Inter‐Strain Variation in Mouse Mitochondrial Genome and Effects of Oxidative Stress
Authors: Verhein Kirsten C , Burkholder Adam , Nichols Jennifer L , McCaw Zachary , Marzec Jacqui , Panduri Vijayalakshmi , Gladwell Wesley , Reeves Nicole , Malphurs Jason , Solomon Greg , Wiltshire Tim , Fargo David , Bell Douglas , Van Houten Bennett , Kleeberger Steven R 
Published: November 2023

Reactive oxygen species contribute to the pathogenesis of many acute and chronic pulmonary disorders, including bronchopulmonary dysplasia (BPD), a respiratory condition affecting preterm infants. BPD treatment often involves respiratory support with high O2, and oxidative stress is an adverse effect associated with vascular damage and impaired lung development and function in a subset of infants. Genetic polymorphisms in a few candidate genes have been associated with BPD susceptibility, however the genetic basis of differential susceptibility remains poorly understood. Understanding the role of genetics is critical to improving treatment strategies and reducing oxidant injury in developing lungs. We developed a neonatal mouse model to mimic many features of BPD, and utilized genome‐wide association (GWA) mapping of quantitative phenotypes to identify susceptibility genes for neonatal hyperoxic lung injury. One day after birth, neonatal mice from 29 inbred strains were exposed for 72 hr to normoxia (room air, 20% O2) or hyperoxia (>95% O2). Hyperoxia induced phenotypes (inflammatory cells/histopathology) that are similar to BPD when compared to normal postnatal lung development in normoxia. We also measured hyperoxia‐induced lesions in nuclear (nuc) and mitochondrial (mt) DNA, and mitochondrial copy number from lung homogenates. Significant inter‐strain variation was found for mtDNA and nucDNA lesions after normoxia and hyperoxia. GWA mapping identified significant quantitative trait loci for mtDNA lesions, nucDNA lesions, and mitochondrial copy number after normoxia and for hyperoxia. We ultra‐deep sequenced neonatal lung mtDNA from mice exposed to normoxia and hyperoxia and found 385 sequence variations in wild‐derived PWD/PhJ inbred mice compared to reference C57BL/6J mice. Higher variation frequency (1/32 bases) was found in the D‐loop region compared to the rest of the mitochondrial genome (1/42 bases). There was also more heteroplasmy in PWD/PhJ mice compared to C57BL/6J. We found five haplotype groups among 27 strains that were sequenced, with classical inbred strains in haplotype 1, NZB and NZO mice in haplotype 2, and PWD/PhJ and PWK/PhJ mice in haplotype 3. Wild derived strains had more variation than classical inbred strains. We conclude that exposure to low (20%) and high (>95%) concentrations of oxygen early in life cause lesions in mouse lung mtDNA and nucDNA that are strain dependent. Ultra‐deep sequencing identified mtDNA sequence variation and differences in heteroplasmy and indels across inbred mouse strains that associate with disease phenotypes. Through these combined approaches, we have identified novel candidate susceptibility genes that may improve our understanding of neonatal lung injury and development.

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