Abstract
Purpose :
Extraocular muscle (EOM) function is required for proper binocular vision, and EOM disorders are common causes of visual dysfunction. Autophagy is a normal cellular mechanism that disassembles and degrades unnecessary or damaged cytosolic components, through a tightly regulated process, to maintain tissue homeostasis and cellular survival. Using an adult zebrafish model, we studied the role of autophagy in regulating the response of EOMs to injury.
Methods :
Standard biochemical, cellular and molecular techniques were used to assess for autophagy activation, including electron microscopy (TEM), qRT-PCR, and antisense morpholino oligonucleotide (MO) knockdown of gene expression. Chloroquine (CQ) was used to block autophagy. Muscle regeneration was measured using a-actin:GFP transgenic zebrafish that facilitated visualization of the injured EOM. The role of FGF signaling in the regulation of autophagy was studied using the hsp70l:dnFGFR1a-EGFP transgenic line.
Results :
The lateral rectus (LR) muscle of adult zebrafish underwent myectomy, which is followed by robust EOM regeneration. Lysotracker staining and LC3 accumulation in the regenerating muscle suggested autophagy activation. TEM revealed that the number of autophagosomes (double-membraned organelles) was greatly elevated in EOMs following myectomy. Among autophagy-related genes, only atg5 was up-regulated, suggesting that autophagy is mainly regulated at the protein level. We found significant inhibition of LR regeneration at 5 days in fish treated with CQ, which was confirmed using atg5 knockdown and TEM. The induction of autophagy was not explained by its role in nutrient recycling, suggesting intrinsic importance to the injury response, and blockage of FGF signaling resulted in reduced induction of autophagy.
Conclusions :
Our results show that injured muscles activate autophagy to degrade the sarcomeric protein machinery and remodel the cytoplasm during the process of EOM regeneration. Proper activation of autophagy required FGF signaling, and was independent of starvation conditions. The study of autophagy in EOM disease and injury response may provide novel insights into strabismic conditions along with new therapeutic targets for intervention.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.