Hindawi
Oxidative Medicine and Cellular Longevity
Volume 2017, Article ID 9169146, 22 pages
https://doi.org/10.1155/2017/9169146
Research Article
Differential Alterations of the Mitochondrial Morphology and
Respiratory Chain Complexes during Postnatal Development of
the Mouse Lung
Natalia El-Merhie,1 Eveline Baumgart-Vogt,1 Adrian Pilatz,2 Susanne Pfreimer,1
Bianca Pfeiffer,1 Oleg Pak,3 Djuro Kosanovic,3 Michael Seimetz,3 Ralph Theo Schermuly,3
Norbert Weissmann,3 and Srikanth Karnati1
1Institute for Anatomy and Cell Biology II, Division of Medical Cell Biology, Justus Liebig University, Giessen, Germany
2Department of Urology, Pediatric Urology and Andrology, Justus Liebig University, Giessen, Germany
3Excellence Cluster Cardio-Pulmonary System (ECCPS), German Lung Center (DZL),
Universities of Giessen and Marburg Lung Center (UGMLC), Justus Liebig University, Giessen, Germany
Correspondence should be addressed to Eveline Baumgart-Vogt; eveline.baumgart-vogt@anatomie.med.uni-giessen.de
and Srikanth Karnati; srikanth.karnati@anatomie.med.uni-giessen.de
Received 21 August 2017; Accepted 28 September 2017; Published 19 December 2017
Academic Editor: Liang-Jun Yan
Copyright © 2017 Natalia El-Merhie et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Mitochondrial biogenesis and adequate energy production in various organs of mammals are necessary for postnatal adaptation to
extrauterine life in an environment with high oxygen content. Even though transgenic mice are frequently used as experimental
models, to date, no combined detailed molecular and morphological analysis on the mitochondrial compartment in different
lung cell types has been performed during postnatal mouse lung development. In our study, we revealed a significant
upregulation of most mitochondrial respiratory complexes at protein and mRNA levels in the lungs of P15 and adult animals in
comparison to newborns. The majority of adult animal samples showed the strongest increase, except for succinate
dehydrogenase protein (SDHD). Likewise, an increase in mRNA expression for mtDNA transcription machinery genes (Polrmt,
Tfam, Tfb1m, and Tfb2m), mitochondrially encoded RNA (mt-Rnr1 and mt-Rnr2), and the nuclear-encoded mitochondrial
DNA polymerase (POLG) was observed. The biochemical and molecular results were corroborated by a parallel increase of
mitochondrial number, size, cristae number, and complexity, exhibiting heterogeneous patterns in distinct bronchiolar and
alveolar epithelial cells. Taken together, our results suggest a specific adaptation and differential maturation of the mitochondrial
compartment according to the metabolic needs of individual cell types during postnatal development of the mouse lung.
1. Introduction and the matrix [3, 4]. The most important role of mitochon-
dria is the production of nicotinamide adenine dinucleotide
Mitochondria, commonly referred to as the “powerhouse” of (NADH) and adenosine triphosphate (ATP) [5–7]. The
the cell, are involved in energy production, β-oxidation of respiratory chain, comprising five protein complexes and
fatty acids, calcium buffering, cell signaling, proliferation residing in the inner mitochondrial membrane (IMM), is
and apoptosis, embryonic development, and general body responsible for the generation of ATP by oxidative
ageing [1, 2]. Structurally, mitochondria differ from other cell phosphorylation (OXPHOS) [8]. Five respiratory chain com-
organelles by possessing four distinct membrane compart- plexes are known as complex I (NADH-coenzymeQ oxidore-
ments: the outer mitochondrial membrane (OMM), the ductase), complex II (succinate-coenzymeQoxidoreductase),
intermembrane space (IMS), the inner mitochondrial complex III (coenzyme Q-cytochrome c oxidoreductase),
membrane (IMM) that forms invaginations called cristae, complex IV (cytochrome c oxidase), and complex V (ATP
2 Oxidative Medicine and Cellular Longevity
synthase) [9]. Most OXPHOS complexes are encoded by both wild-type mice. However, to date, no detailed combined
mitochondrial and nuclear subunits with the exception of molecular and morphological analysis has been performed
complex II, which is solely encoded by nuclear genes [10]. on the mitochondrial compartment during postnatal devel-
Moreover, mitochondria contain in their matrix a circular opment of the mouse lung. Postnatal alterations of the mito-
genome (mtDNA) that is essential for their function in chondrial compartment and its respiratory function have
oxidative phosphorylation. The mtDNA encodes 13 poly- been mainly described in rat liver and muscle [28, 39–44],
peptides of complexes I, III, IV, and V, 2 ribosomal RNAs, pig skeletal muscle [45], rat, rabbit, and bovine hearts
and 22 tRNAs [11]. [43, 46–49], mouse blood lymphocytes [50], and rat and
Mitochondrial metabolic pathways are interlinked, and mouse brain [51–53]. Therefore, here, we analyzed mito-
the whole mitochondria interact with other organelles, such chondrial alterations during postnatal development (new-
as the endoplasmic reticulum and peroxisomes, facilitating born, P15, and adult) of the mouse lung. Our results
intracellular/interorganellar communication and influencing revealed an upregulation of most mitochondrial complexes
cellular metabolic functions [12, 13]. The mitochondrial (complexes I, III, IV, and V) at the protein and the
membrane potential is essential for the normal functioning mRNA levels in the lung tissue samples of P15 and adult
of the mitochondrial respiratory chain, in which even under in comparison to newborn mice. These results were cor-
physiological conditions reactive oxygen species (ROS) are roborated by the upregulation of mRNAs for mtDNA
released [14]. Under pathological conditions, higher genera- transcription machinery genes, mitochondrially encoded
tion of ROS (H2O2) combined with NO leads to the produc- RNA, and mitochondrial DNA polymerase from the lungs
tion of peroxynitrite (ONOO−) or other RNS species [15, 16]. of the P15 and adult mice in comparison to the lungs of
Higher NO levels impair mitochondrial respiration and the newborn mice. Interestingly, a clear difference was
membrane potential and inhibit superoxide dismutase observed in the SDHD protein abundance pattern that
(SOD2), leading to a higher superoxide release [17, 18]. To peaked in the lungs of P15 animals and then decreased
combat higher generation of ROS, mitochondria employ a in the adult animals.
complex network of enzymatic and nonenzymatic defense
systems. These antioxidant systems include enzymes local-
ized within the mitochondrial matrix such as manganese- 2. Materials and Methods
containing superoxide dismutase (MnSOD), glutathione
2.1. Experimental Animals. Three groups of C57BL/6J mice
peroxidase (GPx), glutathione reductase (GR), thioredoxin
with different postnatal ages were used in this study: eighteen
II (Trx II), and peroxiredoxins I and III (Prxs I and III) as
newborn pups (P0.5) of four pregnant dames as well as 18
well as the enzymes localized within the mitochondrial inter-
membrane space such as copper/zinc (Cu/Zn) SOD [19–23]. male mice (9 mice at postnatal day and 15 and 9 adult mice
Mitochondria are also involved in acetyl-CoA production at 12 weeks of age). The mice were obtained from Charles
and in calcium metabolism [24, 25]. River (Sulzfeld, Germany) and were housed after the delivery
in cages at the central animal facility (Zentrales Tierlabor
Many energy-demanding physiological processes are ini-
(ZTL)) of the Justus Liebig University (Giessen, Germany).
tiated at birth, leading to the modification of the metabolic
They were maintained under standard environmental condi-
pathways important for energy production. This modifica-
tions with a 12-hour light/dark cycle at 23°C± 1°C and
tion, which triggers the switch from glycolysis to respiration,
55%± 1% relative humidity. Animals had access to normal
depends on the maturation of mitochondria [26, 27]. It is
laboratory diet and food ad libitum. All animal experiments
known that the acquirement of functional mitochondria after
in this study were approved by the German Government
birth is an important homeostatic process allowing newborn
Commission of Animal Care (RegierungspraesidiumGiessen,
mammals to adapt for the extrauterine life in an environment
Germany, permit number V 54-19 C 20/15 c GI 20/23).
