Mitochondrial DNA variation modulates alveolar development in newborn mice exposed to hyperoxia

Abstract

Hyperoxia-induced oxidant stress contributes to the pathogenesis of bronchopulmonary dysplasia (BPD) in preterm infants. Mitochondrial functional differences due to mitochondrial DNA (mtDNA) variations are important modifiers of oxidant stress responses. The objective of this study was to determine whether mtDNA variation independently modifies lung development and mechanical dysfunction in newborn mice exposed to hyperoxia. Newborn C57BL6 wild type (C57ⁿ/C57^mt, C57WT) and C3H/HeN wild type (C3Hⁿ/C3H^mt, C3HWT) mice and novel Mitochondrial-nuclear eXchange (MNX) strains with nuclear DNA (nDNA) from their parent strain and mtDNA from the other—C57MNX (C57ⁿ/C3H^mt) and C3HMNX (C3Hⁿ/C57^mt)—were exposed to 21% or 85% O₂ from birth to postnatal day 14 (P14). Lung mechanics and histopathology were examined on P15. Neonatal mouse lung fibroblast (NMLF) bioenergetics and mitochondrial superoxide (O₂⁻) generation were measured. Pulmonary resistance and mitochondrial O₂⁻ generation were increased while alveolarization, compliance, and NMLF basal and maximal oxygen consumption rate were decreased in hyperoxia-exposed C57WT mice (C57ⁿ/C57^mt) versus C57MNX mice (C57ⁿ/C3H^mt) and in hyperoxia-exposed C3HMNX mice (C3Hⁿ/C57^mt) versus C3HWT (C3Hⁿ/C3H^mt) mice. Our study suggests that neonatal C57 mtDNA-carrying strains have increased hyperoxia-induced hypoalveolarization, pulmonary mechanical dysfunction, and mitochondrial bioenergetic and redox dysfunction versus C3H mtDNA strains. Therefore, mtDNA haplogroup variation-induced differences in mitochondrial function could modify neonatal alveolar development and BPD susceptibility.

INTRODUCTION

Bronchopulmonary dysplasia (BPD), characterized by impaired alveolar development and pulmonary mechanics, affects 25–35% of prematurely born infants (26). Exposure to supraphysiological oxygen (O₂) and the resultant oxidant stress contribute to BPD pathogenesis. However, susceptibility to hyperoxia-induced hypoalveolarization and pulmonary mechanical impairment and BPD is variable even among infants born at similar gestational age or birth weight. Our limited knowledge of the causes of such variability prevents targeted use of antioxidant BPD therapeutic strategies (24). Therefore, identifying novel mechanisms and therapeutic targets for neonatal hyperoxic hypoalveolarization is critical for accelerated development of novel strategies to ameliorate BPD risk.

Recently, mitochondrial function has emerged as a critical pathogenetic factor for complex multifactorial diseases with differential individual susceptibility and severity. The mitochondrial DNA (mtDNA) encodes 13 proteins that constitute the electron transport chain (ETC) and ATP synthase complexes. Electrons that leak from these complexes and couple directly with oxygen in the mitochondria contribute significantly to cellular oxidant stress (27). Due to its central position in the bioenergetic, redox, and homeostatic domains of cell function, the mitochondrion is considered to be important in oxidant stress-mediated pathology such as chronic obstructive pulmonary disease (3). Neonatal alveolar development and BPD have also been linked to decreased pulmonary ETC complex I activity and mtDNA damage (9, 23).

Inherited mtDNA variations (haplotypes) have been associated with variable severity in diseases such as adult-onset pulmonary hypertension secondary to differences in cellular bioenergetic function (7, 16). Such a role for mtDNA variations in neonatal lung development and BPD pathogenesis has not been explored. Therefore, the aim of this study was to determine whether mitochondrial genetic background differences independently modify neonatal lung development by modulating pulmonary mitochondrial bioenergetics and oxidant generation. We hypothesized that the severity of hyperoxic hypoalveolarization, pulmonary mechanical impairment, and mitochondrial dysfunction would vary based on the mitochondrial genetic background in a novel mouse model of neonatal hyperoxia.

