Am J Physiol Lung Cell Mol Physiol 310: L103–L113, 2016.
First published November 13, 2015; doi:10.1152/ajplung.00320.2015.
Review
TRANSLATIONAL PHYSIOLOGY
Rejuvenating cellular respiration for optimizing respiratory function: targeting
mitochondria
Anurag Agrawal and Ulaganathan Mabalirajan
CSIR Institute of Genomics and Integrative Biology, Delhi, India
Submitted 8 September 2015; accepted in final form 10 November 2015
mitochondria; lung; chronic obstructive pulmonary disease; asthma; pulmonary
hypertension
CELLULAR RESPIRATION IS INEXTRICABLY intertwined with functioning of the respiratory system. Aerobic cellular metabolic
processes transfer the energy of carbon-hydrogen (C-H) bonds
in nutrients to high-energy phosphate bonds of ATP, consuming oxygen and generating carbon dioxide (oxidative phosphorylation, OxPhos). Ventilation, gas exchange, and circulation are tightly coupled to the cellular OxPhos, delivering
oxygen and removing carbon dioxide. Together, they determine the overall respiratory fitness of the organism, measured
as maximal oxygen consumption (V̇O2max) during exercise (57,
83). These well-established principles explain the diminished
exercise capacity in obstructive airway diseases (ventilation),
lung fibrosis (gas exchange), heart failure (circulation), or
deconditioning (cellular aerobic capacity) (42, 57). Mitochondria, often referred to as the powerhouse of the cell, are
typically placed at the end of this hierarchy because they house
the OxPhos reactions. However, the last two decades have seen
an explosion of information pertaining to the multifaceted roles
of mitochondria and their important roles in pathogenesis of
lung diseases beyond generating energy (14, 30, 88, 124).
Importantly, it has become evident that mitochondrial function
is a direct determinant of lung health, and targeting mitochondria is a viable strategy for preventing or treating lung disease
(110). The American Journal of Physiology Lung Cellular and
Molecular Physiology has recently published a perspective by
Schumacker and colleagues (103) highlighting the sentinel role
of mitochondria in regulating cytotoxicity and the intricate
mechanisms of mitochondrial quality control. Here, we will
focus on the role of mitochondrial dysfunction in the pathogenesis of lung disease, especially asthma and chronic obstructive pulmonary disease (COPD), and discuss possible mitochondria-targeted therapeutic strategies for restoring optimal
respiratory function. A brief introduction to the main actors is
provided in the initial section.
Mitochondria
Address for reprint requests and other correspondence: A. Agrawal, 615,
CSIR-IGIB, Mall Rd., Delhi Univ. (North Campus), Delhi, India 110007
(e-mail: [email protected]).
http://www.ajplung.org
Mitochondrial origin and function. Mitochondria are semiautonomous cell organelles, considered to be descendants of
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Agrawal A, Mabalirajan U. Rejuvenating cellular respiration for optimizing respiratory function: targeting mitochondria. Am J Physiol Lung Cell Mol
Physiol 310: L103–L113, 2016. First published November 13, 2015;
doi:10.1152/ajplung.00320.2015.—Altered bioenergetics with increased mitochondrial reactive oxygen species production and degradation of epithelial function
are key aspects of pathogenesis in asthma and chronic obstructive pulmonary
disease (COPD). This motif is not unique to obstructive airway disease, reported in
related airway diseases such as bronchopulmonary dysplasia and parenchymal
diseases such as pulmonary fibrosis. Similarly, mitochondrial dysfunction in
vascular endothelium or skeletal muscles contributes to the development of pulmonary hypertension and systemic manifestations of lung disease. In experimental
models of COPD or asthma, the use of mitochondria-targeted antioxidants, such as
MitoQ, has substantially improved mitochondrial health and restored respiratory
function. Modulation of noncoding RNA or protein regulators of mitochondrial
biogenesis, dynamics, or degradation has been found to be effective in models of
fibrosis, emphysema, asthma, and pulmonary hypertension. Transfer of healthy
mitochondria to epithelial cells has been associated with remarkable therapeutic
efficacy in models of acute lung injury and asthma. Together, these form a 3R
model—repair, reprogramming, and replacement—for mitochondria-targeted therapies in lung disease. This review highlights the key role of mitochondrial function
in lung health and disease, with a focus on asthma and COPD, and provides an
overview of mitochondria-targeted strategies for rejuvenating cellular respiration
and optimizing respiratory function in lung diseases.
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other cellular elements is still evolving, with mtROS as the
most evident and targetable aspect of mitochondrial pathology (24, 30).