with high oxygen levels. Therefore, the induction of many
mitochondrial enzymes occurs during the first hours of
postnatal life [28]. Further, during postnatal lung develop- 2.2. Perfusion Fixation, Sampling, and Tissue Processing for
ment also, mitochondria of nonciliated bronchiolar epithe- Routine Transmission Electron Microscopy (TEM). The
lial cells and type II alveolar epithelial cells (AECII) detailed protocol for perfusion fixation with a 1.5% parafor-
undergo extensive remodeling by significant alterations in maldehyde (PFA)/1.5% glutaraldehyde (GA) in 0.15M
their morphological appearance (volume density, size, HEPES buffer (pH7.4), sampling, and tissue processing of
number, and distribution) [29–32]. lungs for routine transmission electron microscopy (TEM)
It is becoming increasingly clear that mitochondrial was described previously [54]. Fixed lung tissue blocks of
dysfunction may promote or predispose to the onset of lung 1mm3 of the newborn, P15, and adult animals were embed-
diseases. Indeed, mitochondrial dysfunction with reduced ded into the epoxid Agar 100 Resin® (Agar, Essex, England)
levels of respiratory complexes has been shown in COPD and polymerized for 3 days at 60°C. Embedded tissue blocks
and non-COPD smokers as well as in bronchial epithelial were trimmed with a diamond trimmer (Reichert TM 60,
cells in mice suffering from asthma [33–38]. Given the exten- Austria), and then ultrathin sections (80 nm) were cut with
sive usage of wild-type and various genetically modified mice a Leica Ultracut E ultramicrotome (Leica, Nussloch, Ger-
to understand the pathophysiological changes, it is essential many). The cut sections were contrasted with uranyl acetate
to investigate mitochondrial biogenesis, metabolism, and (2min) and lead citrate (45 s) and thereafter examined with
maturation during regular postnatal lung development of a LEO 906 transmission electron microscope (LEO Electron
Oxidative Medicine and Cellular Longevity 3
Table 1: List of primary and secondary antibodies used in this study.
Cat. Dilution Dilution Dilution
Primary antibody against antigen Symbol Host Company
number (WB) (IF) (IEM)
Mitochondrial proteins
NADH:ubiquinone oxidoreductase core subunit 1 Rabbit,
MT-ND1 Abcam ab74257 1 : 500 — —
(complex I) polyclonal
Succinate dehydrogenase complex subunit D SDHD, Rabbit,
Millipore ABT110 1 : 3000 — 1 : 50
(complex II) CybS polyclonal
Mouse,
OXPHOS complex III core 2 subunit (complex III) UQCR2 Invitrogen A11143 — 1 : 250 —
monoclonal
OXPHOS complex IV subunit I/cytochrome c COX1, Mouse,
Invitrogen 459600 1 : 1000 — 1 : 100
oxidase I (complex IV) MT-CO1 monoclonal
OXPHOS complex IV subunit II/cytochrome c COX2, Mouse,
Invitrogen A6404 1 : 1000 — —
oxidase II (complex IV) MT-CO2 monoclonal
ATP synthase, H+ transporting mitochondrial F1 Mouse, Life
ATP5b A21351 1 : 3000 — —
complex, beta subunit (complex V) monoclonal Technologies
MT- Rabbit,
ATP synthase 6 (complex V) Santa Cruz Sc-20946 — — 1 : 50
ATP6E polyclonal
Polymerase (DNA) gamma 2, Mouse,
POLG2 Abcam ab66961 1 : 1000 — —
accessory subunit polyclonal
Goat,
Mitochondrial transcription factor A TFAM Santa Cruz Sc-19050 — — —
polyclonal
Cell-specific markers
Rabbit,
Prosurfactant protein C Pro-SP-C Chemicon ab3786 — 1 : 1000 —
polyclonal
Rabbit,
Club cell protein 10 (CC10) CC10 Santa Cruz sc-25555 — 1 : 1000 —
polyclonal
Loading control
Mouse, HyTest,
Glyceraldehyde 3-phosphate dehydrogenase GAPDH 5G4 1 : 8000 — —
monoclonal Finland
Secondary antibodies
Fab anti-rabbit IgG ultrasmall gold — Goat Aurion 800.255 — — 1 : 400
Fab anti-mouse IgG ultrasmall gold Goat Aurion 800.266 — — 1 : 400
Life
Anti-rabbit-IgG Alexa Fluor 488 — Donkey A21206 — 1 : 1000 —
Technologies
Life
Anti-mouse-IgG Alexa Fluor 555 — Donkey A31570 — 1 : 1000 —
Technologies
Goat, Sigma-
Anti-rabbit-IgG alkaline phosphatase conjugate — A0545 1 : 20,000 — —
polyclonal Aldrich
Goat, Sigma-
Anti-mouse-IgG alkaline phosphatase conjugate — A3562 1 : 20,000 — —
polyclonal Aldrich
Counterstaining of nuclei for immunofluorescence
Life
Hoechst 33342 (1 μg/ml) — — 33342 — 1 : 1000 —
Technologies
Life
TOTO®-3 iodide — — T-3604 — 1 : 1000 —
Technologies
Microscopy, Oberkochen, Germany) equipped with a 2k- ultrathin sections of lung tissue were incubated overnight in a
camera (TRS, Troendle systems). wet chamber with primary antibodies (Table 1) in 0.1% BSA-
c (Aurion) in PBS containing 0.05% Tween 20. On the next
2.3. Postembedding Immunoelectron Microscopy of day, the sections on grids were washed 6 times on a series
Mitochondrial Proteins. The detailed protocol for perfusion of 0.1% BSA-c (Aurion) in PBS containing 0.05% Tween 20
fixation with 4% (w/v) paraformaldehyde (PFA)/0.05% drops and then incubated for 120min with ultrasmall immu-
(v/v) glutaraldehyde (GA)/PBS, processing of lungs, embed- nogold goat anti-rabbit Fab fragments (Aurion) in 0.1%
ding into LR white was described previously [54–56]. Briefly, BSA-c (Aurion), diluted with 1 : 400 in PBS containing
4 Oxidative Medicine and Cellular Longevity
0.05% Tween 20. Then, the grids with sections were rinsed supernatant was discarded. The RNA pellet was washed
shortly (3× 3min) with 0.1% BSA in PBS containing 0.05% twice with 0.5ml of 75% ethanol per ml of supernatant.
Tween 20 followed by the washing with PBS (3× 3min). The RNA was centrifuged at 8000g for 3min at RT, and
Thereafter, the antigen-antibody complexes were fixed for the ethanol was removed. Finally, the RNA pellet was sol-
10min with 2% glutaraldehyde in PBS. The fixative was ubilized in RNase-free water at concentration of 1-2μg/ml.
washed away with a drop series of PBS (3× 3min), followed The purity and quantification of RNA was determined
by (6× 3min) aqua dest. Silver intensification was done with a spectrophotometer.
according to the method of Danscher in a light tight box
for 25min at RT [57]. Thereafter, grids with sections were 2.8. cDNA Synthesis. 1μg total RNA from adult, P15, and
washed for 6 times (3min each) with aqua dest. Sections new born lungs was reverse transcribed to cDNA using 1μl
were contrasted with uranyl acetate (2min) and lead of (dT) 12–18 primer (Invitrogen, Germany) and 1μl of
citrate (45 s) and thereafter examined with a LEO 906 SuperScript™ II Reverse Transcriptase (RT) Kit (Invitrogen,
transmission electron microscope (LEO Electron Micros- Germany) according to the manufacturer’s protocol. The
copy, Oberkochen, Germany) equipped with a 2k-camera reaction was incubated in a Biometra Trio Thermocycler
(TRS, Troendle systems). (The Netherlands). The qRT-PCR of target genes,
described in Table 2, was performed in the iCycler iQ5™
2.4. Immunofluorescence on Paraffin-Embedded Tissue. The Real-Time PCR Detection System (Bio-Rad, USA). The
detailed protocol for perfusion fixation, paraffin embedding, reactions were set up with the SYBR™ Green PCR mix
sectioning of lung tissue, and subsequent immunofluores- (Life Technologies) according to the manufacturer’s proto-
cence for newborn, P15, and adult lungs was described col. The PCR cycle consisted of an initial cycle of 95°C for
previously [55]. Briefly, perfusion-fixed lungs (4% PFA in 3min followed by 42 repeated cycles of 95°C for 15 s,
PBS, pH7.4) were embedded into paraffin (Paraplast, 60°C annealing temperature for 30 s, and the primer
Sigma-Aldrich, St. Louis, MO, USA) using an automated vac- extension at 72°C for 1min. The real-time PCR primer
uum tissue processor (Leica TP 1020) and sections (2-3μm) pairs used in this study are listed in Table 2. All reactions
were cut with Leica RM2135 rotation microtome and proc- were run in triplicates. Calculation of the relative gene
essed for double immunofluorescence. The dilutions of the expression was done by the 2−ddCT method, where dCT=
primary and secondary antibodies used are listed in (CTtarget gene−CTinternal control gene) using GAPDH as
Table 1. Negative controls for secondary antibody reaction an endogenous control.
were processed in parallel by addition of PBST buffer instead
of the first antibodies. Nuclei were visualized with 1μM 2.9. Statistics. Data are expressed as mean± standard
TOTO-3 iodide (molecular probes) for 10min at RT. Sam- deviation. Differences between groups were evaluated by Stu-
ples were analyzed by confocal laser scanning microscopy dent’s unpaired t-test and one-way analysis of variance
(CLSM) with a Leica TCS SP5 (Leica Mikrosysteme Vertrieb (ANOVA) using Tukey’s test. Data were considered statisti-
GmbH, Wetzlar, Germany). All images were processed with cally significant if p < 0 05.