METHODS

All experiments were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham (UAB).

MNX Mice and Hyperoxia

Mitochondrial-nuclear eXchange (MNX) mouse strains were obtained from colonies that are maintained at UAB. The generation of MNX mice, with the mitochondrial genome of one strain and the nuclear genome of the other, using wild-type C57BL/6J (C57ⁿ/C57^mt, C57WT) and C3H/HeN (C3Hⁿ/C3H^mt, C3HWT) mice to produce C57MNX (C57ⁿ/C3H^mt) and C3HMNX (C3Hⁿ/C57^mt) strains and subsequent confirmation of their mtDNA haplotypes through PCR for single nucleotide polymorphisms unique to each strain’s mtDNA have been described previously (8).

Hyperoxia exposure.

Newborn mice were exposed to room air (normoxia) or 85% O₂ (hyperoxia) from birth to postnatal day 14 (P14) as described previously (13).

Lung Function and Histology

Histopathology.

Photomicrographs of mice lung sections captured at ×100 magnification were used to quantify pulmonary alveolarization by calculating mean linear intercepts (MLI) and radial alveolar counts (RAC) using the Scion Image software as described previously (18).

FlexiVent measurements.

Pulmonary compliance and resistance were measured in P14 mice using the FlexiVent system as described previously (19). Neonatal mouse lung fibroblasts (NMLF) obtained from mice exposed to normoxia were exposed to 21% O₂ while cells from hyperoxia-exposed mice continued to be exposed to 85% O₂ before they were used for bioenergetic and oxidant generation assays as described below.

Mitochondrial Bioenergetic Function

NMLF were isolated from mice as described previously, and a Seahorse XF24 analyzer (Agilent, Santa Clara, CA) was used for oxygen consumption rate (OCR) measurements (17). Cell density and mitochondrial inhibitor concentrations were optimized before the actual analyses that were conducted using 30,000 NMLF per well in multiwell plates as described previously. Briefly, basal OCR was measured in the absence of any treatment following which 4 μg/mL oligomycin (to block mitochondrial ATP generation), 1 μM FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, to stimulate mitochondrial respiration to maximal levels), and 10 μM antimycin A/1 μM rotenone (to completely inhibit ETC complexes I and III) were sequentially injected to measure OCR at various states of mitochondrial respiration (5, 8). A minimum of 12 wells were averaged for each measurement.

Mitochondrial Superoxide Generation

NMLF mitochondrial superoxide (O₂⁻) production was measured using the MitoSOX Red (ThermoFisher Scientific, Waltham, MA) fluorescent probe. Cells (30,000 NMLF per well) were incubated with 0.5 mL of 5 μM MitoSOX Red at 37°C for 10 min and placed in a microplate reader to measure O₂⁻ specific MitoSOX Red oxidation (400/10 nm excitation and 595/35 nm emission) as described previously (28).

Statistical Analyses

Results are expressed as median ± interquartile range. Scheirer–Ray–Hare modification of the Kruskal-Wallis test was followed by the post hoc Dunn test to test for significant differences among individual pairwise comparisons. Bonferroni correction for multiple comparisons was used and P < 0.05 was considered significant. Linear regression models were created using sex, weight, length, oxygen exposure, and nuclear/mitochondrial genotypes as predictors and lung development and mitochondrial function measurements as dependent variables. R statistical software and associated packages were used for all analyses (22).