MtROS. ROS production is an unavoidable consequence of
OxPhos. High-energy electrons pass through the mitochondrial
electron transport chain (ETC), culminating in the reduction of
oxygen to water in a reaction catalyzed by mitochondrial
cytochrome c oxidase (63). Energy transfer from electrons is
used to pump protons across the mitochondrial inner membrane into the intermembrane space, creating a measurable
electrochemical proton gradient (⌬⌿), which is in turn used by
ATP synthase to generate ATP (Fig. 1). Some electrons directly leak to oxygen at intermediary steps, resulting in formation of the superoxide anion radical (O2·⫺), which is further
decomposed to hydrogen peroxide (H2O2) by superoxide dismutase that is present at very high concentrations within
mitochondria (20, 109, 116). This is further decomposed by
catalase to water and oxygen. Although most mtROS are thus
scavenged within mitochondria, some leak to cytosol via
poorly understood mechanisms (132). Mitochondrial ROS release is thus dictated by primary factors such as redox state,
integrity of ETC components, rate of electron flow, and oxygen
availability, among others, as well as secondary factors, such
as local availability of antioxidants and influence of drugs or
chemicals (118). It is important to emphasize here that low
Fig. 1. Generation of reactive oxygen species (ROS) during electron transport. During aerobic respiration, electrons from NADH and FADH2 are transferred to
molecular oxygen via a chain of redox enzymes (Complex I–IV) embedded in the inner mitochondrial membrane (the dashed line indicates electron flux). The
energy is used to pump protons across the inner membrane, as shown, generating an electrochemical gradient (⌬⌿). This proton gradient is converted to ATP
by ATP synthase. Some electrons leak and react with molecular oxygen to generate superoxide (O2·⫺), especially at complexes I and III. The superoxide is further
converted into hydrogen peroxide (H2O2) by superoxide dismutase (SOD). H2O2 may give rise to hydroxyl radicals (●OH) or be decomposed to water and oxygen
by catalase. It can be seen that electron flux in excess of available oxygen at complex IV or reduced integrity of ETC would promote electron leak/ROS.
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endosymbiotic proteobacteria that, not only house metabolic
processes of OxPhos and fatty acid metabolism, but also
regulate cellular danger and damage responses. Positioned at
the hub of cellular metabolic flux, mitochondria are uniquely
adapted to communicate with the nucleus to bring about
cellular as well as extracellular responses to perceived threats
(88). These threats may range from altered availability of
nutrients or oxygen, sensed through changes in electron flow,
to presence of viral ds-RNA sensed through mitochondrial
antiviral signaling protein (124). Mitochondria respond
through release of reactive oxygen species (mtROS), changes
in mitochondrial membrane polarity, and signaling through
mitochondria-associated membranes and proteins. Initiation of
stress response pathways, NF-␬B, or interferon signaling cascades critically depends on such mitochondrial signals. Mitochondrial damage, with release of mitochondrial proteins or
DNA, is a powerful activator of immune signaling as well as a
trigger for apoptosis (30, 124). These innate cellular defense
systems are well-preserved aspects of innate immunity in most
species and are now well recognized to be associated with a
full spectrum of health and disease (24, 30, 124). Of these,
mtROS production is probably the best studied, especially in
the context of respiratory diseases, and will be discussed in
more detail. It should be emphasized here that the understanding of bidirectional mitochondrial communication with
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depolarized nonfunctional mitochondria are marked for selective degradation attributable to reduced ⌬⌿-dependent translocation of PTEN-induced kinase 1 (PINK1) from the outer to
inner mitochondrial membrane. This initiates a cascade of
events, leading to arrest of mitochondrial motility and capture
by a phagophore (mitophagy) (122). Thus an intricate system
of homeostasis maintains optimal mitochondrial function in
healthy cells, the failure of which leaves the cell susceptible to
mtROS-mediated injury (Fig. 2).
Connecting Mitochondrial Dysfunction with Lung Disease
Mitochondrial energy drives the functioning of more than 40
types of cells that compose the lung (70). Some of these, like
type II alveolar epithelial cells that secrete surfactant or ciliated
epithelial and Clara cells that line airways, are highly metabolically active and rich in mitochondria. Dysfunctional cellular bioenergetics leads to epithelial fragility, reduced barrier
function, impaired secretion, and increased propensity to inflammation (121). Bronchopulmonary dysplasia, asthma,
Fig. 2. Mitochondrial homeostasis and its
failure in disease. Mitochondrial dysfunction
may be a consequence of a wide variety of
external stressors acting via multiple pathways, as depicted, usually leading to oxidative stress. Homeostatic mechanisms like
mitochondrial biogenesis and fusion, with
elimination of defective mitochondrial components, maintain mitochondrial function,
promoting cellular recovery. Failure of mitochondrial homeostasis leads to cell death.
