MyoD is required for the differentiation of Myo/Nog cell progenitors of myofibroblasts in explants of human lens tissue

Jackie Gerhart¹Mara Crispin¹Brian Heists¹Keith Mathers²Joseph Infanti¹David Venuti¹Joseph Richards¹Steven Morency¹Cathy J Hatcher¹Mindy George-Weinstein^1*

• ¹Philadelphia College of Osteopathic Medicine (PCOM), Philadelphia, United States
• ²Bala Eye Care, Bala Cynwyd, PA, United States

Posterior capsule opacification (PCO) is a complication of cataract surgery that impairs vision. Clouding and distortion of the posterior capsule occur as a result of cell migration, deposition of exrtracellular matrix proteins and contractions of myofibroblasts. The subpopulation of Myo/Nog cells differentiate into myofibroblasts in the lens. Myo/Nog cells express MyoD, Noggin and brain-specific angiogenesis inhibitor (BAI1). Depletion of Myo/Nog cells in the rabbit lens during cataract surgery prevented the emergence of myofibroblasts and reduced PCO to below clinically significant levels. A requirement for MyoD in the differentiation of Myo/Nog cells to myofibroblasts was explored in explant cultures of human anterior lens tissue. Tissue was incubated with MyoD or non-targeting siRNA. MyoD and Noggin mRNAs were co-expressed in control cultures. The number of MyoD mRNA-positive (+) cells was reduced by 90% after treatment with MyoD siRNA. The Noggin mRNA+ population was significantly increased with MyoD knockdown. Nearly all cells with BAI1 contained MyoD protein and all had Noggin. The MyoD family members Myf5 and Myogenin were also synthesized in Myo/Nog cells. Most cells with BAI1 contained alpha smooth muscle actin (α-SMA) and striated muscle myosin (SMM). MyoD siRNA nearly eliminated Myogenin and SMM, and significantly reduced the number of BAI1+ cells with Myf5. Expression of α-SMA was unaffected by MyoD knockdown. The numbers of BAI1+ cells was increased in response to treatment with MyoD siRNA. Noggin and muscle proteins were not detected in LECs in control explants or after MyoD knockdown. Cell free areas of the capsule were surrounded by differentiated Myo/Nog cells in control cultures. Wrinkles in the capsule were visible within these areas. Wrinkles were reduced by approximately 75% after MyoD knockdown. These results indicate that MyoD lies upstream of Myogenin, Myf5 and SMM in Myo/Nog cells of the lens. Contractions that deform the anterior capsule are dependent on SMM but not α-SMA. This study demonstrates that MyoD drives Myo/Nog cell differentiation to contractile myofibroblasts in cultures of human anterior lens tissue. The resulting increase in the progenitor population indicates that temporary knockdown of MyoD is not a therapeutic option for preventing of PCO.