RESULTS

General Characteristics

MNX strains exposed to hyperoxia had similar respiratory patterns and survival rates versus their WT counterparts. Survival rates were similar across all groups (>90%). No differences in growth were observed for any of the four strains between hyperoxic mice and normoxic room air controls. As illustrated in the figures and to clarify the results, comparisons were made between 1) normoxic versus hyperoxic groups from each strain, 2) strains with similar nDNA but different mtDNA - C57WT (C57ⁿ/C57^mt) versus C57MNX (C57ⁿ/C3H^mt) and C3HMNX (C3Hⁿ/C57^mt) versus C3HWT (C3Hⁿ/C3H^mt), and 3) strains with different nDNA but similar mtDNA—C57WT (C57ⁿ/C57^mt) versus C3HMNX (C3Hⁿ/C57^mt) and C3HWT (C3Hⁿ/C3H^mt) versus C57MNX (C57ⁿ/C3H^mt).

C57 mtDNA-Carrying Strains Are Susceptible While C3H mtDNA Strains Are Resistant to Hyperoxic Hypoalveolarization

Normoxic mice with C57 nDNA had similar lung structure (Fig. 1, A and C) as did normoxic mice with C3H nDNA (Fig. 1, E and G). However, when exposed to hyperoxia, lung structure and function varied significantly between mice with similar nDNA but different mtDNA. Hyperoxic mice with C57 mtDNA— C57WT and C3HMNX—had increased alveolar size (MLI), decreased alveolar count (RAC), and poor alveolarization as well as decreased lung compliance and increased resistance versus their normoxic controls. In contrast, hyperoxic mice with C3H mtDNA— C57MNX and C3HWT— had similar MLI, RAC, alveolarization, compliance, and resistance versus their normoxic controls (Fig. 2, A–D).

Fig. 1.

Alveolar development in newborn mice. Representative photomicrographs of hematoxylin and eosin-stained sections of lungs from newborn mice exposed to normoxia or hyperoxia from birth to postnatal day 14 (calibration bars = 100 μm). Mice with C57 mtDNA [C57WT (A and B) and C3HMNX (G and H)] had poor alveolarization when exposed to hyperoxia. Lung structure was relatively preserved in mice with C3H mtDNA: C57MNX (C and D) and C3HWT (E and F). MNX, mitochondrial nuclear exchange (MNX); WT, wild type.

Fig. 2.

Lung structure and function in newborn mice. Box-whisker and dot plots representing mean linear intercept (A) and airway resistance (D) that were increased and radial alveolar count (B) and lung compliance (C) that were decreased in hyperoxic mice with C57 mtDNA versus mice with C3H mtDNA. Overall, hyperoxic hypoalveolarization was least severe in C3HWT (C3Hⁿ/C3H^mt) mice, most severe in C57WT (C57ⁿ/C57^mt) mice, and intermediate in the C57MNX (C57ⁿ/C3H^mt) and C3HMNX (C3Hⁿ/C3H^mt) strains. n = 16 per group per exposure, box indicates 25th–75th centiles, central bar represents median, and whiskers indicate 5th–95th centiles. MNX, mitochondrial nuclear exchange (MNX); NS, not significant; WT, wild type. *P < 0.05, **P < 0.005, and ***P < 0.0005.

Hyperoxic Hypoalveolarization Is Most Severe in C57ⁿ/C57^mt Mice and Attenuated in C3Hⁿ/C3H^mt Mice

Mice with similar nDNA but different mtDNA were compared. Normoxic C57WT and C3HMNX strains had similar MLI, RAC, compliance, and resistance values versus C57MNX and C3HWT, respectively. However, hyperoxic C57WT and C3HMNX strains had higher MLI and resistance as well as lower RAC and compliance versus C57MNX and C3HWT strains, respectively.

Mice with different nDNA but similar mtDNA were also compared. Normoxic C57WT mice had lower MLI and compliance, similar RAC, and higher airway resistance versus C3HMNX mice. No differences in these measures were noted between these strains when exposed to hyperoxia. C3HWT mice had similar RAC, higher MLI and compliance, and lower resistance when exposed to either normoxia or hyperoxia versus C57MNX mice (Fig. 2, A–D).

