Corneal edema in inducible Slc4a11 knock out is mediated by mitochondrial oxidative stress and Src Kinase activation

Abstract

Purpose : The inducible Slc4a11 KO leads to corneal edema by disruption of the pump and barrier functions of the corneal endothelium (CE) (IOVS 202; 62(8):807). The loss of Slc4a11 NH₃-activated mitochondrial uncoupler leads to mitochondrial membrane potential hyperpolarization induced oxidative stress. The goal of this study is to investigate the link between oxidative stress and failure of pump and barrier functions and test different approaches to revert the process.

Methods : Mice homozygous for Slc4a11 Flox and Estrogen receptor –Cre Recombinase fusion protein alleles at 8 weeks of age were fed Tamoxifen (Tm) enriched chow (0.4 g/Kg) for 2 weeks, and controls were fed normal chow. Ten µl of mitochondrial-targeted antioxidant Visomitin (1.5 µM) or Src kinase inhibitor eCF506 (1 µM) or PBS were applied to either cornea 3 times/day starting on the same day as Tm-chow feeding. Corneal thickness (CT) was measured by OCT. Pump function was tested by stromal lactate retention, barrier function by fluorescein permeability and Na-K-ATPase by ouabain-inhibitable ATPase activity. Mitochondrial ion superoxide (O₂^-) was detected with MitoSOX, tight junction integrity by ZO-1 and cortical cytoskeleton by F-actin staining. Levels of proteins were compared with Protein Simple Jess.

Results : Tm induced 84% and 96% Slc4a11 knock down in the CE at 7 and 14 days of treatment, respectively. O₂^- levels increased by day 7. CT and fluorescein permeability increased at day 14. Tight junction structure and cortical cytoskeleton were disrupted at day 14, but not at day 7. At day 14 we observed decreased level of Cldn1 and an increase in its tyrosine phosphorylation. Lactate accumulated and Na-K-ATPase activity decreased 40% at 14 days. Expression of lactate transporters MCT2 and MCT4 was decreased. Src kinase was activated but not Rock, PKCα, JNK or P38Mapk. Visomitin and eCF506 significantly inhibited the increase in CT (PBS: 153.5±9.2 µm;Vis: 126.4±7.7, p<0.0001;eCF: 126.3±14.0, p<0.0001, n=24), with improved barrier function, tight junction and cortical cytoskeleton integrity, decreased lactate retention, Src activation, Cldn1 downregulation and tyrosine phosphorylation and improved MCT2 and MCT4 expression.

Conclusions : Slc4a11 KO-induced CE oxidative stress increased Src kinase activity that results in perturbation of the pump and barrier functions of the CE.

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