Mitophagy appears to be a dual-edged sword
in lung epithelium (see Refs. 9, 84). ADMA,
asymmetric di-methyl arginine.
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levels of mtROS are important in maintaining OxPhos and are
considered normal (34). Balance between ROS production and
antioxidant defense is a critical aspect of this healthy state.
Mitochondrial quality control. Excessive levels of ROS
production coupled with depletion of ROS scavengers are
associated with damage to the mitochondrial machinery, which
further accelerates ROS production (34, 118). Left unchecked,
this leads to degradation of mitochondrial function and fall in
the electrochemical gradient, ⌬⌿. Also, there is degradation of
mitochondrial DNA (mtDNA), which is much more susceptible than nuclear DNA to oxidative damage and lacks sophisticated repair machinery (11, 12). Mitochondrial dynamics,
referring to fission or fusion of mitochondria, enables mitochondria to maintain high functional capacity in adverse conditions. Fission leads to fragmented mitochondria that rapidly
diverge in their mtDNA and respiratory capacity, whereas
mitochondrial fusion promotes mixing and protects mtDNA
function through several distinct mechanisms. It maintains
mtDNA levels, preserves mtDNA fidelity, and enables cells to
tolerate high levels of mtDNA mutations (26). When all fails,
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such 13-S-hydroxyoctadecadienoic acid and asymmetric dimethyl arginine (ADMA) appear to be critical in epithelial
injury (5, 6, 77, 78). These may be endogenously generated in
response to inflammation or be exogenously delivered in the
case of systemic metabolic diseases that precipitate asthma (2,
3, 117). Overall, most lines of data converge to show that
mitochondrial dysfunction is an important part of epithelial
fragility in asthma. Multifarious mechanisms, as shown in Fig.
2, are implicated.
The evidence for an important role of mitochondrial abnormality in altered airway smooth muscle (ASM) function is
more conflicted. Trian et al. (112) reported about a twofold
increased mitochondrial number in ASM of patients with
severe persistent asthma, presumably attributable to increased
biogenesis. However, a more recent study, looking mostly at
fatal young asthma cases, did not find such an increase, even in
ASM surrounded by inflammatory cells (112). In cultured
human ASM, inflammation alters mitochondrial calcium flux
and inhibits calcium buffering, which raises cytoplasmic calcium, promotes ASM contraction, and may alter mitochondrial
metabolism or biogenesis (35). The Ca2⫹-channel blocker
gallopamil was found to decrease the proliferation of ASM
cells from patients with severe asthma and also reduced their
exacerbations (49). Thus ASM contractility, inflammation,
calcium flux, and mitochondrial function are intricately interlinked, and different subtypes of asthma may show different
patterns. Furthermore, based on the possibility of mitochondrial transfer in vivo, another possibility is that increased
mitochondrial biogenesis in ASM is a compensatory response
to epithelial mitochondrial loss. In support, we have shown the
ability of smooth muscle cells or fibroblasts to donate mitochondria to AEC with mitochondrial dysfunction (8). Although
we have not visualized such a transfer in vivo, peri-bronchial
increase in myofibroblasts and mitochondrial biogenesis is
attenuated by exogenous mitochondrial delivery. Although
much remains to be done, cross talk between AEC and ASM
mitochondrial function is an intriguing possibility.
Data on mitochondrial function of immune cells in asthma
are largely lacking. These cells may use alternative pathways
such as glycolysis or NADPH oxidases for sudden bursts of
energy requirement or ROS production, and the exact role of
mitochondria is highly variable. Eosinophils, the prototypic
inflammatory cells of allergic asthma, are thought to have
relatively few mitochondria, which are possibly unimportant in
cellular bioenergetics but critical for apoptosis regulation with
a delicately poised balance of proapoptotic Bid/Bax and antiapoptotic Mcl-1. The exquisite efficacy of glucocorticoids in
eosinophilic allergic asthma may be due to their ability to
inhibit Mcl-1 while accelerating Bid processing, causing eosinophil apoptosis (59). Although a general sentinel apoptotic
role of mitochondria may be common to other short-lived
granulocytes, the finer details vary greatly. In neutrophils,
glucocorticoids induce Mcl-1 expression, which may prolong
neutrophil survival, as seen in neutrophilic steroid-resistant
asthma (101). In contrast to granulocytes, mitochondria are
likely to be a primary energy source in lymphocytes. However,
the relative contribution of mitochondrial respiration vs. glycolysis is variable and critically influences T cell lineage
specification, especially along the Th17/Treg axis (17). Dominance of Th17 over Treg is associated with glycolytic dominance and is seen in severe asthma. Systemic metabolic factors
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COPD, and pulmonary hypertension have been strongly associated with mitochondrial dysfunction in multiple studies (14,
110). The mitochondrial defect is typically characterized by
increased mtROS production, abnormal morphology, and disordered homeostatic processes. A recent review by Aravamudan et al. (14) describes this in greater detail. More recently,
impaired mitochondrial homeostasis attributable to deficiency
of PINK1 has been found to be associated with idiopathic
pulmonary fibrosis (19). Defective mitochondria with impaired
fatty acid oxidation have been associated with reduced lung
compliance in mice, possibly attributable to long-chain acylcarnitine-mediated effects on lung surfactant (90). Furthermore, several genetic defects associated with impaired mitochondrial respiration are present with pulmonary hypertension
(PH) (58, 108, 125). Thus the thread of mitochondrial dysfunction and altered mitochondrial homeostasis runs through a wide
variety of lung pathologies. Asthma and COPD are common
lung diseases in which the connection with mitochondria is
best established. These are discussed in greater detail, with a
focus on targetable pathology.