Adobe Photoshop CS5.
2.5. Preparation of Whole Lung Homogenates for Protein 3. Results
Analysis (Western Blotting). For Western blotting, 3 lungs
3.1. Abundance of Mitochondrial Proteins in Lung
each from the newborn, P15, and adult mice were col-
− ° Homogenates. To characterize the differential abundance oflected and stored at 80 C prior to homogenization. The
mitochondrial encoded proteins, immunoblot analysis of
lung tissue of each group was cut into small pieces and
distinct mitochondrial proteins in lung homogenates from
homogenized in 2ml ice-cold homogenization buffer as
newborn, P15, and adult animals was performed. The results
previously described [55].
revealed a significant and continuous increase in the abun-
2.6. Western Blotting. ForWestern blot analysis, 50μg of lung dance of polymerase gamma 2 (POLG2), ATP synthase
homogenates from the newborn, P15, and adult mice was (ATP5b), and cytochrome oxidase subunit II (COX2) from
separated on 10% SDS-polyacrylamide gels using a Bio-Rad the newborn to adult lungs with high levels of abundance
gel electrophoresis apparatus (Bio-Rad, München, Germany) in adult lungs in comparison to the low levels of abundance
as previously described [55]. Dilutions of primary antibodies in the lungs of newborn animals (Figures 1(a) and 1(b)). Sim-
used are listed in Table 1. ilarly, cytochrome oxidase subunit I (COX1) also showed a
continuous upregulation as was observed in P15 and adult
2.7. RNA Isolation. For RNA isolation, 100mg of frozen lungs in comparison to the newborn lungs. However, the
lung tissue was homogenized with an IKA T 25 ULTRA protein abundance of COX1 detected in the newborn was
TURRAX (IKA, Germany) in 1ml RNAzol (RNAzol® higher than the ones for COX2 as well as ATP synthase and
RT, Sigma-Aldrich). Then, 0.4ml of RNase-free water per POLG2. Interestingly, the expression of NADP dehydroge-
ml of RNazol was added and left for 15min at RT. The nase complex I subunit I (MT-ND1) was only present in very
lysate was centrifuged at 12,000g for 15min and the low amounts in the lungs of newborns but increased thereaf-
supernatant was transferred to a fresh tube to which an ter to a still relatively low level in adults. The succinate dehy-
equal volume of 100% isopropanol was added. After that, drogenase complex II subunit D (SDHD) was differently
the lysate was centrifuged at 12,000g for 10min and the altered. It exhibited a higher abundance level in the newborn
Oxidative Medicine and Cellular Longevity 5
Table 2: List of primers used in this study for qRT-PCR (the annealing temp was 60°C).
Gene Sense primer (5′–3′) Antisense primer (5′–3′) Size of
Full name
target 20–23 mers 20–24 mers product
Mitochondrially encoded NADH:ubiquinone GCTTTACGAGCCGTAG GGGTCAGGCTGGCAGA
mt-Nd1 147
oxidoreductase core subunit 1 CCCA AGTAA
Mitochondrially encoded NADH:ubiquinone CCTCCTGGCCATCGTA GAATGGGGCGAGGCCT
mt-Nd2 124
oxidoreductase core subunit 2 CTCA AGTT
Mitochondrially encoded NADH:ubiquinone TAGTTGCATTCTGACT GAGAATGGTAGACGTG
mt-Nd3 100
oxidoreductase core subunit 3 CCCCCA CAGAGC
Mitochondrially encoded NADH:ubiquinone CGCCTACTCCTCAGTT TGATGTGAGGCCATGT
mt-Nd4 112
oxidoreductase core subunit 4 AGCCA GCGA
Mitochondrially encoded NADH:ubiquinone AGCTCCATACCAATCC GGACGTAATCTGTTCC
mt-Nd4l 109
oxidoreductase core subunit 4L CCATCAC GTACGTGT
Mitochondrially encoded NADH:ubiquinone GGCCCTACACCAGTTT AGGGCTCCGAGGCAAA
mt-Nd5 134
oxidoreductase core subunit 5 CAGC GTAT
Mitochondrially encoded NADH:ubiquinone CTTGATGGTTTGGGAG ACCCGCAAACAAAGAT
mt-Nd6 138
oxidoreductase core subunit 6 ATTGG CACC
GCTCGAGCTCTCCTAC GCTTGGTGACAGGTGA
Succinate dehydrogenase complex subunit D Sdhd 117
TCC ATGT
TCCTTCATGTCGGACG AATGCTGTGGCTATGA
Mitochondrially encoded cytochrome b mt-Cytb 100
AGGC CTGCG
mt-Co1, TCAACATGAAACCCCC GCGGCTAGCACTGGTA
Mitochondrially encoded cytochrome c oxidase I 100
Cox1 AGCCA GTGA
mt-Co2, ACCTGGTGAACTACGA TCCTAGGGAGGGGACT
Mitochondrially encoded cytochrome c oxidase II 121
Cox2 CTGCT GCTC
mt-Co3, CCAAGGCCACCACACT GGTCAGCAGCCTCCTA
Mitochondrially encoded cytochrome c oxidase III 150
Cox3 CCTA GATCA
AGCTCACTTGCCCACT AAGCCGGACTGCTAAT
Mitochondrially encoded ATP synthase 6 mt-Atp6 114
TCCT GCCA
ACACCTTGCCTAGCCA GTGGCTGGCACGAAAT
Mitochondrially encoded 12S RNA mt-Rnr1 112
CACC TTACCA
ACACCGGAATGCCTAA ATACCGCGGCCGTTAA
Mitochondrially encoded 16S RNA mt-Rnr2 148
AGGA ACTT
GGCTGAGAGACTTGTA AGGTGCACCACTCCTA
Transcription factor B1, mitochondrial Tfb1m 150
GCCACT CATCAA
TTTGGCAAGTGGCCTG ACTGATTCCCCGTGCT
Transcription factor B2, mitochondrial Tfb2m 109
TGAC TTGACT
GCCCGGCAGAGACG GCCGAATCATCCTTTG
Mitochondrial transcription factor A Tfam 137
GTTAAA CCTCC
ACAACACCGTGATGCT GAACATCCTGGTCCCT
Polymerase (RNA) mitochondrial Polrmt 150
TGGC GCGT
CTGGTTGCGTCATCGG TGCTTCCCTTGCGTCC
Polymerase (DNA) gamma 2, accessory subunit Polg2 101
CTTC CAAT
TGGCAAAGTGGAGATT AAGATGGTGATGGGCT
Glyceraldehyde 3-phosphate dehydrogenase GAPDH 156
GTTGCC TCCCG
in comparison to all other mitochondrial proteins. Similar to 3.2. Relative mRNA Expression of Different Complex I Gene
other mitochondrial proteins, it was upregulated at P15 lungs Subunits during Postnatal (Newborn, P15, and Adult)
but decreased thereafter to an intermediate level in adult ani- Development of the Mouse Lung. To ascertain the expression
mals (Figures 1(a) and 1(b)). The reason for not presenting of genes encoding mitochondrial proteins during postnatal
blots for complex III is that the antibody did not work development, total RNA was isolated from 3 lung tissue
in Western blots, whereas it worked perfectly well in mor- samples of newborn, P15, and adult mice and subse-
phology for immunogold labelling (Figures 2(d)–2(f)), quently analysed by quantitative polymerase chain reaction
suggesting that this antibody mainly detected the native (qRT-PCR). These results revealed a continuous and sig-
(nondenatured) protein. nificant increase in the expression of all complex I genes
6 Oxidative Medicine and Cellular Longevity
Complex NB P15 AL ⁎⁎
1.5 ⁎ ⁎ 4 ⁎⁎⁎⁎ ⁎⁎⁎
I MT-ND1⁎⁎
1.0 3
II SDHD
⁎
2
0.5
COX1⁎⁎
1
IV
0.0 0
MT-ND1 SDHD
IV ⁎⁎COX2
8 ⁎⁎⁎⁎⁎⁎⁎⁎ 8 ⁎⁎⁎⁎⁎⁎⁎⁎
⁎⁎⁎ ⁎⁎
V ATP synthase ⁎ 6 6
4 4
⁎
POLG2
2 2
GAPDH 0 0
COX1 COX2
6 ⁎⁎⁎⁎ 8 ⁎⁎⁎⁎
⁎⁎⁎
⁎⁎ ⁎⁎⁎⁎
6 ⁎⁎
4
4
2
2
0 0
ATP synthase POLG2
Newborn
P15
Adult
(a) (b)
Figure 1: Western blot analyses of distinct mitochondrial proteins in lung homogenates from newborn, P15, and adult mice. (a) Following
the homogenization, 50 μg of protein isolated from the lungs of each group (NB, P15, and adult) was resolved by 10% SDS-polyacrylamide gel
electrophoresis, and the blots were immunostained with anti-MT-ND1, anti-SDHD, anti-COX1 (OXPHOS complex IV subunit I),
anti-COX2 (OXPHOS complex IV subunit II), anti-ATP synthase (ATP5b), and anti-POLG2. GAPDH was used as a loading
control. ∗Nuclear-encoded protein. ∗∗Mitochondrially encoded protein. (b) Bar graphs summarizing normalized data. p values were
calculated by the one-way ANOVA using Tukey’s test. n = 3; ∗p ≤ 0 05, ∗∗p ≤ 0 01, ∗∗∗p ≤ 0 001, and ∗∗∗∗p ≤ 0 0001.
