Swapping mitochondria: a key to understanding susceptibility to neonatal chronic lung disease

Preterm birth and its most commonly associated chronic lung disease, bronchopulmonary dysplasia (BPD), are significant causes of morbidity and mortality worldwide (12). BPD is associated with long-term complications such as pulmonary hypertension, neurodevelopmental impairment, obstructive lung disease, and long-lasting pulmonary function deficits (4). There are multiple superimposed postnatal risk factors for BPD including intrauterine or postnatal infection, nutritional deficits, mechanical ventilation, and supplemental oxygen exposure, all of which can exacerbate the conditions surrounding preterm birth (16). Additionally, multiple studies have hinted at a genetic predisposition to BPD recognizing male sex, race, and other host factors such as mitochondrial dysfunction in its pathogenesis (7, 14). As we enter the era of increased personalized medicine, there is a continued need to understand how subtle genetic differences can risk-stratify preterm infants susceptible for BPD. Such knowledge could produce novel therapies designed to improve health of former preterm infants, and reduce the enormously high economic and public health burdens associated with preterm birth. In the current issue of American Journal of Physiology-Lung Cellular and Molecular Physiology, a clever study that involved swapping mitochondria between mice that are sensitive and resistant to hyperoxia by Kandasamy and colleagues (6) may aid our understanding of how genetic predisposition, at the level of the cell’s other genome (i.e., mitochondrial DNA), can drive susceptibility to BPD.

The authors took advantage of previous studies showing that C57BL/6 mice are susceptible to hyperoxia while C3H mice are more tolerant (1). In that study, genetic linkage analysis revealed that strain-specific susceptibility to hyperoxia was associated with reduced expression and a nucleotide substitution in the promoter for nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor that promotes the expression of antioxidant enzymes that detoxify reaction oxygen species (ROS). Because mitochondria are a major source of ROS, this finding raises the question of whether genetic variability in mitochondrial DNA that affects the production of ROS might also impact susceptibility to hyperoxia. The human mitochondrial genome is ~16.5 kilobases in length and contains 37 genes, of which 13 encode proteins and the rest produce rRNAs and tRNAs. Mitochondrial function is absolutely dependent on the expression of these genes as well as many other nuclear encoded genes. But, unlike nuclear encoded genes that undergo meiotic recombination of parental genomes during fertilization, the mitochondrial genome is usually inherited entirely from the mother. This direct inheritance of mitochondrial DNA through the maternal lineage provides an opportunity to test how swapping mitochondria between sensitive and tolerant strains of mice might impact how the developing lung responds to hyperoxia. Since there are hundreds to thousands of mitochondria per cell, the procedure actually involves isolating the embryonic pronucleus from one strain of mice and injecting into the enucleated embryo of another strain (8). In this case, the nucleus from a C57BL/6 mouse was injected into the cytoplasm of an enucleated C3H mouse and vice versa (Fig. 1). The mitochondrial-nuclear exchanged (MNX) zygotes were cultured overnight and implanted into surrogate CD-1 females as two-cell embryos. The nuclear and mitochondrial DNA of progeny was then verified by single nucleotide polymorphisms and complete sequencing of mitochondrial DNA. Four groups of mice used in the study were the original parental C57BL/6 (C57ⁿ/C57^mt) and C3H (C3Hⁿ/C3H^mt) wild-type mice and the two mitochondrial-nuclear exchange mice containing C57 nuclear and C3H mitochondrial (C57ⁿ/C3H^mt) and C3H nuclear and C57 mitochondrial (C3Hⁿ/C57^mt) genomes.

Fig. 1.

Cartoon model showing how mitochondrial heteroplasmic mice were created. Fertilized oocytes were collected from C57BL/6 (gray) and C3H/HeN (brown) mice. The pronuclei were removed from the C57BL/6 cells and injected into C3H/HeN cells. The fertilized oocytes were then implanted into surrogate females to generate the C57ⁿ/C3H^mt mitochondrial nuclear exchange (MNX) mouse (gray mouse derived from the gray C57BL/6 nucleus with brown mitochondria derived from C3H oocyte). The reciprocal procedure produced C3Hⁿ/C57^mt MNX mice (brown mouse derived from the brown C3H nucleus with gray mitochondria derived from C57BL/6 oocyte).

