Effect of loss of Pitx2 expression on eye movements in the mdx mouse model of muscular dystrophy.

Abstract

Purpose : The extraocular muscles (EOM) are both morphologically and physiologically spared in various muscular dystrophies. One potential explanation is that their elevated numbers of myogenic precursor cells allow the EOM to be maintained despite the absence of dystrophin. Pitx2 is a transcription factor that is required for EOM formation in development, and when knocked out in otherwise normal EOM, a more limb-like morphology results. We examined the changes caused by loss of Pitx2 expression at birth in the mdx mouse model of muscular dystrophy, specifically analyzing EOM morphologically and functionally.

Methods : Mice examined were normal controls, mdx4cv, or mdx4cvMCK-Cre^+/-;Pitx2^fl/fl (mdx/Pit2KO), where Pitx2 expression is turned off at birth when muscle creatine kinase is expressed, at ages 3 months to 24 months of age. Eye movements were assessed using optokinetic nystagmus (OKN) performed using an ISCAN device and analyzed using R. Animals were euthanized at selected ages and prepared for histological examination. Muscle fiber number and size were determined morphometrically and significance was determined using ANOVA.

Results : The mdx mice displayed OKN, except in the extremely aged. Muscle fiber number and size were normal, but these began to decrease over the age of 20 months. The mdx/PitxKO mice showed variability in the OKN traces. Some mice at 9 months were unaffected, while others showed a significant decrease in OKN amplitude, with irregular movements. By 20 months and older, no eye movements were detected in these mice. This correlated with a significant loss in muscle fiber number and myofiber cross-sectional areas in their EOM, with some of the muscles more affected than others. The muscle fibers that remained were centrally nucleated.

Conclusions : The absence of Pitx2 expression at birth in the mdx mice resulted in a significant loss of eye movement function, but only in the aging EOM. This suggests that even with a decrease in fiber number and the presence of centrally nucleated myofibers, normal eye movements are maintained until the muscle loss has become significant. There was considerable variation between mice, suggesting that other factors play an important role in maintaining EOM function.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.