Mitochondrial DNA control of gene expression and cell function in HeLa cybrids of HeLa ρ0 cells with transferred mtDNA of A549 cells

Asthma is a disease characterized by airway inflammation and hyperreactivity. Mitochondria have their own DNA (mtDNA) that encodes essential components of Electron Transport Chain complexes and is inherited exclusively maternally. Variants in mtDNA (haplogroups) are linked to risks and severity of other diseases but have not been studied in asthma. We hypothesized that mitochondrial genome can regulate expression of nDNA genes important in asthma pathophysiology. Haplogroup H mtDNA from A549 lung epithelial cells was transferred to mtDNA-deleted HeLa cells (ρ0) to create HeLa[H] cybrids that have identical nuclei but varied mitochondrial genome as compared to HeLa cells with haplogroup L mtDNA. Cells were analyzed for mtDNA copy number by quantitative polymerase chain reaction (qPCR), energy metabolism by monitoring the cellular respiration and glycolysis using Seahorse analyzer, and mtDNA sequencing for haplogroup. Single-cell RNA sequencing technology, SeqGeq bioinformatics program, and western blot were used to analyze differential gene expression and involved pathways among these cells. ρ0 cells have mtDNA copy number <1, while cybrids have mtDNA copy number similar to A549 cells and different from HeLa cells {mtDNA copy number (mean±SD), HeLa 636±115, ρ0 0.13±0.08, cybrid 1956±354, A549 1738±504, n≥2 replicate experiments, ANOVA p<0.001}. ρ0 have no mitochondrial oxygen consumption and maintain energy generation by increased glycolysis, whereas cybrids have similar oxygen consumption and glycolytic rates to HeLa and A549 {mitochondrial basal oxygen consumption pmole/min/mg protein (mean±SD), HeLa 7.78±0.43, ρ0 0.51±0.56, cybrid 6.76±2.34, A549 4.86±0.41, n≥3, ANOVA p<0.001; glycolysis mpH/min/mg protein (mean±SD), HeLa 0.91±0.08, ρ0 1.96±0.16, cybrid 0.52±0.14, A549 0.72±0.48, n≥3, ANOVA p<0.001}. Compared to HeLa, ρ0 cells lack expression of all 13 mtDNA-encoded proteins and have changes in nuclear-encoded DNA (nDNA) gene expression (4,047 nDNA up, and 5,552 nDNA down). Switching from L to H haplogroup results in greater changes in gene expression (9,320 nDNA up, 4,376 nDNA down, 9 mtDNA up, and 4 mtDNA down). Compared to HeLa, cybrids have greater expression of genes important in bioenergetics and asthma, i.e., TCA pathway genes (aconitase, isocitrate dehydrogenase, oxoglutarate dehydrogenase, succinate dehydrogenase), cytokines, and inducible NO synthase (iNOS) pathway genes. Overall, these studies suggest that mitochondria are not only important for energy generation but relevant to cell signaling, inflammation, and metabolism in asthma. This work is supported by the National Heart, Lung, and Blood Institute HL081064, HL103453. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.