(mt-Nd1, mt-Nd2, mt-Nd3, mt-Nd4, mt-Nd4l, mtNd5, and (by approximately 2 and 2.8 times) in P15 and adult lungs,
mt-Nd6) (Figures 3(a)–3(g)). This mRNA upregulation respectively, in comparison to mt-Nd3, mt-Nd4l, and mtNd5
was observed in the lungs from both P15 and adult ani- (Figures 3(c), 3(e), and 3(f)). The expression of complex I
mals in comparison to the lungs from the newborn mice. subunit 1 gene mt-Nd1 (Figure 3(a)) was the lowest among
Similarly, the mRNA expression of these genes showed a the other complex I subunits by only showing an increase
significant elevation in the lungs from adult animals in of by 1.5 and 2 times in the P15 and adult animals, respec-
comparison to the lungs from the P15 mice. Interestingly, tively, in comparison to the newborns.
the mt-Nd5 (Figure 3(f)) showed the highest expression
among the other complex I subunits, where an increase 3.3. Relative Gene Expression Levels for Complex II–VmRNAs
of 2.5 and 6 times was observed in the lungs from P15 during Different Stages of the Postnatal Mouse Lung
and adult animals, respectively, as compared to the neonates. Development. Real-time PCR results of the total lung RNA
The second highest increase in the gene expression was content revealed a continuous increase in the expression of
detected for mt-Nd3 (Figure 3(c)), mt-Nd4l (Figure 3(e)), the mRNA for the nuclear-encoded succinate dehydrogenase
and mt-Nd6 (Figure 3(g)) genes where a 2 and 4 times subunit D (Sdhd) (Figure 4(a)), mitochondrially encoded
increase in the mRNA levels was observed in the lungs cytochrome b (mt-Cytb) (Figure 4(b)), mitochondrially
of the 15-day and 12-week animals, respectively. The encoded cytochrome c oxidase subunits I and II (mt-Co1
real-time PCR results showed that mt-Nd2 and mt-Nd4 and mt-Co2) (Figures 4(c) and 4(d)), and mitochondrially
(Figures 3(b) and 3(d)) genes were much less upregulated encoded ATP synthase 6 (mt-Atp6) (Figure 4(f)) from
Ratio to GAPDH Ratio to GAPDH Ratio to GAPDH
Ratio to GAPDH Ratio to GAPDH Ratio to GAPDH
Oxidative Medicine and Cellular Longevity 7
NB UQCR2 P15 UQCR2 AL UQCR2
S
(a) (b) (c)
NB COX1 P15 COX1 AL COX1
S
(d) (e) (f)
NB ATP6E P15 ATP6E AL ATP6E
(g) (h) (i)
Figure 2: Electron micrographs showing immunogold labelling for mitochondrial proteins in ultrathin sections of club cells in newborn
(NB), P15, and adult (AL) animals. Lung tissue processed for immunoelectron microscopy was incubated with gold-labelled secondary
antibody particles and thereafter contrasted with uranyl acetate and lead citrate prior to analysis by transmission electron
microscopy. (a–i) Immunogold labelling in mitochondria of club cells for (a–c) complex III (UQCR2), (d, e) complex IV (COX1),
and (g, h) complex V (ATP6E). S, secretory granule. Bars: a, c, e, g, and i = 0.5μm and b, d, f, and h = 0.25 μm.
newborn to adult stage. Complex II Sdhd (Figure 4(a)) exhib- only increased by 1.5 and 2.5 times in P15 and adult animals,
ited the lowest but still significant increase of gene expression respectively, and these expression levels were the lowest com-
among all mRNAs for respiratory complexes. The mRNA pared to the ones for complex III subunits II (mt-Co2) and III
expression level for complex III (mt-Cytb) (Figure 4(b)) was (mt-Co3). When compared to the mRNAs for respiratory
strongly upregulated (4 times) in the lungs of adults in com- complex III subunits, the mRNA level of mt-Co3 is not
parison to the lungs of the newborns. Interestingly, the induced significantly between the values of P15 and adult
expression of the mRNAs for the three mitochondrially animals. The mRNA levels for mt-Atp6 (Figure 4(f)) exhib-
encoded complex IV subunits were altered differently. The ited a 1.5 and 2.5 times elevation in the lungs from P15 and
mt-Co2 (Figure 4(d)) was the most highly expressed among adult mice in comparison to newborn animals.
the other 2 subunits since it already increased 3 times in the
lungs from P15 animals and up to 4 times in the adult lungs 3.4. Expression of mtDNA Transcription Machinery,
in comparison to the situation in the lung samples from the Mitochondrially Encoded RNAs, and Mitochondrial DNA
newborns. The mRNA level for mt-Co1 (Figure 4(c)) was Polymerase Genes. Real-time PCR results revealed that the
Club cells
8 Oxidative Medicine and Cellular Longevity
⁎⁎⁎ ⁎⁎⁎
2.5 ⁎⁎ 3 ⁎ 5 ⁎⁎⁎⁎ ⁎⁎ ⁎⁎
⁎
2.0 4
2
1.5 3
1.0 1 2
0.5 1
0.0 0 0
mt-Nd1 mt-Nd2 mt-Nd3
Newborn Newborn Newborn
P15 P15 P15
Adult Adult Adult
(a) (b) (c)
⁎⁎ ⁎⁎
4 ⁎ 6 ⁎⁎ 8 ⁎⁎⁎
⁎ ⁎ ⁎⁎
⁎⁎
3 6
4
2 4
2
1 2
0 0 0
mt-Nd4 mt-Nd4l mt-Nd5
Newborn Newborn Newborn
P15 P15 P15
Adult Adult Adult
(d) (e) (f)
⁎⁎
5 ⁎
4 ⁎⁎
3
2
1
0
mt-Nd6
Newborn
P15
Adult
(g)
Figure 3: mRNA expression of the mitochondrially encoded complex I genes in the new born, P15, and adult lung tissues as determined via
quantitative real-time PCR (qRT-PCR) analysis. Total RNA was extracted from the lung tissues of newborn (NB), P15, and adult (AL) mice
using RNAzol, reverse transcribed and subjected to qRT-PCR using specific primers for the mRNAs for distinct subunits of the
mitochondrially encoded complex I (mt-Nd1, mt-Nd2, mt-Nd3, mt-Nd4, mt-Nd4l, mt-Nd5, and mt-Nd6). The bar graphs represent relative
levels of the transcripts in three independent experiments. p values were calculated by the one-way ANOVA using Tukey’s test. Statistical
significance is indicated by ∗p < 0 05, ∗∗p < 0 01, and ∗∗∗p < 0 001. The mRNA levels were normalized to the ones of GAPDH.
rRNA expression patterns of genes encoding mitochondrial gene was observed in the P15 lungs in comparison to the
RNA were distinct from each other. A 1.4 and 2.4 times newborn and adult animals. Tfb1m and Tfam (Figures 5(c)
increase in the mt-Rnr1 (Figure 5(a)) expression was and 5(e)), the genes encoding transcription factors impli-
observed in the lungs from P15 and adult mice, respectively. cated in the mitochondrial DNA (mtDNA) transcription
The second mitochondrial encoded rRNA, mt-Rnr2 machinery, were regulated in a similar pattern with 1.7 times
(Figure 5(b)), only showed a 1.4 times increase in the lungs higher expression levels in the lungs of P15 and adult animals
of adult animals in comparison to the lungs of newborn mice when compared to the newborn mice. No significant changes
whereas no significant change in the rRNA expression of this in the expression levels of these genes were observed between
Relative mRNA levels
Relative mRNA levels
Relative mRNA levels Relative mRNA levels Relative mRNA levels
Relative mRNA levels Relative mRNA levels
Oxidative Medicine and Cellular Longevity 9
(a) (b) (c)
(d) (e) (f)
Figure 4: mRNA expression of the complex II–V genes in new born, P15, and adult lung tissues as determined by real-time PCR analyses.