Asthma. A critical role for mitochondria in the pathogenesis
of asthma emerges from observed genetic associations and
experimental studies. The mitochondrial haplogroup U is associated with increased IgE (97) and asthma (131). There is
also greater transmission of maternal asthma for some asthma
phenotypes, and altered mitochondrial gene expression was
reported in the placenta of mothers with asthma (31, 54).
Sex-specific associations of polymorphisms in mitochondrially
encoded genes, cytochrome B (males) and NADH dehydrogenase 2/16S RNA (females), have been seen in the only genome-wide study so far, conducted in a sample of 372 asthmatic children and 395 healthy children (40). These genes are
strongly associated with ROS production. Notably, this study
typed 16,158 mitochondrial single-nucleotide polymorphisms
using the DNA-pooling technique for reducing costs. More
than 30 very highly significant associations were seen despite
stringent statistical correction and modest sample size. Because
mitochondria lack the sophisticated nuclear DNA repair system, some of these may be a consequence of increased ROS
production in asthma, rather than a cause. In more precise
experimental systems, mitochondrial dysfunction, induced by
deficiency of UQCRC2 in airway epithelium of mice, potentiated allergic airway inflammation, mucin secretion, and bronchial hyperresponsiveness (4). Together, this fits a model in
which genetic predisposition toward increased mtROS production or increased susceptibility to oxidative damage increases
asthma risk. Histological or biochemical evidences of mitochondrial dysfunction have been consistently reported in airway biopsies or cultured airway epithelial cells (AEC) from
patients with asthma, as well as in experimental models (73,
79). However, the etiology of such mitochondrial dysfunction
and its role in the key cellular mediators of asthma pathogenesis, namely airway epithelium, smooth muscle, and immune
cells, is less clear.
AEC from mouse models of asthma and human biopsies
typically show mitochondrial swelling and fragmentation, increased oxidative damage, and activation of apoptotic pathways (73, 79). Cultured AEC from patients with asthma exhibit
preserved total cellular mitochondrial respiration through increased mitochondria with abnormal morphology and high
ROS production. The mitotoxic effects of metabolic products
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lateralis muscle of hospitalized patients with COPD showed
that several genes involved in the mitochondrial respiration
chain process were downregulated, whereas apoptosis pathway
genes were upregulated (33). Skeletal muscle from patients
with COPD has decreased mitochondrial content, decreased
capacity to generate new mitochondria, reduced activity and
coupling of the ETC, and increased production of ROS. Weakness of inspiratory muscles-like diaphragm, which may precipitate respiratory failure, is also associated with mitochondrial
dysfunction and attributed to hypoxia, hypercapnia, inflammation, and acidosis (82).
Genetic associations of COPD with mitochondria are not
well described, but common polymorphisms in the isocitrate
dehydrogenase (IDH) gene family and peroxiredoxins (PRDX)
interacted with smoking to predict FEV1/FVC in both African
Americans and European Americans (18). Many of these,
namely IDH2, IDH3, and PRDX5, are localized to mitochondria and are critical regulators of redox function. The effect
sizes were almost 5%, which is sizable. Interestingly, hypercapnia induces miR-183, which represses mitochondrial IDH2,
reducing ␣-ketoglutarate and OxPhos (120). Blood levels of
miR-183 are more than threefold elevated in patients with
COPD (22). We are not aware of mitochondrial genome-wide
association studies for COPD, but the copy number of mitochondrial genome is almost 50% lower in leukocytes of patients with COPD, compared with healthy smokers or controls
(72). This is further associated with reduced serum glutathione
and oxidative stress. Genetic variations in response to oxidative stress may be additionally important because the
rs1806649 minor allele of nuclear factor erythroid 2-related
factor 2 (Nrf2), the master antioxidant regulator, is associated
with a marked reduction in COPD mortality (39).