NMLF From Hyperoxic Mice With C57 mtDNA Have Increased Mitochondrial Bioenergetic Dysfunction

NMLF from hyperoxic mice with C57 mtDNA—C57WT and C3HMNX—had decreased basal OCR versus their normoxic controls. In contrast, basal OCR of NMLF from hyperoxic mice with C3H mtDNA— C57MNX and C3HWT— was similar to that of their normoxic controls. Maximal OCR of NMLF from hyperoxic mice of all four strains was lower versus their normoxic controls (Fig. 3, A and B). NMLF ATP-linked OCR [expressed as median [quartile (Q) 1, Q3] in pmol·min⁻¹·3 × 10⁴ cells⁻¹] was calculated by deducting OCR measured after oligomycin infusion from basal OCR. It was lower in hyperoxic C57WT [60(56, 62) versus 135(128,143)] and C3HMNX strains [107(104,112) versus 148(147,149), P < 0.05 for both comparisons], similar in C3HWT strains [161(158,164) versus 198(194,206)], and increased in C57MNX strains [164(162,169) versus 121 (119,125), P < 0.005] versus their normoxic controls.

Fig. 3.

Neonatal mouse lung fibroblast (NMLF) oxygen consumption. Box-whisker and dot plots representing basal oxygen consumption rate (OCR; A), maximal OCR (B), and mitochondrial O₂⁻ generation (C) of NMLF that were decreased in hyperoxic mice with C57 mtDNA versus mice with C3H mtDNA. Overall, NMLF bioenergetics was least attenuated and O₂⁻ generation lowest in C3HWT (C3Hⁿ/C3H^mt) mice, most affected in C57WT (C57ⁿ/C57^mt) mice, and intermediate in the hyperoxic C57MNX (C57ⁿ/C3H^mt) and C3HMNX (C3Hⁿ/C3H^mt) strains. n = 16 per group per exposure, box indicates 25th–75th centiles, central bar represents median, and whiskers indicate 5th–95th centiles. MNX, mitochondrial nuclear exchange (MNX); NS, not significant; WT, wild type. *P < 0.05, **P < 0.005, and ***P < 0.0005.

Hyperoxia-Induced NMLF Bioenergetic Dysfunction Is Most Severe In C57ⁿ/C57^mt Mice and Attenuated In C3Hⁿ/C3H^mt Mice

Mice with similar nDNA but different mtDNA were compared. Basal and maximal OCR of NMLF from C57WT and C3HMNX strains were similar to those from C57MNX and C3HWT strains, respectively, when exposed to normoxia but lower when exposed to hyperoxia. NMLF ATP generation-linked OCR [median (Q1, Q3), pmol·min⁻¹·3 × 10⁴ cells⁻¹] was similar in normoxic C57WT versus C57MNX [135(128,143) versus 121(119,125)], and decreased in C3HMNX versus C3HWT [148(147,149) versus 198(194,206), P < 0.005]. It was lower in hyperoxic C57WT versus C57MNX and further decreased in C3HMNX versus C3HWT [60(56, 62) versus 164(162,169) and 107(104,112) versus 161(158,164), respectively, P < 0.005 for both comparisons].

Mice with different nDNA but similar mtDNA were also compared. Basal and maximal OCR of NMLF from normoxia and hyperoxia-exposed C57WT mice were similar to those from C3HMNX mice. NMLF from C3HWT mice had higher basal OCR (when exposed to normoxia or hyperoxia) and maximal OCR (only with normoxia) versus those from C57MNX mice (Fig. 3, A and B). ATP-linked OCR was similar between NMLF from normoxic and hyperoxic C57WT and C3HMNX strains [135(128,143) versus 148(147.149) and 60(56, 62) versus 107(104,112)]. It was higher in the normoxic C3HWT versus C57MNX mice [198(194,206) versus 121(119,125), P < 0.0005], but no difference was identified with hyperoxia exposure [161(158,164) versus 164(162,169)].