Total RNA was extracted from lung tissue of newborn (NB), P15, and adult (AL) mice using RNAzol, reverse transcribed and
subjected to qRT-PCR using specific primers for mRNAs of (a) the nuclear-encoded succinate dehydrogenase complex II subunit D
(Sdhd), (b) the mitochondrially encoded complex III (mt-Cytb), (c–e) the different complex IV subunits (mt-Co1, mt-Co2, and mt-
Co3), and (f) the mitochondrially encoded complex V (mt-Atp6). The bar graphs represent relative levels of the transcripts in three
independent experiments. p values were calculated by the one-way ANOVA using Tukey’s test. Statistical significance is indicated by
∗p < 0 05, ∗∗p < 0 01, ∗∗∗p < 0 001, and ∗∗∗∗p < 0 0001. The mRNA levels were normalized to the ones of GAPDH.
the P15 and adult stages. However, the mRNAs for Tfb2m different stages of postnatal development. The micrographs
and Polrmt (Figures 5(d) and 5(f)), which encode proteins of the ciliated cells (left panel) from the newborns
involved in the mtDNA transcription machinery, showed a (Figures 6(a) and 6(b)) showmany mitochondria with lamel-
significant continuous postnatal elevation in the mRNA lar cristae in the apical part of the cell directly underneath of
expression. The expression of mitochondrial DNA polymer- the basal bodies to which cilia are attached. Additionally, the
ase Polg2 (Figure 5(g)) was differentially altered, exhibiting pools of glycogen were observed in the cytoplasm of the cili-
only a significant 2-time elevation in the mRNA levels in ated cells from the newborns. Starting from P15, the mito-
the adult lungs in comparison to the ones of newborn and chondria changed to larger and elongated structures with
P15 stages. more prominent cristae and a decline in the glycogen content
was well observed postnatally.
3.5. Imaging of Mitochondrial Structural Alterations by Figure 6 (middle panel) reveals the typical organelle
Transmission Electron Microscopy (TEM). To visualize the distribution in the club cells. The micrographs of the club
mitochondrial ultrastructure in the uranyl acetate and lead cells from the newborns (Figures 6(g) and 6(h)) revealed
citrate, contrasted ultrathin lung tissue sections from new- elongated mitochondria possessing lamellar cristae. Besides
born, P15, and adult mice transmission electron microscopy the mitochondria, a substantial part of the cytoplasm in club
(TEM) were applied. The lung tissue from the newborn (NB), cells of newborn animals was filled with glycogen deposits.
P15, and adult (AL) animals exhibited normal ultrastructure Almost no evidence of secretory activity was detected in the
revealing alterations in the organization of mitochondria in club cells of the newborns, whereas secretory granules were
the ciliated cells (left panel), club cells (middle panel), and present in P15 (Figures 6(i) and 6(j)) animals. These data
alveolar epithelial type II (AECII) cells (right panel) in show distinct cell-autonomous ultrastructural changes in
10 Oxidative Medicine and Cellular Longevity
⁎⁎ ⁎
3 ⁎⁎ 2.0 ⁎⁎ 2.0
⁎⁎
1.5 1.5
2
1.0 1.0
1
0.5 0.5
0 0.0 0.0
mt-Rnr1 mt-Rnr2 Tfb1m
Newborn Newborn Newborn
P15 P15 P15
Adult Adult Adult
(a) (b) (c)
(d) (e) (f)
(g)
Figure 5: mRNA expression of the mtDNA transcription machinery genes, mitochondrially encoded RNA, and mitochondrial DNA
polymerase in the new born, P15, and adult lung tissues as determined via quantitative real-time (qRT-PCR) analysis. Total RNA was
extracted from the lung tissues of newborn (NB), P15, and adult (AL) mice using RNAzol, reverse transcribed and subjected to qRT-PCR
using specific primers of the mtDNA transcription machinery (Polrmt, Tfam, Tfb1m, and Tfb2m), mitochondrially encoded RNAs
(mt-Rnr1 and mt-Rnr2), and mitochondrial DNA polymerase (Polg2) involved in mitochondrial DNA replication. The bar graphs
represent relative levels of the transcripts in three independent experiments. Statistical significance is indicated by ∗p < 0 05, ∗∗p < 0 01,
and ∗∗∗p < 0 001. The mRNA levels were normalized to the ones of GAPDH.
the club cells of newborn (Figures 6(g) and 6(h)) and P15 amounts—appeared to be reduced. Electron-dense secretory
(Figures 6(i) and 6(j)) animals. The mitochondria in these granules (S) were clearly observed in the club cells of P15
cells were more numerous but contained fewer cristae. More- animals suggesting secretory activity. The micrographs of
over, the cytoplasmic glycogen—although still present in high the club cells in adult animals (Figures 6(k) and 6(l))
Relative mRNA levels
Relative mRNA levels
Relative mRNA levels
Oxidative Medicine and Cellular Longevity 11
Ciliated cells Club cells AECII cells
NB NB NB NB NB NB
L
N
(a) (b) (g) (h) (m) (n)
P15 P15 P15 P15 P15 P15
L
S
(c) (d) (i) (j) (o) (p)
AL AL AL AL AL AL
S L
(e) (f) (k)  (l) (q) (r)
Figure 6: Transmission electronmicrographs of mitochondrial ultrastructure in the ciliated cells, club cells, and alveolar epithelial type II cells
(AECII) from the lungs of newborn (NB), P15, and adult (AL) animals. Ultrathin sections of lung samples for routine electron microscopy
were contrasted with uranyl acetate and lead citrate prior to analysis by transmission electron microscopy. The left panel (a–f) represents
the TEM images of ciliated cells, the middle panel (g–l) represents the club cells, and the right panel (m–r) represents the AECII cells.
Higher magnifications of selected areas (b, d, f, h, j, l, n, p, and r). L, lamellar body; N, nucleus; S, secretory granule. Bars: a, c, e, g, i, k, m,
o, and q = 0.5μm and b, d, f, h, j, l, n, p, and r = 0.25μm.
revealed large and elongated mitochondria in the club- lamellar bodies were detected in the cytoplasm of AECII at
formed apex which were almost devoid of cristae. More- this stage. Starting from P15 (Figures 6(o) and 6(p)), the
over, these micrographs demonstrated an increase in the mitochondria changed in shape into a more elongated
abundance of secretory granules and a strong decrease and branched structures with a more complex lamellar
in the proportion of cytoplasmic glycogen in the adult cristae as was also seen in adult animals (Figures 6(q)
club cells in comparison to the 2 previously mentioned and 6(r)). In addition to this, alveolar epithelial type II
postnatal stages. cells of P15 to adult animals showed a gradual decline in
The micrographs of the right panel depict typical the cytoplasmic glycogen amount. Despite a decline in gly-
ultrastructure of the alveolar epithelial type II cells (AECII). cogen content, the lamellar body number, size, and mature
Similar to the ultrastructure of club cells, AECII of the new- appearance with parallel lamellae gradually increased up to
born animals (Figures 6(m) and 6(n)) showed an abundant the adult stage.
glycogen content in their cytoplasm. The mitochondria,
during this stage of postnatal development, appeared as sin- 3.6. Immunofluorescence Staining for Complex IV Subunits I
gle, spherical structures. Moreover, only few whirl-shaped and II. Immunofluorescence preparations of lung tissue
12 Oxidative Medicine and Cellular Longevity
NB Pro-SP-C NB Complex IV-I Overlay
(a) (b) (c)
P15 Pro-SP-C NB Complex IV-I Overlay
(d) (e) (f)
AL Pro-SP-C AL Complex IV-I Overlay
(g) (h) (i)
Figure 7: Representative immunofluorescence analysis of mitochondrial complex IV subunit I protein in AECII from lung tissue sections of
newborn (NB), P15, and adult (AL) animals. Lung tissue samples from the three postnatal stages were embedded into paraffin. Thereafter,
3μm paraffin sections were cut with a rotation microtome and processed further for indirect double immunofluorescence. The lung
sections were incubated overnight for double labelling with primary antibodies against mitochondrial complex IV subunit I and pro-SP-C,
a marker for AECII (Table 1). The following morning, the sections were washed and incubated with the secondary antibodies (Table 1)
for 2 h at room temperature. Double fluorescence samples were analyzed by confocal laser scanning microscopy (CLSM) with a Leica TCS
SP5. (a, d, and g) Double IF stainings of AECII with their marker protein pro-SP-C. (b, e, and h) IF preparations for the mitochondrial
complex IV subunit I. (c, f, and i) Double IF overlay for complex IV subunit I combined with pro-SP-C. NB, newborn; P15, postnatal day
15; AL, adult. Bars represent 20μm.
samples from newborn (NB), P15, and adult (AL) mice in comparison to the lungs from P15 (Figures 7(d)–7(f))
showed an increase in the abundance of the mitochon- and newborn mice (Figures 7(a)–7(c)). Despite lower
drial complex IV subunit I in mitochondria in AECII abundance of mitochondrial complex IV subunit I, when
during postnatal development. The abundance of complex compared to the mitochondrial complex IV subunit II,
IV subunit I gradually increased exhibiting the highest levels a gradual increase was observed in the newborn
in the type II cells of the adult lungs (Figures 7(g)–7(i)) (Figures 8(a)–8(c)) and P15 animals (Figures 8(d)–8(f)).