Together, these data strongly support a central role for
mitochondria in the pathogenesis of asthma and COPD, most
likely through chronically increased mtROS production that
persists beyond the initiating environmental triggers. The
mechanisms for such mitochondrial phenotypes merit exploration. We speculate that deficiencies in antioxidant defense
predispose toward earlier development of disease and poor
reversibility. At a mitochondrial level, this may be because of
genetic variations or low mtDNA copy number. Importantly,
such mtDNA defects can be inherited or acquired through
chronic ROS-mediated damage. This is especially relevant in
the context of a reemerging view that asthma and COPD
represent different phenotypes of a continuum of processes,
with overlapping features common and severe chronic asthma
very similar to COPD (89). The asthma-COPD overlap syndrome (ACOS) is now a distinct clinical entity with clinical
features of both asthma and COPD. It is expected that, in
coming years, we will see greater emphasis on systematic
characterization of patients at various levels, rather than COPD
vs. asthma label generation (96). We believe that mitochondrial
dysfunction is part of the continuum of processes that manifest
as asthma or COPD or ACOS and expect that mitochondrial
function assessment will contribute toward better characterization of subjects with suspected respiratory disease.
Targeting Mitochondria
Preceding sections define the important role of mitochondrial dysfunction in the genesis of lung diseases and support
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like nutritional state, redox balance, hypoxic response, and
insulin, among others, are expected to modulate shifts between
glycolytic and mitochondrial respiration. Of these, hypoxic
response is known to be important in Th17 specification, and
strong induction of the hypoxic response led to a severe
phenotype of asthma in mice, characterized by increased inflammatory cell infiltrate and remodeling (7). Importantly,
induction of hypoxic response does not require actual cellular
hypoxia and can be induced by mtROS or metabolic products
like methyl-arginines. These have potentially important roles
in the emerging link between the cardiometabolic syndrome
and asthma and are reviewed elsewhere (3). However, despite
these tantalizing possibilities that may link mitochondrial respiration of inflammatory cells to asthma and associated comorbidities, little is actually known at an experimental or clinical
level. This has important global implications, beyond asthma,
and should be an important avenue of research in coming
years.
COPD. The susceptibility of mtDNA to environmental oxidative damage, such as by cigarette smoke (CS), is thought to
be an important aspect of COPD pathogenesis. It has been
shown that primary bronchial epithelial cells, from ex-smokers
with COPD, have highly abnormal mitochondrial morphology,
similar to cultured bronchial epithelial cells (BEAS2B) exposed to CS extract. This suggests lasting mitochondrial damage from CS (56). Wood smoke, an important cause of nonsmoking COPD, has been similarly shown to be toxic to
mitochondria (51, 102). CS induces mitochondrial fragmentation through increased expression of dynamin-related protein 1
(Drp1) and decreased expression of mitofusin-2 (15). In lung
epithelial cells, CS induced phosphorylation of Drp1 appears to
be a critical step toward mitochondrial fragmentation, accelerated mitophagy, and cellular necroptosis (15).
The effect of CS is not restricted to epithelial cells because
ASM from patients with COPD also show reduced mitochondrial respiration and attenuated reserve capacity (126). Such
ASM cells have higher levels of mtROS than healthy smokers
or normal controls, secrete greater quantities of proinflammatory cytokines, and exhibit greater proliferation in response to
transforming growth factor-␤ (TGF-␤). These changes persisted undiminished through several passages in culture but
could be attenuated by a mitochondria-targeted antioxidant
(MitoQ) or a mitochondria-localized antioxidant (Tiron). Contemporaneous work from the Choi laboratory (84) found similar changes in lung epithelial cells and further showed that CS
stress-mediated effects on mitochondrial respiration are aggravated, rather than rescued, by activation of mitophagy. They
posit that activation of mitophagy in this context leads toward
necroptosis of lung epithelial cells and eventual emphysema.
Conflicting data from the Rahman laboratory (8), however,
shows that defective mitophagy leads to CS stress-induced
lung cellular senescence, and restoring mitophagy delays cellular senescence. Importantly, mitochondria-targeted ROS
scavengers, such as MitoQ or Mito-Tempo, inhibited the effects of CS stress in both studies.