Mitochondrial O₂⁻ Generation Is Higher In NMLF From Hyperoxic Mice With C57 mtDNA

MitoSOX Red fluorescence intensity of NMLF from hyperoxic mice of all four strains was higher versus their normoxic controls. Mice with similar nDNA but different mtDNA were compared. MitoSOX fluorescence intensity of NMLF from normoxic C57WT and C3HMNX mice was similar to those from C57MNX and C3HWT mice, respectively. It was higher in hyperoxic C57WT and C3HMNX mice versus C57MNX and C3HWT mice, respectively. Mice with different nDNA but similar mtDNA were also compared with one another. MitoSOX Red fluorescence intensity of NMLF from normoxic or hyperoxic C57WT and C3HWT was similar to that from C3HMNX and C57MNX, respectively (Fig. 3C).

Mouse Sex Did Not Affect Lung Development or Mitochondrial Function

Mouse sex and length did not significantly modify alveolar development as measured by MLI and RAC, lung mechanics as measured through compliance and resistance estimates or NMLF bioenergetic or mitochondrial O₂⁻ generation. Additionally, these models indicated that, as weight increased, total lung resistance was lower in these mice.

Both C3H mitochondrial and nuclear DNA were associated with significant protection against hyperoxic lung injury as well as NMLF mitochondrial functional impairment (Tables 1 and 2).

Table 1.

Linear regression estimates of measures of neonatal alveolar development in hyperoxia-exposed mice with C57 and C3H nuclear and mitochondrial DNA

	Predictor
	MLI, µm		RAC (count)		Compliance, L/cmH2O		Resistance, cmH2O·s−1·mL−1
	Estimate	P	Estimate	P	Estimate	P	Estimate	P
Intercept	111.6 (86.4 – 136.7)	<0.001	4.8 (2.0 – 7.6)	0.002	1.7 (−1.0 – 4.4)	0.231	6.9 (4.7 – 9.1)	<0.001
Sex (female)	0.3 (−4.3 – 4.9)	0.894	0.0 (−0.5 – 0.5)	0.972	0.2 (−0.3 – 0.7)	0.428	0.1 (−0.3 – 0.5)	0.582
Weight, g	−2.0 (−4.6 – 0.6)	0.132	0.1 (−0.2 – 0.4)	0.643	−0.1 (−0.4 – 0.2)	0.475	0.4 (0.2 – 0.6)	0.001
Length, cm	6.7 (−4.3 – 17.7)	0.237	−0.1 (−1.4 – 1.1)	0.850	0.3 (−0.9 – 1.5)	0.596	−0.3 (−1.3 – 0.6)	0.511
nDNA (C3H)	11.7 (4.8 – 18.5)	0.002	0.1 (−0.7 – 0.8)	0.890	4.6 (3.8 – 5.3)	<0.001	−3.1 (−3.7 – −2.5)	<0.001
mtDNA (C3H)	−28.7 (−34.6 – −22.8)	<0.001	2.9 (2.2 – 3.5)	<0.001	5.6 (5.0 – 6.3)	<0.001	−4.1 (−4.6 – −3.6)	<0.001
Observations	64		64		64		64
R2/R2 adjusted	0.798/0.781		0.698/0.672		0.937/0.931		0.904/0.896

While increased body weight was associated with higher resistance, no other significant relationship was observed between sex, weight, or length of mice and alveolar development and lung mechanics. Both C3H nDNA and mtDNA were associated with improved alveolar development and lung mechanics when compared with C57 nDNA and mtDNA. MLI, mean linear intercept; RAC, radial alveolar count. P values < 0.05 are indicated in boldface.

Table 2.

