferentiation

## TL;DR

This study explores the role of the MyoD gene in corneal fibrosis and shows that silencing MyoD reduces fibrosis markers in corneal cells.

## Contribution

The study is the first to demonstrate MyoD's role in corneal myofibroblast dedifferentiation and fibrosis regulation.

## Key findings

- MyoD expression is significantly higher in fibrotic corneas compared to non-fibrotic ones.
- Silencing MyoD in corneal myofibroblasts reduces fibrotic gene expression and promotes a fibroblast phenotype.
- In vivo experiments show reduced corneal scarring with MyoD gene silencing.

## Abstract

Myogenic differentiation (MyoD), a class II basic helix–loop–helix transcription factor, regulates multiple cell functions, including fibrosis in many organs, but remains unknown in the cornea. This study characterized the expression of MyoD in non-fibrotic and fibrotic rabbit and human donor corneas and investigated the effects of MyoD gene silencing on corneal myofibroblast dedifferentiation in vitro and fibrosis in vivo.

New Zealand White rabbits, human donor corneas, human corneal stromal fibroblasts (CSFs), and human corneal myofibroblasts (CMFs) were used. MyoD shRNA or scrambled shRNA was delivered into CMFs via Lipofectamine 3000 and rabbit cornea via 2-kDa polyethylenimine conjugated to gold nanoparticles (PEI2-GNPs). Quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence, and western blotting quantified the expression of profibrotic genes, intermediate filaments, fibroblast-specific protein 1 (FSP1), and proliferation marker Ki67 in CMF–/+ MyoD gene silencing.

MyoD expression was detected in human and rabbit corneal epithelium, stroma, and endothelium. MyoD levels were significantly greater in fibrotic human corneas (P < 0.001) and rabbit corneas (P < 0.0001) than corresponding non-fibrotic corneas. Also, CMFs had significantly greater MyoD levels than CSFs (P < 0.0001). MyoD shRNA–silenced CMFs acquired a characteristic fibroblast phenotype and showed reduced fibrotic genes, alpha smooth muscle actin (αSMA; P < 0.0001), fibronectin (FN; P = 0.0015), collagen type I (ColI; P = 0.0011), and collagen type IV (ColIV; P = 0.0053) mRNA levels compared to the scrambled shRNA–delivered CMFs. Further, immunofluorescence demonstrated increased vimentin (P < 0.001), FSP1 (P < 0.01), and Ki67 expression (P = 0.0349) and decreased desmin expression (P < 0.001) in MyoD shRNA–silenced CMFs than the scrambled shRNA–delivered CMFs. MyoD shRNA–delivered rabbit corneas had less scarring in vivo (P = 0.368).

MyoD is expressed in cornea and could amend CMF dedifferentiation and fibrosis in an injured cornea. Additional studies are warranted to define the potential of MyoD in corneal fibrosis management.