The role of mitochondria in COPD pathogenesis may extend
beyond the lung. COPD is sometimes considered a systemic
disease. Limb and respiratory striated muscle dysfunction is
commonly seen in COPD exacerbations and is thought to be an
important part of the reduced maximal respiratory capacity and
exercise tolerance in COPD (45). Gene profiling in vastus
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exposed mice (126). MitoTEMPO was effective in attenuating
mtROS, TGF-␤, and collagen deposition in cultured human
airway cells or mouse models of allergic inflammation (61).
Although these appear translatable to bedside and MitoQ is
now sold over the counter as a dietary supplement, some
caution is warranted because even vitamin E and other untargeted antioxidants were found to be beneficial in experimental
models (74, 75, 80). Nevertheless, some clinical data exist for
CoQ10 that can be reasonably extrapolated to MitoQ, its more
efficient form (113). In an open-label crossover study of 41
steroid-dependent patients with asthma, supplementation with
a daily antioxidant cocktail, consisting of CoQ10 (120 mg) ⫹
400 mg ␣-tocopherol ⫹ 250 mg vitamin C, was associated
with a reduction in steroid usage (46, 52). CoQ supplementation has also been variably found to be beneficial in other
diseases characterized by mitochondrial dysfunction (16).
However, supplementation with other nonspecific antioxidants
like ␣-tocopherol and vitamin C may interfere with mitochondrial signaling and biogenesis (50). An important alternate
approach toward optimizing oxidant-antioxidant balance is to
enhance Nrf2, the master antioxidant defense transcription
factor that has been shown to be cytoprotective in lungs (111).
The prototypical Nrf-stimulating molecule, sulforaphane, is
found in natural dietary products such as broccoli, and other
analogs are in development (37, 62, 64). Sulforaphane has been
Fig. 3. The 3 Rs of mitochondria-targeted therapeutic strategies. Failing mitochondrial respiration may be restored through 1) repair via scavenging harmful ROS,
2) reprogramming via regulatory pathways, and 3) replacement by exogenous healthy mitochondria. MitoQ and MitoTEMPO are triphenylphosphonium-tagged
antioxidants that target mitochondria. Sulforaphane, found in broccoli, stimulates nuclear factor erythroid 2-related factor 2 (Nrf2). Vanadia nanowires act as the
cytoprotective antioxidant enzymes as shown. Microbial molecules that activate Nrf2 or pyrroloquinoline quinone (PQQ), a powerful antioxidant that also
stimulates mitochondrial biogenesis, seem to be important. Mitochondrial biogenesis may also be promoted by reprogramming through diet, exercise, metformin
(Metf), L -arginine (L-Arg)/nitric oxide (NO) or antagomirs to mitotoxic miRNAs. Finally, mitochondria can replaced by microinjection of isolated mitochondria,
peptide tagging of naked mitochondria, tunnelling nanotubes (TNT)/microvesicle-mediated transfer of mitochondria from donor cells. PGC-1␣, peroxisome
proliferator-activated receptor-␥ coactivator-1␣; TFAM, mitochondrial transcription factor A.
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mitochondria-targeted therapeutic strategies in patients with
lung disease. Such strategies can be broadly considered in a 3R
model: repair via scavenging harmful ROS, reprogramming via
regulatory pathways, and finally replacement by exogenous
healthy mitochondria (Fig. 3).
Repair. MtROS have a physiological as well as pathological
role. Clinical trial data suggest that untargeted antioxidants
such as ␣-tocopherol (vitamin E) lack beneficial effects and
may worsen the disease (98). MitoQ, developed by Murphy
and Smith (86), is the prototypical mitochondria-targeted form
of ubiquinol or CoQ10, which is a component of the ETC and
can therefore exist in both fully oxidized and reduced states.
Although CoQ10 is a powerful mitochondrial antioxidant, a
very small fraction of CoQ10 supplements will reach the
mitochondria. In MitoQ, CoQ10 is conjugated to a lipophilic
triphenylphosphonium cation group that leads to mitochondrial
accumulation and can more efficiently prevent mitochondrial
oxidative damage and mitochondrial dysfunction (86). MitoTEMPO is a similar triphenylphosphonium mitochondria-targeted antioxidant that has also been extensively validated.
MitoQ and MitoTEMPO have both been found to be effective
in protecting mitochondrial function during CS exposure (9,
84). MitoQ and Tiron, another mitochondria-targeted antioxidant, were effective in reversing defective mitochondrial function in ASM cells from patients with COPD and ozone-
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elevated in COPD, and may be a relevant therapeutic target.
The effects of mitomirs are not restricted to mitochondria and
may be relevant to comorbidities of asthma/COPD. For example, miR-183 downregulates the expression of large-conductance, Ca2⫹-activated K⫹ channel (BKCa␤1) that determines
arterial tone, possibly increasing risk for systemic and pulmonary hypertension (22).
