Hybrid molecule SA-2 improves both mitochondrial respiration and glycolysis in primary human trabecular meshwork cells




Amankwa, Charles E.
Gondi, Sudershan
Stankowska, Dorota
Acharya, Suchismita


0000-0002-1120-7053 (Amankwa, Charles E.)

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Purpose: Oxidative stress (OS) caused by hypoxia/hyperoxia environment results in progressive loss of trabecular meshwork (TM) cells in primary open angle glaucoma (POAG). Our previous report demonstrated; a hybrid nitric oxide (NO) donor-antioxidant molecule SA-2 protect primary human (h) TM cells against t-butyl hydrogen peroxide (TBHP) -induced cell death and increased superoxide dismutase enzyme level. Here we investigated the effect of SA-2 on mitochondrial energy metabolism by measuring the respiration status, glycolysis rate and energy production. Methods: Primary hTM cells obtained from human donor eyes were seeded in 24-well culture plates (Seahorse XFe 24 Cell Mito Stress test kit, Agilent), and starved for 24h before treatment with SA-2 (1 µM,10µM,100µM, and 1mM). In a separate experiment, the cells were pretreated with TBHP (150µM) for 30 minutes, followed by the addition of SA-2 (10µM,100µM). After 24h, the mitochondrial complex inhibitors and uncoupling reagents (oligomycin, FCCP, rotenone/antimycin A) were added. The plate was analyzed for changes in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse XFe24 analyzer following the manufacturer's instructions. Results: The mean OCR was significantly decreased (>70%) followed by increase in the mean ECAR (~3-fold) after treatment with TBHP compared to oligo/FCCP/rot treated cells, hereafter called as negative control. Treatment with SA-2 at 1 µM,10µM,100µM and 1mM concentrations increased both oligomycin/FCCP induced decrease in ATP production and maximal mitochondrial respiration followed by an increase in the mean ECAR compared to negative control. The mean OCR was higher in SA-2 (100µM) +TBHP treated cells followed by an increase in ECAR in SA-2 (10µM or 100µM) +TBHP treated cells than TBHP and negative control treated cells. N =2-3. Conclusion: Mitochondrial respiration was impaired after TBHP treatment to hTM cells following cell death. While most of the mitochondrial targeting anti-oxidant compounds increase OCR but not ECAR, we found the hybrid NO donor-anti-oxidant compound SA-2 increases ATP production, maximal mitochondrial respiration and increases glycolytic energy production in hTM cells. This finding provides a novel direction for further investigation into the effect of SA-2 and mitochondrial bioenergetics during OS-induced cell death.