Oxidative Medicine and Cellular Longevity 13
NB Pro-SP-C NB Complex IV–11 Overlay
(a) (b) (c)
P15 Pro-SP-C NB Complex IV–11 Overlay
(d) (e) (f)
AL Pro-SP-C AL Complex IV–11 Overlay
(g) (h) (i)
Figure 8: Representative immunofluorescence analysis of mitochondrial complex IV subunit II protein in AECII from lung tissue sections of
newborn (NB), P15, and adult (AL) animals. Lung tissue samples from the three postnatal stages were embedded into paraffin. Thereafter,
3μm paraffin sections were cut with a rotation microtome and processed further for indirect double immunofluorescence.The lung
sections were incubated overnight for double labelling with primary antibodies against mitochondrial complex IV subunit II and pro-SP-
C, a marker for AECII (Table 1). The following morning, the sections were washed and incubated with the secondary antibodies (Table 1)
for 2 h at room temperature. Double fluorescence samples were analyzed by confocal laser scanning microscopy (CLSM) with a Leica TCS
SP5. (a, d, and g) Double immunofluorescence stainings of AECII with their marker protein pro-SP-C. (b, e, and h) IF preparations for
the mitochondrial complex IV subunit II. (c, f, and i) Double IF overlay for complex IV subunit II combined with pro-SP-C. NB,
newborn; P15, postnatal day 15; AL, adult. Bars represent 20μm.
High levels of complex IV subunit II were noted in mito- 3.7. Postembedding Immunoelectron Microscopy for Different
chondria of AECII in adult animals (Figures 8(g)–8(i)). Mitochondrial Respiratory Complexes. In order to analyze
These differences in complex IV subunit abundance as the abundance of mitochondrial respiratory complex
observed in the IF preparations completely corroborated proteins on the ultrastructural level in individual mito-
the Western blot results (Figure 1(a)) in lung tissue chondria in lung tissue samples from newborn, P15, and
homogenates. adult mice, LR white-embedded lung ultrathin sections
14 Oxidative Medicine and Cellular Longevity
NB UQCRB P15 URCRB AL UQCRB
L L
L
L
L
L
L
(a) (b) (c)
NB COX1 P15 COX1 AL COX1
L L
N
L
L
(d) (e) (f)
NB ATP6E P15 ATP6E AL ATP6E
L L
L L
L
L L
(g) (h) (i)
Figure 9: Electron micrographs showing immunogold labelling for mitochondrial proteins in ultrathin sections of AECII in newborn (NB),
P15, and adult (AL) animals. Lung tissue processed for immunoelectron microscopy was incubated with gold-labelled secondary antibody
particles and thereafter contrasted with uranyl acetate and lead citrate prior to analysis by transmission electron microscopy. (a–i)
Immunogold labelling in mitochondria of AECII for (a–c) complex III (UQCR2), (d, e) complex IV (COX1), and (g, h) complex V
(ATP6E). S, secretory granule; L, lamellar bodies. Bars represent 0.5 μm.
were processed by postembedding immunocytochemistry newborn mice (Figures 2(a), 2(b), and 2(d)). A continuous
with the protein immunogold method and thereafter gradual increase of the gold particle-labelling density
analyzed by TEM. As shown in (Figures 2, 9 and 10), gold occurred during later stages of postnatal development of
particles clearly label mitochondria in the vicinity of their AECII exhibiting the highest labelling density in the stain-
cristae, suggesting a high specificity of the antibodies used. ings with the antibody against complex III (UQCR2)
Due to the low abundance of many respiratory complexes (Figures 9(b) and 9(c)). In contrast to AECII, club cell
in newborn animals and the use of the postembedding (Figures 2(b) and 2(c)) and ciliated cell (Figures 10(b)
technique allowing mainly surface labelling, only few gold and 10(c)) mitochondria showed significantly lower labelling
particles were present in mitochondria of AECII and ciliated for UQCR2 especially in the adult mice. We also noticed a
cells in newborn mice (Figures 9-10(a), 10(d), and 10(j)). The postnatal gradual increase of the gold particle-labelling
gold particle number in mitochondria of club cells was even density for complex IV subunit I (COX1) in club cells
lower, resulting in labelling of few club cell mitochondria in (Figures 2(e) and 2(f)), AECII (Figures 9(e) and 9(f)), and
AECII cells
Oxidative Medicine and Cellular Longevity 15
NB UQCRB P15 URCRB AL UQCRB
(a) (b) (c)
NB COX1 P15 COX1 AL COX1
(d) (e) (f)
NB ATP6E P15 ATP6E AL ATP6E
(g) (h) (i)
Figure 10: Electron micrographs showing immunogold labelling for mitochondrial proteins in ultrathin sections of ciliated cells in newborn
(NB), P15, and adult (AL) animals. Lung tissue processed for immunoelectron microscopy was incubated with gold-labelled secondary
antibody particles and thereafter contrasted with uranyl acetate and lead citrate prior to analysis by transmission electron
microscopy. (a–i) Immunogold labelling in mitochondria of ciliated cells for (a–c) complex III (UQCR2), (d, e) complex IV (COX1),
and (g, h) complex V (ATP6E). Bars represent 0.5 μm.
ciliated cells (Figures 10(e) and 10(f)). The intensity of the 4. Discussion
labelling for complex IV in all the three cell types was the
lowest in comparison to that of complexes III and V. In Nearly all cell types in the lung depend on the metabolic
addition to this, the gold particle labelling for complex V activity of the mitochondria for their energy supply that is
(ATP6E) as well increased postnatally in all the three cell generated via the mitochondrial respiratory chain and oxida-
types with the intensity of the labelling being very high in tive phosphorylation (OXPHOS) [58, 59]. Hence, mitochon-
AECII (Figures 9(h) and 9(i)) in comparison to club cells drial dysfunction can contribute to the pathophysiology of
(Figures 2(h) and 2(i)) and ciliated cells (Figures 10(h) and various pulmonary diseases such as bronchopulmonary
10(i)). The negative controls of Figures 2, 9 and 10 are dysplasia [60–62], chronic obstructive pulmonary disease
shown in Supplementary Figure 1 where no primary anti- (COPD) [63–65], lung cancer [66, 67], cystic fibrosis
body was used and the micrographs remained completely [68, 69], and asthma [70, 71]. This understanding of
devoid of staining suggesting a high specificity of the the human normal lung functioning and the mechanisms
secondary antibody. behind lung disease comes often from studies utilizing
Ciliated cells
16 Oxidative Medicine and Cellular Longevity
lung samples or animal models. Nowadays, frequently, such as biosynthesis of secretory products [30]. The cellular
mice are employed in lung research due to the advantages constituents of the club cells undergo significant shifts in
that this species provides [72, 73]. Surprisingly, only few organelle and glycogen deposit during differentiation, sug-
reports are available from the literature concerning gesting a metabolic shift from glycolysis to higher respiration.
mitochondrial biogenesis, mitochondrial function, and the In addition to these signs of cellular maturation, we detected
regulation of mtDNA genes during the postnatal lung devel- large and dense mitochondria which became more elongated
opment in mice. Most of the previously published data on the but lost their cristae in the adult mouse club cells as com-
postnatal development of the mitochondrial compartment pared to the P15 and newborn groups which is in agreement
were described in other organs such as the liver, heart, blood with a study in rat lung reporting that the mitochondria from
lymphocytes, brain, skeletal muscle, or cell types. Therefore, adult club cells were more devoid of cristae in comparison to
we investigated the differential expression and cell type the neonates [79]. Newborn and P15 animals in our study
specific differences of distinct mitochondrial respiratory showed a comparable ultrastructure, except for the appear-
chain proteins and factors of the mitochondrial transcription ance of secretory granules and a decrease in the number of
and translation machinery during postnatal development of cristae in the mitochondria of the club cells of P15 mice.
