In this project, we aimed to determine functional cellular differences at basal conditions between trans-mitochondrial cybrids generated from African and European mtDNA backgrounds. 

Human mitochondrial DNA (mtDNA) heritage significantly influences disease sensitivity, but the cellular mechanisms and functional distinctions among mtDNA lineages are not fully understood. MtDNA is strictly maternally inherited and encodes for transcripts responsible for the synthesis of respiratory electron transport chain complexes. Accordingly, changes in mtDNA sequence affect the efficiency of oxidative phosphorylation and influence cellular energetics. Ancient human migration out of Africa, across continents, and into new environments led to the accumulation of regional, non-pathogenic mtDNA variants that conferred metabolic advantages specific to their environments, forming mtDNA haplotypes. These haplotypes group into phylogenetically distinct haplogroups, which are characterized by unique energetic profiles and have been identified as key modulators of sensitivity to chronic and transmissible diseases. 

We hypothesize that these non-pathogenic mtDNA variants – haplogroups – confer functional differences between transmitochondrial (cybrid) cell lines carrying mtDNA from African and European haplogroups. We generated cybrids using A549 lung carcinoma epithelial cells harboring mtDNA from European haplogroups H, J, and Uk, as well as the African haplogroup L4. We then evaluated differences in global transcriptome using RNA-sequencing and cellular energetics using Seahorse Assay between the African and European-derived cybrids. We found significant transcriptional and energetic differences between the European and African-derived cybrids. RNA sequencing analysis and pathway analysis revealed enrichment of gene sets responsible for amino acid metabolism, the integrated stress response, and antioxidant defense in African cybrids relative to European cybrids at basal conditions. The Mitostress Test, an energetic assay, also revealed differences in extracellular acidification rate and non-mitochondrial oxygen consumption rate between the African and European cybrids.

This study demonstrates the profound effects of subtle mtDNA differences between the African and European haplogroups on the nuclear transcriptome and cellular energetics, and indicates the efficacy of the cybrid approach for studying haplogroup differences.