AMPK orchestrates mitochondrial homeostasis via dynamics and mitophagy to combat hypoxia-induced energetic crisis in keratocytes.

Abstract

Background: Hypoxia disrupts corneal stromal physiology and alters cellular energy metabolism. This study aimed to elucidate the molecular role of the cellular energy sensor AMP-activated protein kinase (AMPK) in modulating the energy metabolism of human keratocytes (HKs) under hypoxic conditions and to explore a potential therapeutic strategy.

Methods: In vitro, HKs were exposed to 1% O₂ for 2 h and 24 h to simulate hypoxia. In vivo, a mouse corneal alkali burn model was established, in which hypoxia is recognized as an important contributing factor to tissue injury. The expression of AMPK and its influence on downstream pathways related to mitochondrial metabolism were investigated. The AMPK agonist AICAR was administered in these models to assess its effects. Key molecular pathways, including AMPK/MFF/DRP1, AMPK/MTFR1L/OPA1 (mitochondrial dynamics), and AMPK/ULK1/PINK1/PARKIN (mitophagy), were analyzed to understand the mechanisms of action.

Results: In vitro, hypoxia shifted HKs energy production from oxidative phosphorylation to anaerobic glycolysis over time, accompanied by AMPK upregulation. AICAR restored mitochondrial function, increased OCR, reduced ECAR and ROS, and improved mitochondrial membrane potential. AMPK knockdown abolished these protective effects. In vivo, AICAR treatment of alkali-burned mouse corneas significantly reduced corneal opacity and fluorescein staining scores, restored mitochondrial respiration and glycolysis balance. Mechanistically, AICAR activated AMPK to regulate mitochondrial dynamics via the AMPK/MFF/DRP1 and AMPK/MTFR1L/OPA1 pathways and enhanced mitophagy through the AMPK/ULK1/PINK1/PARKIN axis. This coordinated action increased mitochondrial oxidative phosphorylation capacity.

Conclusions: Activation of AMPK via AICAR ameliorates hypoxia‑induced energy imbalance in HKs in vitro and in mouse corneal alkali burn in vivo by restoring mitochondrial homeostasis. This mechanism represents a novel therapeutic target for hypoxia-related keratopathy and suggests a promising approach for energy replacement therapy.