Linear regression estimates of measures of mitochondrial bioenergetics and mitochondrial O₂⁻ generation in hyperoxia-exposed mice with C57 and C3H nuclear and mitochondrial DNA

	Predictor
	Basal OCR, pmol·min−1·3 × 104 cells−1		Maximal OCR, pmol·min−1·3 × 104 cells−1		MitoSOX Red, AFU
	Estimate	P	Estimate	P	Estimate	P
Intercept	100 (83 – 116)	<0.001	206 (124 – 287)	<0.001	190,939 (150,435 – 231,444)	<0.001
Sex (female)	1 (−2 – 5)	0.340	−8 (−23 – 7)	0.277	904 (−6,506 – 8,315)	0.812
Weight, g	0 (−1 – 2)	0.569	−1 (−10 – 7)	0.794	4,543 (387 – 8,699)	0.036
Length, cm	0 (−7–8)	0.903	−11 (−47 – 24)	0.537	4,649 (−13,096 – 22,394)	0.610
nDNA (C3H)	22 (18 – 27)	<0.001	46 (24 – 69)	<0.001	−52,897 (−63,961 – −41,834)	<0.001
mtDNA (C3H)	113 (109 – 117)	<0.001	176 (157 – 195)	<0.001	−78,803 (−88,256 – −69,351)	<0.001
Observations	64		64		64
R2/R2 adjusted	0.990/0.990		0.910/0.902		0.908/0.900

While increased body weight was associated with higher MitoSOX Red fluorescence, no other significant relationship was observed between sex, weight, or length of mice and neonatal mouse lung fibroblast (NMLF) mitochondrial function. Both C3H nDNA and mtDNA were associated with increased protection against hyperoxia-induced mitochondrial bioenergetic and redox dysfunction when compared with C57 nDNA and mtDNA. AFU, arbitrary fluorescence units; OCR, oxygen consumption rate. P values < 0.05 are indicated in boldface.

DISCUSSION

Strain-specific variation in hyperoxic lung injury severity has been described in in-bred mice with C3H strains considered to be relatively resistant and with C57 strains considered to be susceptible (2, 12). By exposing susceptible newborn C57BL6 and resistant C3H/HeN mice and their corresponding MNX strains to hyperoxia, we were able to assess the independent contribution made by mtDNA variations to differences in hyperoxic hypoalveolarization. We showed that alveolarization and lung compliance were significantly decreased in hyperoxic mice carrying C57 mtDNA (C57WT and C3HMNX) but not in mice carrying C3H mtDNA (C3HWT and C57MNX). However, we also noted that C57MNX mice had increased hyperoxic hypoalveolarization versus C3HWT mice. Additionally, at basal state (normoxia), mice with similar nDNA had similar lung structure irrespective of their mtDNA background. Therefore, our results suggest that, in addition to nuclear genome-mediated effects, mtDNA variations can also modify neonatal alveolar proliferation and BPD susceptibility and that mtDNA-mediated effects become especially pronounced when these mice are exposed to hyperoxia.

We have previously shown that mitochondrial function of human umbilical venous endothelial cells (HUVECs) obtained from preterm infants at their birth strongly predicts their risk for BPD. HUVEC mitochondrial basal and maximal OCR were higher in African-American infants (mtDNA haplotype H) versus Caucasian infants (mtDNA L haplotype) (15). In this study, NMLF basal and maximal OCR were only mildly decreased in hyperoxic C3HWT mice but markedly decreased in C57WT mice. Similar to hyperoxic hypoalveolarization severity noted in these strains, NMLF OCR was lower and O₂⁻ generation higher in neonatal mice with C57 mtDNA (C57WT and C3HMNX) versus mice with C3H mtDNA (C57MNX and C3HWT). These findings support the hypothesis that mtDNA haplotype variation-induced bioenergetic functional changes lead to differences in neonatal hyperoxic hypoalveolarization and BPD susceptibility.

