Abstract
Purpose :
Although RPE metabolic dysfunction has been observed in multiple forms of macular degeneration (MD), the precise trigger of the metabolic dysfunction remains obscure. During MD, Bruch’s membrane (BrM) undergoes many alterations including changes to biomechanical stiffness. We set out to determine the impact of extracellular matrix (ECM) stiffness on RPE metabolism.
Methods :
Primary porcine RPE cells were plated on polydimethylsiloxane (PDMS) matrices of different stiffnesses (2ka, 16kPa, 64kPa) and allowed to differentiate for 1 week. Subsequently, metabolism was directly monitored through steady state[13C5] glutamine and [13C6] glucose isotopic tracing for 24 hours followed by semi-targeted metabolomics. Gas chromatography-mass spectrometry (GC-MS) was used to quantify relative levels of central carbon metabolites. Mass isotopic distributions were calculated to assess pathway activity and nutrient contribution.
Results :
13C6 glucose tracing showed decreased M3 and M4 TCA cycle intermediates in cells grown on soft (2kPa) substrates. In addition, intracellular glutamine levels were decreased by 50% in cells grown on soft substrates (2kPa) and increased by 50% in cells grown on stiff (64kPa) substrates. 13C5 glutamine labeling showed decreased M5 enrichment of glutamate, proline, citrate, and alpha-keto glutarate with decreased M4 enrichment of citrate, succinate, fumarate, malate, and aspartate at lower stiffness (2kpa, 16kpa) indicating that softer ECM reduces glutamine anaplerosis in RPE cells.
Conclusions :
Biomechanical properties of the RPE extracellular matrix can regulate RPE cell metabolism. These data suggest that reduced matrix stiffness causes metabolic rewiring in which RPE cells shift away from glutamine and towards glucose utilization for the TCA cycle and energy production.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.