Because the lungs are easy to target specifically through
aerosol delivery, antagomirs to miR-210 and miR-183 may be
specifically valuable for treating pulmonary hypertension or
hypercapnic lung disease, respectively, as discussed previously. Extensive preclinical testing across multiple models will
be necessary because these miRNAs have multiple targets and
functions. For example, miR-210 is associated with increased
stem cell survival in hypoxic conditions and may mediate other
cytoprotective functions of the hypoxic response.
Replacement. There is now extensive evidence of mitochondrial transfer as a mechanism to replace defective mitochondria. Methods for mitochondrial transfer to cells are diverse,
including microinjection (29, 36, 65, 92), incubation with
mitocytoplasts or intact purified mitochondria (66, 67), gap
junctional channel-mediated cell attachments (60), and direct
transfer from donor cells like mesenchymal stem cells (MSC)
to recipient cells via cytoplasmic bridges called tunneling
nanotubes (TNT) (8, 48, 93, 94, 100). Each of these methods
has its own advantages and disadvantages. Microinjection of
isolated mitochondria was pioneered by Tatum’s group (36)
and was later used by many other groups to generate transmitochondrial cell lines that have provided significant insights
into the survival of foreign mitochondria in recipient cells (29,
65, 87). Clark and Shay (29) demonstrated the spontaneous
incorporation of isolated mitochondria into recipient cells.
Delivery of isolated autologous mitochondria through injection
into ischemic heart tissue was able to improve tissue ATP
content and postinfarct cardiac function (81). Further work
from the same group found this to be an actin-dependent
uptake, presumably endocytosis, but how mitochondria escape
the endosome remains unclear (91). Although this avoids the
unknown long-term effects of MSC therapy and benefits from
the relatively short time to isolate the organelles than to grow
stem cells, others have been less successful with this approach
(107). Cell-penetrating peptide tags for efficient cell penetration of mitochondria may represent the next generation of
naked mitochondrial transfer (25). However, mitochondria are
also powerful triggers of the innate immune response, and the
risk of damaged mitochondria enhancing inflammation should
be carefully excluded. MSC are found in the connective tissue
of multiple organs and have limited stem potential, earning
them an alternate name, mesenchymal stromal cells. Prockop’s
group (107) showed for the first time that mitochondria or
mtDNA shuttled from human MSC to neighboring rodent cells
with nonfunctional mitochondria, leading to a bioenergetic
rescue. This discovery extended the potential clinical application of MSC to a wide array of disease associated with
mitochondrial dysfunction (1, 8, 27, 53, 60, 71, 94, 114, 130).
Coculture of MSC or fibroblast cells with mitochondria-depleted (␳0) lung alveolar epithelial cells (A549) showed rescue
of proliferative capacity, even in restricted media. Genetic
screening of both the mitochondrial and nuclear DNA from the
revived cells confirmed that the mitochondria in the revived
cells were derived from either of the donor cells (human MSC
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shown to protect against COPD (44, 55). Although mitochondrial function was not specifically looked for in the COPD
studies, sulforaphane has been shown to enhance mitochondrial antioxidant defense and protect against a panel of oxidant
stressors (129). However, the enthusiasm for activating Nrf2 as
a protective strategy is somewhat dampened by concerns about
potentially increased cancer risk (47).
Reprogramming. Mitochondrial function is importantly governed by lifestyle. Nutrient overload and metabolic changes
associated with obesity are highly prevalent causes of mitochondrial stress. Exercise, diet, and possibly metformin remain
the most reliable methods of reprograming mitochondria. Increased mitochondrial biogenesis is a well-known consequence
of exercise. Exercise has been found to be beneficial in reducing asthma exacerbations and is a cornerstone of pulmonary
rehabilitation to improve overall respiratory performance in
COPD (43). Chemical stimulators of mitochondrial biogenesis
include resveratrol and nitric oxide. Resveratrol or L-arginine
supplementation improved mitochondrial function and attenuated asthma features in experimental models of allergic inflammation (10, 76). Similar effects were obtained by inhibition of
arginase or ADMA (5). Metformin shows anti-asthma potential
in experimental models and also attenuates the increased allergic inflammatory tendencies of obese mice (21). These
approaches may be particularly relevant in targeting mitochondrial dysfunction in patients with obese-asthma or COPD (3).
Clinical trials of metformin in COPD show some promise
toward reducing dyspnea and increasing exercise capacity, but
results of more comprehensive studies are awaited (104).
