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Celia Bisbach, Rachel Hutto, Susan Brockerhoff, James Hurley; Increasing mitochondrial Ca2+ in cones stimulates α-ketoglutarate dehydrogenase. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1680.
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© ARVO (1962-2015); The Authors (2016-present)
Photoreceptors have higher TCA cycle activity in darkness, which coincides with high Ca2+ levels. We hypothesized that Ca2+ could help photoreceptors increase energy production in darkness by stimulating mitochondrial dehydrogenases to increase flux through the TCA cycle. To investigate this, we measured TCA cycle flux in photoreceptors overexpressing or lacking expression of the mitochondrial Ca2+ uniporter (MCU).
Cone-specific MCU OE zebrafish were generated by injecting MCU cDNA under control of a cone-specific promoter (gnat2) into zebrafish embryos. Rod-specific MCU KO mice were generated by crossing mice with loxP sites flanking exons 5 and 6 of MCU (MCUfl) with mice expressing Cre-recombinase under control of a rod-specific opsin promoter (iCre-75).MCU OE and MCU KO retinas from both genders were incubated in KRB buffer containing U-13C-glucose or U-13C-glutamine in darkness. Metabolites were extracted with MeOH, derivatized, and quantified using an Agilent 5975 GC-MS. Each data point represents the mean ± SD of 3 retinas from 3 separate animals.
MCU OE cones have 3.9±0.3 -fold higher mitochondrial Ca2+ levels compared to WT. MCU OE retinas fed with 13C-glucose accumulate labeled succinate, fumarate, malate, and aspartate at higher levels compared to WT. This is consistent with faster flux through the Ca2+-sensitive enzyme α-ketoglutarate dehydrogenase (αKGDH), so 13C-glutamine was used to more directly fuel αKGDH. MCU OE retinas supplied with 13C-glutamine produce labeled metabolites downstream of αKGDH at twice the rate of WT retinas (p≤0.01, figure C provides exact values for each metabolite). This analysis was repeated on MCU KO retinas, where TCA cycle flux was unaltered.
Increasing mitochondrial Ca2+ in cones stimulates αKGDH, indicating that Ca2+ has the capacity to increase TCA cycle activity in photoreceptors. Yet, we did not observe lower αKGDH activity in MCU KO rods. To integrate these results, we will measure mitochondrial Ca2+ levels in MCU KO photoreceptors. If mitochondrial Ca2+ levels are not lower, there may be a compensatory Ca2+ influx mechanism that maintains normal mitochondrial Ca2+ levels after MCU KO.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
Labeled TCA cycle metabolites in MCU OE (A) and KO (B) retinas fed with U-13C-glucose
Labeled TCA cycle metabolites in MCU OE (C) and KO (D) retinas fed with U-13C-glutamine (* indicates p≤.01) Labeling pattern for U-13C-glucose (E) and U-13C-glutamine (F)
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