the mouse lung. The total number and volume of the club cells, the abun-
dance and expression of the club cell secretory protein
4.1. General Ultrastructure and Alterations of the (CC10), and the volume of lung and bronchioles increased
Mitochondrial Compartment during Postnatal Development postnatally in P15 and adult mice in comparison to the
of Different Cell Types in the Lung as Revealed by newborns suggesting that club cell maturation occurs post-
Transmission Electron Microscopy. Clear differences were natally [54]. Collectively, all these findings reveal the post-
noted in general ultrastructure and the development of the natal differentiation and maturation of the club cells in the
mitochondrial compartment in distinct lung cell types (cili- mouse lung.
ated, club, and AECII) of the bronchiolar or the alveolar epi- Ultrastructural analysis of AECII also revealed a gradual
thelium during postnatal mouse lung development. Similar decrease in glycogen amounts during the postnatal develop-
to the club and AECII cells, TEM analysis of the ciliated cells ment. Additionally, our micrographs showed an increase in
revealed an elongation and an increase in the size of mito- the number of lamellar bodies from the newborns to the
chondria starting from P15 in comparison to the newborns. P15 and adult animals. These results are in accordance with
In addition, this postnatal development of the ciliated cells other findings on the development and maturation of AECII
was accompanied by a decrease in the cytoplasmic glycogen in the rat model [29]. Moreover, our data showed an increase
deposits. These morphological changes could imply the post- in the mitochondrial length where more elongated mito-
natal development of the ciliated cells. It was reported by chondria with longer and more densely packed lamellar cris-
Francis and coworkers [74] that mice are born with few cili- tae were observed in AECII from P15 and adult mice as
ated cells in the trachea and that there is a postnatal increase compared to the newborn mice. These results are in agree-
in the ciliated cell density and cilia-generated flow in the ment with published data on mitochondrial shape and vol-
trachea of the C57BL/6 mice from postnatal day 0 to day ume in AECII of the lungs of Sprague-Dawley rats [29]. In
28. Another study by Sorokin [75] described that the earliest previous articles using different animal species, it was sug-
ciliary motion was observed at postnatal days 5 to 7 in culture gested that this increase in the volume of mitochondria and
series of fetal rat lungs. Additionally, ciliary vibration after change in its shape throughout the postnatal stages of devel-
first being detected in the trachea and largest bronchi appears opment could be associated with the growing metabolic
in the epithelium of smaller bronchial branches coinciding demands of the cell as well as with the production of a greater
with the pattern of epithelial differentiation in the fetal area-to-volume ratio [29, 32]. Furthermore, also in other
lung [76]. Furthermore, our micrographs showed the dis- organs, significant alterations of mitochondria were observed
tribution of mitochondria directly underneath the apical during postnatal development. For instance, Sato and col-
part of the cell, below the cilia. This distribution of mito- leagues detected larger and elongated mitochondria in the
chondria is necessary for the mammalian ATP-dependent brain cortex of Wistar rats from postnatal day 5 with cristae
ciliary beating [77]. reaching the maximal complexity in 15- and 21-day-old rats
TEM analysis of club cell ultrastructure in adult animals [52]. They speculated that this increase in the cristae com-
which showed a clear decrease in the proportion of cytoplas- plexity is correlated with the increase of respiratory enzyme
mic glycogen and a marked increase in the abundance of activities in the membrane of the mitochondria. This change
secretory granules in comparison to the club cells of the new- in the mitochondrial structure and shape in AECII and club
borns reflecting their higher degree of maturation in compar- cells is associated with morphological and functional differ-
ison to newborn club cells. This finding is in accordance with entiation of the cells as well as it is correlated with the total
studies from Plopper et al. [30] on the rabbit lung as well as lung volume enlargement during the postnatal development.
from Baskerville [78] on the pig lung, reporting a decrease Similar to the club cells, AECII differentiate and mature post-
in the cytoplasmic glycogen abundance and a significant natally where it was shown in rats that there is an increase in
increase in the amount of electron-dense secretory granules the fraction volume composition of AECII subcellular organ-
postnatally. Plopper and coworkers suggested that the glyco- elles (cytoplasm, mitochondria, and lamellar bodies) in adult
gen degradation would be required for energy production in Sprague-Dawley rats in comparison to the newborns [29].
order for a cell to initiate the important biogenesis processes Another study confirming the postnatal maturation of AECII
Oxidative Medicine and Cellular Longevity 17
in mice has shown that from E17.5 to P5 differentiation and are in agreement with the study by Valcarce and colleagues
maturation of AECII occurs that is associated with the ability [28] that showed a strong increase in the postnatal SDH
of the cells to secrete surfactant [80]. activity in the liver of albino Wistar rats until postnatal hour
Indeed, in our study, we were able to prove that mito- 6, and afterwards, the activity of the SDH started to decline. It
chondrial respiratory complexes, as well as transcription fac- was suggested that this increase in mitochondrial enzyme
tors and mitochondrial ribosomal RNA, were strongly activity is due to the increased rate of protein synthesis for
upregulated during postnatal development. In the following, mitochondrial enzymes after birth [28]. In a more recent
our results on alterations of respiratory complexes are com- study, Sato and colleagues [52] demonstrated as well an
pared in details to the international literature. upregulation in the activity of SDH, from theWistar rat brain
cortex that increased at 15 days postnatal, maintained at 21
4.2. Complex I. Complex I is known as the largest complex of days, and then decreased in mitochondria of adult animals.
the mitochondrial respiratory chain which transfers electrons The reason behind this is not clearly understood, but several
from NADH to coenzyme Q and uses this free energy to other studies showed a similar pattern of SDH activity. For
pump protons from the mitochondrial matrix into its inter- instance, Sieck and Blanco [87] reported in their study on
membrane space [81, 82]. The results of our study show a sig- the postnatal changes in the succinate dehydrogenase activity
nificant upregulation of mitochondrially encoded complex I in the diaphragm of cats that the muscle fiber SDH activity
gene as well as protein (MT-ND1) starting from the lungs increased between 3 and 6 weeks postnatally declining there-
of P15 mice and reaching the highest peak in the lungs of after to adult values. In addition, in a study on the postnatal
the adult mice. Comparable to our results, Bates and col- development of complex II in mitochondria isolated from
leagues [51] detected a significant increase in the activity of rat brain synaptosomes, an increase in the activities of com-
complex I in the rat brain from postnatal day 1 to day 21, plex II from day 10 to day 15, without significant alterations
suggesting that the increase is due to the high demand for of SHD activity thereafter, was reported [88].
mitochondrial ATP during the brain development. Interest-
ingly, not only mitochondrially encoded complex I subunits 4.4. Complex III. Complex III constitutes the central part of
increased in the expression during the postnatal development the mitochondrial respiratory chain and is composed of 11
as was shown by our study but also the nuclear-encoded different subunits where the cytochrome b gene mt-Cyb is
mitochondrial subunits as was revealed by Wirtz and the only complex III subunit encoded in the mitochondrial
Schuelke [53]. In their study on the expression of the 33 DNA (mtDNA) [89, 90]. Our data revealed a significant
nuclear-encoded complex I genes during postnatal develop- gradual upregulation in the expression of themt-CytbmRNA
ment of the C57BL/6J mouse brain, they found the rise of in the lungs of the mice starting from postnatal day 15 (P15)
expression intensity of the complex I around P11 in compar- in comparison to the lungs from the newborn animals. Our
ison to earlier stages and this coincided with the synaptogen- findings are in agreement with the results of the study from
esis [53, 83–85]. A more recent study of the mitochondria in Marin-Garcia and colleagues [49] where they detected a 3-
the intrinsic muscle of Wistar rat tongue demonstrated a and 4-fold increase in the mRNA expression of mt-Cytb in
gradual increase in the mRNA expression of the mitochond- the bovine hearts of late fetal and young adult stages, respec-
rially encoded complex I subunit mt-Nd1 from birth to 15 tively, in comparison to early fetal stages of development
days of age reaching the highest expression at 21 days of which correlated with the increase in mtDNA copy number.
