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Cristina Kenney, Marilyn Chwa, Shari Atilano, Payam Falatoonzadeh, Deepika Malik, Claudio Ramirez, S Michal Jazwinski, Miceli Michael, Baruch Kuppermann; Cybrids with Different mtDNA Haplogroups Show Differential Expression of Respiratory Complex Genes. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5005.
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© ARVO (1962-2015); The Authors (2016-present)
Mitochondrial DNA (mtDNA) haplogroups represent different human populations and are defined by accumulation of specific SNPs. The J haplogroup is high risk for age-related macular degeneration (AMD) while the H haplogroup is protective for AMD. Cybrids (cytoplasmic hybrids) are an excellent model to study mitochondrial functions because the cells have identical nuclei and vary only in the mtDNA content. This study was designed to analyze the oxygen consumption rates (OCR representing OXPHOS) and extracellular acidification rates (ECAR representing glycolysis) and the expression of mtDNA encoded genes that are associated with the electron transport chain complexes I, III, IV, and V.
Cybrids were created by fusing Rho0 ARPE-19 cells (depleted of mitochondria) with platelets isolated from haplogroup H or J subjects (n=3 each). The Seahorse FX24 extracellular flux analyzer was used to measure OCR and ECAR in H vs J cybrids. These measurements allow for evaluation of cellular metabolic activity, mitochondrial function, and energy expenditure. Q-PCR was performed on H cybrids vs J cybrids using primers for the mtDNA genes of complexes I, III, IV, and V. mtDNA copy numbers were measured by comparing nDNA:mtDNA ratios (18S:MT-ND2).
The J cybrids had a significantly lower OCR/ECAR ratio than the H cybrids (14±2.7 vs 24 ±2.5, p<0.05) and lower expression levels for 7 of the mtDNA genes (MT-ND1, p=0.02; MT-ND2, p=0.003; MT-ND3, p=0.01; MT-ND4/ND4L, p=0.03; MT-CO2, p=0.003; MT-CO3, p=0.05; MT-ATP6, p=0.04). The mtDNA copy numbers were similar to each other in the H vs J cybrids (1.34±0.0228 vs 1.36±0.017, p=0.59).
The OCR/ECAR ratio confirms that the H cybrids have increased OXPHOS while the J cybrids use glycolysis. This finding is supported by lower expression levels of the mtDNA encoded genes in complex I (MT-ND1, MT-ND2, MT-ND3, MT-ND4/ND4L), IV (MT-CO2, MT-CO3) and V (MT-ATP6). Our findings show that mtDNA variants can mediate energy production pathways and mtRNA expression which in turn may alter the signaling pathways and stress-response patterns of the cells.
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