Polymorphisms that differentiate C57BL6 and C3H/HeN mtDNA have been identified in genes that encode for the ND3 subunit of complex I (nucleotide 9461 T/A → C, methionine replaced by isoleucine as first amino acid), subunit III of complex IV (nucleotide 9348 G → A, valine is replaced by isoleucine as amino acid 248), and tRNA^Arg (TA insert at nucleotide 9818). While the ND3 mutation has not been associated with changes in the levels or activity of complex I between these mice, C-IV activity in cardiac mitochondria exposed to volume overload was higher in mice with the C57 mtDNA haplotype versus C3H mtDNA (8). Both developmental differences in expression (ND3 subunit) as well as clinical phenotypes due to mutations associated with functional oxidative phosphorylation differences (the tRNA^Arg gene) have been previously described (6, 25). Therefore, hyperoxia-induced differential expression of these ETC polymorphisms and conformational changes in the structure of the tRNA secondary to the TA base insertion are plausible candidate mechanisms for the decreased NMLF ATP-linked OCR and increased hyperoxic hypoalveolarization noted in mice with C57 mtDNA and require further study. In addition to isolated effects caused by mtDNA polymorphisms, studies that utilized MNX mice have also noted differences in nuclear genomic expression in mice with different mtDNA, indicating potential retrograde signaling effects caused by mitochondrial variations that may also be active in our animal model (4).

We used NMLF to assess pulmonary mitochondrial function since fibroblasts contribute to the fibrosis and inhibition of alveolar development that accompanies hyperoxia-induced BPD. Using Raman spectroscopy our group has previously observed that hyperoxic NMLF develop progressive intracellular acidosis and proteomic changes associated with hyperoxic lung injury (20). In the current study, hyperoxic mice carrying C57 mtDNA had higher NMLF mitochondrial O₂⁻ generation versus C3H mtDNA strains. Mitochondrial oxidant production, which increases linearly with intracellular Po₂, is considered to be the most important source of oxidants in the initial phases of hyperoxic lung injury (14). Certain mtDNA haplotypes lead to increased oxidant generation and mtDNA damage. Extrusion of mtDNA damage-associated molecular patterns (DAMPs) increases inflammatory lung injury and activates apoptotic mechanisms, which, in the developing lung, could result in dysregulated development at a critical phase of alveolar growth (1, 10). Connective tissue stains and hydroxyproline assays to quantify pulmonary fibrosis could therefore provide a direct link between NMLF mitochondrial dysfunction, increased fibrosis, and pulmonary hypoalveolarization.

Limitations of our study include lack of specific tests of glycolysis and ATP generation and the use of fluorescence-based methods to detect O₂⁻, which can be prone to measurement errors (which we mitigated by measuring relatively O₂⁻ specific wavelengths) (11). Finally, as mtDNA is highly susceptible to somatic mutations, deep sequencing of mtDNA is required to determine the impact of heteroplasmy on the phenotypical and functional differences noted in the lungs of these mice. Strengths of the study include using a well-established model of neonatal hyperoxia in the novel MNX mouse model to investigate the independent contribution of mtDNA in lung development and using a whole cell approach to study bioenergetic function rather than isolated mitochondria.

In summary, our study indicates that mtDNA variation plays a significant role in modulating the severity of neonatal hyperoxic hypoalveolarization and BPD in in-bred mouse strains. MtDNA modification using strategies such as targeted endonucleases is already being tested in some disease models (21). Therefore, investigating the mechanisms behind our results and firmly identifying the mitochondrial genome as a potentially useful and innovative therapeutic target could be useful for BPD. Replicating and expanding our mitochondrial functional assessments in human infant-derived cells is a logical next step.

GRANTS

This work is supported by funding from the Kaul Pediatric Research Institute Foundation of Children’s of Alabama and the American Thoracic Society (J. Kandasamy) and National Institutes of Health National Heart, Lung, and Blood Institute Grant R01 HL092906 (N. Ambalavanan).

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

J.K., G.R., S.W.B., and N.A. conceived and designed research; J.K., G.R., and T.J. performed experiments; J.K., G.R., T.J., and N.A. analyzed data; J.K., G.R., T.J., S.W.B., and N.A. interpreted results of experiments; J.K. prepared figures; J.K. drafted manuscript; J.K., T.J., S.W.B., and N.A. edited and revised manuscript; J.K., G.R., S.W.B., and N.A. approved final version of manuscript.