Emerging molecular strategies for metabolic reprogramming
also include manipulating the let-7/lin28 axis that has been
shown to critically determine cellular OxPhos capacity and is
shown to enhance tissue repair or reduce aging (106). Let-7
miRNAs have been implicated in asthma pathogenesis (68, 69,
95). Other miRNAs also appear to modulate mitochondrial
function, especially two classes, hypoxamirs and mitomirs.
Hypoxamirs are, as the name suggests, induced by hypoxic
response (32). This is relevant to both asthma and COPD.
MiR-210 is one of the best understood hypoxamirs, with a
highly conserved hypoxia response element in its promoter. It
is also inducible by nuclear NF-␬B, which is known to be
increased in severe asthma and COPD. The iron-sulfur (Fe-S)
cluster assembly homologue (ISCU) 1/2, which enables redox
reactions in the mitochondrial ETC, is one of the direct targets
of miR-210 (23). Foxp3, the master transcription factor for
lineage specification of the anti-inflammatory regulatory T
cells, is also targeted by miR-210, supporting a proinflammatory role of chronic hypoxic response (128). Modulation of
miR-210 is not yet attempted in COPD but has been shown to
attenuate pulmonary hypertension that can secondarily complicate COPD or severe asthma.
Mitomirs are a specific set of miRNAs present in the
mitochondria that are closely linked to mitochondrial functions
like energy metabolism and mitochondrial dynamics (38).
They are more extensively reviewed elsewhere (38), but,
briefly, miR-149 increases mitochondrial biogenesis through
poly(ADP-ribose) polymerase-2, Sirt-1, and peroxisome proliferator-activated receptor-␥ coactivator-1␣ (PGC-1␣) (85).
MiR-761 and miR-30 members modulate mitochondrial fission
(38). MiR-183, which is discussed in greater detail in the
section on COPD, inhibits OxPhos by targeting IDH2, is
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REJUVENATING RESPIRATION: REPAIR, REPROGRAMMING, REPLACEMENT
or fibroblasts). Interestingly, an attempt to revive the A549 ␳0
cells with isolated mitochondria had failed, suggesting that
MSC-mediated mitochondrial donation may be more efficient
(107). Together, these studies provided strong evidence for
MSC-mediated mitochondrial transfer that appeared suitable
for therapeutic purposes. Such therapeutic effects were further
proven in in vivo lung injury models by Jahar Bhattacharya’s
laboratory and our group (8, 60). Islam et al. (60) found that
MSCs could transfer mitochondria to damaged alveolar epithelial cells, via TNT and microvesicles, in a mouse model of
sepsis-related acute lung injury, restoring organ function and
improving survival. We found that Miro1, a calcium-sensitive
GTPase that regulates intracellular mitochondrial transport in
neurons, also regulated intercellular mitochondrial transport
from MSC to lung epithelial cells, via TNT (8). Miro1 levels
were positively associated with mitochondrial transport and
therapeutic efficacy in models of asthma and lung injury. In a
CS model of COPD, induced pluripotent stem cell-derived
MSC attenuated lung damage through mitochondrial transfer
and were more effective than bone marrow-derived MSC
(127). Together, these studies provide a mechanistic basis for
MSC therapy in diseases characterized by mitochondrial dysfunction and inflammation. A number of phase I and II clinical
trials are in progress for the evaluation of safety and therapeutic efficacy of MSC for the aforementioned diseases (13, 41,
123). Important factors that would determine success include
retention of MSC at the site of injury, dose of MSC, and
mitochondrial donor efficiency. Despite some potential concerns about long-term safety, cellular donors like MSC have
the advantage of an innate capacity to hone in at the damaged
site, contribute to the tissue repair, and inhibit inflammation
(130).
ROS bursts without altering the basal antioxidant system.
Although these remain untested outside cell lines and the
ability to penetrate mitochondria is unclear, this represents an
exciting new frontier in the field.
New Directions in Mitochondrial Medicine
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Cellular respiration is a reflection of global respiratory
health, and disordered mitochondrial homeostasis confers increased risk for lung diseases. Mitochondrial dysfunction initiates cytotoxic events in AEC and leads to persistent remodeling of ASM. Although much remains to be understood,
targeting mitochondria appears to be a viable strategy for
optimizing respiratory health. A greater focus on the role of
mitochondria in lung cellular and molecular physiology appears warranted.
ACKNOWLEDGMENTS
We thank Kritika Khanna and Rituparna Chaudhuri for invaluable assistance in improving the manuscript.
GRANTS
The authors received funding support from Council of Scientific and
Industrial Research, India, grant MLP 5502 and Swarnjayanti fellowship (A.
Agrawal).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: A.A. conception and design of research; A.A. and
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