By exposing newborn mice to 75% oxygen for 14 days, Kandasamy and colleagues found that mice with C57BL/6 mtDNA produce more superoxide, have more alveolar simplification, and worse pulmonary function (increased resistance and decreased compliance) compared with mice with C3H mtDNA—in each case regardless of the nuclear DNA component. This is an important finding because it suggests mitochondrial-encoded genes confer susceptibility (or tolerance) to oxygen-induced neonatal lung disease. A potential molecular mechanism arising from the use of MNX mice is induction of the mitochondrial unfolded protein response (UPR^mt) (5). The UPR^mt can be triggered by mitonuclear protein imbalance, caused by a mismatch in the stoichiometry of nDNA and mtDNA encoded subunits of the respiratory chain. While blunt induction of such an imbalance can be achieved by simply disrupting the mitochondrial ribosome using tetracycline antibiotics (10), it is unclear whether other types of respiratory subunit mismatch, such as that expected in MNX mice, would be capable of inducing a UPR^mt. This is important, because recent work has shown that prophylactic induction of UPR^mt can be protective against stress (18). Furthermore, such protection via “mito-hormesis” is also cell nonautonomous, such that mitochondrial stress can induce protection in distant tissues (20). It is tempting to speculate that UPR^mt induction in MNX mice may have conferred protection in the study of Kandasamy et al. (6); however, it should be noted that such protection if caused by mitonuclear imbalance would be expected in both MNX strains (C3Hⁿ/C57^mt and C57ⁿ/C3H^mt), rather than just the C3H MNX strain (C57ⁿ/C3H^mt).

It is important to also note that a potential limitation is the long 14-day duration of oxygen exposure used in this study. This period spans the saccular, early alveolar, and bulk alveolar stages in murine lung development, which is beyond the typical comparable exposure for preterm human infants in the neonatal intensive care unit. It is therefore difficult to gauge if the observed lung function differences between mitochondrial haplotypes at postnatal day 14 are due to differences in the immediate post-birth proliferative properties of mitochondria (19), or due to alterations in mitochondrial function that are unrelated to this early proliferation response, such as activating Nox proteins (2). Regardless, comparing the mitochondrial genomes of these two strains of mice should identify haplotypes that influence how the lung responds to hyperoxia. Such knowledge may someday aid understanding of genetic susceptibility to neonatal chronic lung disease and adult lung diseases in general. Mitochondria play an important role in promoting survival of alveolar type 2 cells during Staphylococcus aureus infections (15) and limiting the severity of oxygen-induced lung disease in adult mice (11). They mediate how airway smooth muscle cells respond to inflammatory cytokines like TNF-α (3) and bitter taste molecules (13). And they may play an important role in the pathogenesis of plexiform lesions seen in pulmonary arterial hypertension (9). The advent of mitochondrial transfer therapy for inherited metabolic disorders (already approved in the United Kingdom) also raises the possibility of enhanced lung disease in offspring born from such n vitro fertilization approaches (17). Swapping mitochondria may have therefore unlocked the door to a room that is full of new research opportunities.

GRANTS

This work was supported in part by National Heart, Lung, and Blood Institute Grants R01 HL-091868 (to M. A. O’Reilly) and R01 HL-071158 (to P. S. Brookes), which support our own pursuit of how oxygen and mitochondrial respiration affect cardiopulmonary health and disease.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

M.A.O. prepared figure; A.M.D., P.S.B., and M.A.O. drafted manuscript; A.M.D., P.S.B., and M.A.O. edited and revised manuscript; A.M.D., P.S.B., and M.A.O. approved final version of manuscript.