Hypoxic Regulation of Notch1 Promotes Mitochondrial Fission and Scleral Remodeling in Myopia

Background: The progression of myopia is typically accompanied by hypoxia-induced remodeling of the scleral extracellular matrix (ECM). In this study, we focused on the regulatory mechanisms underlying Notch signaling pathway activation of mitochondrial dynamics under conditions of scleral hypoxia, along with its effects on the scleral ECM.

Methods: Three-week-old male guinea pigs were used to establish form-deprived myopia (FDM) models, and human scleral fibroblasts (HSFs) were cultured in a hypoxic environment. To examine the intrinsic associations among factors, we used short hairpin RNAs (shRNAs) to independently knock down hypoxia-inducible factor alpha (HIF-1α) and Notch1. Expression of key molecules was assessed by western blotting, quantitative real-time polymerase chain reaction (qPCR), and immunofluorescence (IF) analyses. Additionally, mitochondrial morphology, membrane potential (ΔΨm), and Reactive Oxygen Species (ROS) levels were assessed, and the specific dynamin-related protein 1 (DRP1) inhibitor Mdivi-1 was used to determine its regulatory effects on scleral ECM.

Results: In the FDM model, we detected a marked upregulation of scleral HIF-1α and Notch1, along with abnormal mitochondrial fission and ECM remodeling. Hypoxia-cultured HSFs were found to be characterized by mitochondrial fragmentation, a reduction in ΔΨm, elevated levels of ROS and α-smooth muscle actin (α-SMA), and a reduction in type I Collagen [markers indicative of fibroblast-myofibroblast transition (FMT) and ECM remodeling]. Notably, we observed that knockdown of HIF-1α was associated with a reduction in Notch1 levels, and a subsequent knockdown of Notch1 inhibited the expression of DRP1 and attenuated abnormal mitochondrial fission. Furthermore, pharmacological inhibition of mitochondrial fission using Mdivi-1 contributed to an amelioration of the aberrant mitochondrial morphology and reduced the expression of FMT markers in vitro.

Conclusion: Collectively, our findings indicate a potential link between scleral hypoxia and mitochondrial fragmentation, which may involve activation of Notch1 signaling and subsequent changes in the composition of the scleral ECM. In vitro, inhibition of mitochondrial fission appeared to mitigate the transformation of human scleral fibroblasts toward a myofibroblast-like phenotype. These findings provide evidence of a novel hypoxia-associated cellular pathway that warrants further investigation to establish its causal role and therapeutic relevance in myopic scleral remodeling.