age [44]. Comparable results were observed from Fujita and In addition to this, Schagger et al., in 1995, showed that the
Sato [44] for the level of NADH-O2 oxidoreductase activity total protein amount of complex III and its catalytic activity
in the intrinsic muscle of theWistar rat tongue. They hypoth- was increased in the heart and liver tissue samples of Wistar
esized that the hyperactivity in the NADH enzyme is indis- rats during transition from fetal to adult stage correlating
pensable for the transition from swallowing to mastication with the higher demands of oxidative phosphorylation activ-
and that the increase in mt-Nd1 could be a result of this ities in proliferating tissues [47]. Complex III activity was as
hyperactivity [44]. well significantly upregulated in the mitochondria from the
rat brain from postnatal day 1 to day 60 as was reported by
4.3. Complex II. Complex II is composed of 4 subunits which Bates and colleagues [51].
are encoded solely by nuclear DNA, with succinate dehydro-
genase (SDH) being the largest subunit. This complex forms 4.5. Complex IV. Complex IV transfers the electrons from
a direct link between the tricarboxylic acid (TCA) cycle and cytochrome c to molecular oxygen driving downstream
the respiratory chain [86]. Surprisingly, the only exception ATP biosynthesis. It consists of 14 subunits, three of which
of the general mitochondrial maturation pattern was (mt-Co1, mt-Co2, and mt-Co3) are encoded by mitochon-
observed in our study for succinate dehydrogenase subunit drial genome [91]. In our study, we analyzed the three mito-
D (SDHD) of complex II. We found that SDHD protein chondrially encoded subunits (subunits I–III) of complex IV
abundance peaked in the lung tissue samples from P15 mice at the mRNA (mt-Co1, mt-Co2, and mt-Co3) and protein
and then decreased in the lungs from the adult animals. (COX1, COX2) level and we found that the mRNA expres-
Moreover, although the mRNA expression of Sdhd was sion and abundance of this complex is drastically increased
found to gradually increase starting from the lungs of P15 starting from the lungs of P15 mice. However, the increase
animals, the relative increase of this expression was the low- in the abundance of these subunits, as shown by immunoflu-
est among all the 5 complexes studied. Interestingly, our data orescence and Western blot results, exhibited a different
18 Oxidative Medicine and Cellular Longevity
pattern of upregulation. The abundance of complex IV sub- 2 months, followed by a moderate decline at 5 months
unit I gradually increased displaying the highest levels in and then an increase again by 15 months of age [98]. This
the type II cells of the adult lungs in comparison to the lungs increase in mtDNA level is associated with periods of
from P15 and newborn mice. Similarly, a gradual increase in increased susceptibility of the tissues to oxidative stress
mitochondrial complex IV subunit II was as well observed; and injury during the early life and advance stages [98].
however, newborn and P15 animals exhibited lower levels Likewise, an increase in the expression of the mitochondrial
of this protein in comparison to the labelling for subunit I. transcription factor (TFB2M) and the mitochondrial RNA
The increase in the expression and abundance of complex polymerase (POLRMT) in growing rat hearts was shown
IV was the highest in comparison to other mitochondrial in adult and aged animals [99] suggesting of an increase
respiratory complexes. Moreover, we detected that complex is the mitochondrial biogenesis in maturing cardiomyocytes
IV, although upregulated in club and AECII cells, showed which fits the role of TFB2M as an accessory subunit of
different levels of upregulation between these lung cell types POLRMT required for promoter recognition [100, 101].
as was shown by electron microscopy. There was a continu- However, in contrast to our study, they showed that RNA
ous gradual increase of the gold particle labelling during later expression and protein abundance of TFAM remained
stages of postnatal development; however, in contrast to constant throughout life and this might be due to the fact
AECII, club cell mitochondria exhibited significantly lower that TFAM levels are correlated with the amount of
labelling for complex IV especially in adult mice. Our results mtDNA in the cell [99]. TFAM is an mtDNA-binding pro-
are in accordance with other studies which as well detected tein required for the transcription of mtDNA and is thought
the elevation of the complex IV during postnatal develop- to act as mtDNA copy number regulator [102, 103]. It was
ment of distinct species such as in the (1) Wistar rat suggested, in this study, that rats, apparently, do not need
hearts by Schagger and colleagues [47], (2) rat brain by an increase in mtDNA in order to cope with increased mito-
Almeida et al. [88], (3) Wistar rat heart homogenates by chondrial biogenesis during the postnatal heart development.
Drahota and colleagues [48], (4) bovine heart tissues by In addition to this, Pohjoismäki and colleagues [99] also
Marin-Garcia and colleagues [49], and (5) liver and muscle showed an elevation in the expression of Polg2 in 10-day-
COX activity in human samples by Pejznochova and col- old rats as compared to neonates. Mitochondrially encoded
leagues suggesting that the maturation of the mitochondrial RNA (rRNA) is necessary for mitochondrial protein bio-
compartment is important for the differentiation of tissues synthesis. After mtDNA is transcribed, mitochondria trans-
and organs in mammals [92]. late their mRNA on mitochondrial ribosomes consisting of
2 mtDNA-encoded rRNAs and nuclear-encoded proteins
4.6. Complex V. Mitochondrial complex V, ATP synthase, is [104]. Our results report an increase in the expression of
responsible for the synthesis of ATP from the ADP in the mitochondrial rRNA during the postnatal development of
mitochondrial matrix using the provided energy from the murine lungs. This increase in the expression is reasonable
proton electrochemical gradient [93, 94]. Nearly no labelling as it emphasizes the upregulation of the mitochondrial
for complex V was detected in the club cells of the newborn translation machinery and mitochondrial expression sys-
mice in comparison to the AECII in the newborns. Moreover, tems during the postnatal development showing that the
complex V showed a lower labelling in the club cell mito- cells are still proliferating.
chondria of the adult mice as compared to the labelling in
the AECII of adult animals. Our data on complex V are in 5. Conclusions
agreement with a study in which isolated brain mitochondria
of rats showed a significant increase in the activity of complex In this article, we show that the alterations of the mitochon-
V starting from postnatal day 10 in comparison to the post- drial compartment with special focus on mitochondrial
natal day 1 [51]. Similarly, Marin-Garcia and colleagues respiratory complexes and associated mitochondrial tran-
showed an increase in the expression of ATP-β Synthase scription machinery as well as mitochondrial rRNAs exhib-
mRNA in the bovine heart tissue from the late fetal and ited a specific adaptation and differential maturation of the
young adult animals [49]. mitochondrial compartment according to the metabolic
needs of individual cell types during postnatal development
4.7. Associated Mitochondrial Transcription Machinery and of the mouse lung.
Mitochondrial rRNAs. It is known that the initiation of mito-
chondrial transcription needs nucleus-encoded proteins, Conflicts of Interest
such as POLRMT, auxiliary factors for promoter recognition,
such as TFB1M and TFB2M, and promoter activation like The authors declare that they have no conflicts of interest.
Tfam [95–97]. These factors are required for the transcrip-
tion of mtDNA that controls the rRNA/mRNA ratio. We Authors’ Contributions
have noticed that the regulation of the machinery required
for the maintenance and expression of mtDNA showed a Eveline Baumgart-Vogt and Srikanth Karnati designed the
significant increase from postnatal day 15 to the adult stage research. Natalia El-Merhie, Susanne Pfreimer, Bianca
in comparison to the newborn mice. These results are in Pfeiffer, Oleg Pak, Djuro Kosanovic, Michael Seimetz,
accordance with earlier study showing a postnatal increase and Srikanth Karnati performed the research. Eveline
in the mtDNA content in the mouse lung during the first Baumgart-Vogt, Adrian Pilatz, Ralph Theo Schermuly,
Oxidative Medicine and Cellular Longevity 19
Norbert Weissmann, and Srikanth Karnati contributed Nature Reviews Molecular Cell Biology, vol. 15, no. 10,
reagents, materials, and analysis tools. Eveline Baumgart- pp. 634–646, 2014.
Vogt and Adrian Pilatz contributed the budget to the [12] V. Soubannier, G. L. McLelland, R. Zunino et al., “A vesicular
study. Natalia El-Merhie, Susanne Pfreimer, Bianca transport pathway shuttles cargo from mitochondria to lyso-
Pfeiffer, and Srikanth Karnati acquired the data. Natalia somes,” Current Biology, vol. 22, no. 2, pp. 135–141, 2012.
El-Merhie, Eveline Baumgart-Vogt, and Srikanth Karnati [13] R. Bravo-Sagua, A. E. Rodriguez, J. Kuzmicic et al., “Cell
analyzed and interpreted the data. Natalia El-Merhie, death and survival through the endoplasmic reticulum-
Eveline Baumgart-Vogt, and Srikanth Karnati wrote the mitochondrial axis,” Current Molecular Medicine, vol. 13,
paper. Eveline Baumgart-Vogt and Srikanth Karnati con- no. 2, pp. 317–329, 2013.
ceived and supervised the study. [14] E. Gottlieb, S. M. Armour, M. H. Harris, and C. B. Thompson,
“Mitochondrial membrane potential regulates matrix config-
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This study was supported by LOM (Leistungsorientierte [15] P. Ghafourifar and C. Richter, “Nitric oxide synthase activity
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Germany. drial calcium uptake stimulates nitric oxide production in
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pp. C406–C415, 2004.
Supplementary Figure 1: Electron micrograph of the negative [17] R. Radi, A. Cassina, R. Hodara, C. Quijano, and L. Castro,
control (NC) for Figures 8–10. (A–C) Ciliated cells, (D–F) “Peroxynitrite reactions and formation in mitochondria,”
club cells, and (G–I) AECII of newborn (NB), P15, and adult Free Radical Biology & Medicine, vol. 33, no. 11, pp. 1451–
(AL) animals. (Supplementary Materials) 1464, 2002.
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