Mitochondrial bioenergetics and pulmonary dysfunction: Current progress and future directions

Paediatric Respiratory Reviews xxx (xxxx) xxx

Contents lists available at ScienceDirect

Paediatric Respiratory Reviews

Review

Mitochondrial bioenergetics and pulmonary dysfunction: Current progress and future directions Vadim S. Ten a,⇑, Veniamin Ratner b a b

Division of Neonatology, Department of Pediatrics, Columbia University Medical Center, New York, NY, United States Division of Neonatology, Department of Pediatrics, Icahn Mount Sinai School of Medicine, New York, NY, United States

Educational aims The reader will come to appreciate that:
Mitochondria are essential cellular organelles responsible for cellular energy production, oxygen sensing, generation of ROS, Ca2+ handling, signaling, and death.
Mitochondrial bioenergetics dysfunction results in decreased generation of ATP by any of the following mechanisms: inhibition of mitochondrial respiratory chain, mitochondrial uncoupling, failure of Ca2+ handling, generation of ROS and self-oxidation.
Mitochondrial bioenergetic dysfunction can be viewed as one of the mechanisms of pulmonary insufficiency associated with different lung diseases in children.
Novel therapeutic strategies may be developed by targeting cellular bioenergetics and other mitochondria-related mechanisms depending on the nature of the lung disease.

a r t i c l e

i n f o

Keywords: Mitochondria Bioenergetics failure Oxidative stress Hyperoxia Bronchopulmonary dysplasia Pulmonary hypertension

a b s t r a c t This review summarizes current understanding of mitochondrial bioenergetic dysfunction applicable to mechanisms of lung diseases and outlines challenges and future directions in this rapidly emerging field. Although the role of mitochondria extends beyond the term of cellular ‘‘powerhouse”, energy generation remains the most fundamental function of these organelles. It is not counterintuitive to propose that intact energy supply is important for favorable cellular fate following pulmonary insult. In this review, the discussion of mitochondrial dysfunction focuses on those molecular mechanisms that alter cellular bioenergetics in the lungs: (a) inhibition of mitochondrial respiratory chain, (b) mitochondrial leak and uncoupling, (c) alteration of mitochondrial Ca2+ handling, (d) mitochondrial production of reactive oxygen species and self-oxidation. The discussed lung diseases were selected according to their pathological nature and relevance to pediatrics: Acute lung injury (ALI), defined as acute parenchymal lung disease associated with cellular demise and inflammation (Acute Respiratory Distress Syndrome, ARDS, Pneumonia), alveolar developmental failure (Bronchopulmonary Dysplasia, BPD or chronic lung disease in premature infants), obstructive airway diseases (Bronchial asthma) and vascular remodeling affecting pulmonary circulation (Pulmonary Hypertension, PH). The analysis highlights primary mechanisms of mitochondrial bioenergetic dysfunction contributing to the disease-specific pulmonary insufficiency and proposes potential therapeutic targets. Ó 2019 Elsevier Ltd. All rights reserved.

Mitochondrial dysfunction and its potential mechanistic role in the evolution of lung diseases have become increasingly recog-

⇑ Corresponding author at: 3959 Broadway CHN 1201, New York, NY 10032, United States. E-mail address: [email protected] (V.S. Ten).

nized as an important and translationally promising research field. The interest in mitochondrial biology has emerged from observational studies reporting mitochondrial dysfunction in cultured cells, in patient lung tissue and animal models of respiratory diseases. Several well-assembled reviews summarized data on the potential contribution of mitochondria (signaling, mitophagy,

https://doi.org/10.1016/j.prrv.2019.04.001 1526-0542/Ó 2019 Elsevier Ltd. All rights reserved.

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pro-apoptotic role, biogenesis, genome, oxygen-sensing, quality control) to the pathogenesis of lung diseases in humans [1–4]. However, mitochondrial dysfunction discussed in these reviews was not directly related to bioenergetics. Because oxidative phosphorylation remains the most fundamental mitochondrial func-

tion, we decided to focus on those types of mitochondrial dysfunction that affect cellular bioenergetics, potentially leading to pulmonary injury. We defined the type of mitochondrial dysfunction according to the initial mechanism negatively affecting generation of adenosine tri-phosphate (ATP) in cells (Fig. 1).

Fig. 1. Schematic presentation of mitochondrial respiratory chain function in normal (A), mitochondria with inhibited complexes (B), mitochondria with proton leak and uncoupled respiration (C), and mitochondria with physiological Ca2+ flux (D) and Ca2+ overload (D, arrow to C). Fig. 1E and F depict ROS release from RC during forward electron transfer supported by NADH-linked respiration (E) or during reverse electron transfer fueled by FADH2-linked substrates (F). Fig. 1G is actual tracing of H2O2 fluorescence changes over time recorded using Amplex ultra-red assay in isolated murine (p7) lung mitochondria (0.05 g/ml) fueled with either NADH-linked substrates, glutamate-malate (10 mM and 5 mM) generating forward electron transfer or succinate (10 mM) generating reverse and forward electron transfer. Note robust elevation of H2O2 release during succinate-supported respiration.

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MITOCHONDRIAL BIOENERGETICS FAILURE MECHANISMS Inhibition of mitochondrial respiratory chain The respiratory chain (RC) is the most important structure for the generation of energy in cells. RC is tightly integrated into the mitochondrial matrix and consists of five complexes (Cx): CxI – NADH-dehydrogenase; CxII – succinate-dehydrogenase; CxIII – cytochrome c-reductase; CxIV – cytochrome c-oxidase and CxV – ATP-synthase. The primary function of these complexes is to create a H+-proton gradient across the inner (matrix) mitochondrial membrane, which generates a mitochondrial membrane potential (Wm). This electrochemical membrane potential is a source of proton motive force for ATP production by ATPsynthase. To pump H+ from the matrix into mitochondrial intermembrane space, complexes use the energy released in the electron transfer from the Krebs cycle metabolites, NADH or FADH2, to CxI or CxII, then to CxIII and CxIV, and finally to oxygen. The critical role of this process in life support is evidenced by the massive scale, 50 kg per day of ATP production in healthy humans [5]. Inhibition of any of mitochondrial Cx will decrease cellular energy production (Fig. 1A and B). Mitochondrial membrane leak and uncoupling Mitochondrial membrane leak occurs when the intra-matrix H+ flux across the inner membrane bypasses ATP-ase and does not participate in energy production (Fig. 1C). While the outer mitochondrial membrane is freely ion-permeable, the ionconductance of the inner membrane is very low. A physiological example of this enhancement of mitochondrial conductance is the action of uncoupling protein 1 (UCP1) in brown adipose tissue. This protein facilitates non-shivering thermogenesis in mammals by generation of a proton leak across the inner mitochondrial membrane, short-circuiting ATP-synthase [6]. Pathological mitochondrial leak can occur by opening the Ca2+-induced and ROSfacilitated permeability transition pore (mPTP), which collapses the H+ gradient and generation of ATP (discussed in the following section). Less dramatic is the effect of ionophores, like 20 40 dinitrophenol, free fatty acids, etc. These molecules actively transport cations across the inner mitochondrial membrane by binding H+ or other cations in the intermembrane space and releasing them into the matrix without activation of ATP-ase [7]. While membrane leak is not necessarily proton selective and (as in case of mPTP) leads to flux of many ions, the net effect is to dissipate the mitochondrial H+ gradient and membrane potential. To attempt to restore a proton motive force, the RC intensifies H+ pumping into the intermembrane space, which accelerates electron transfer up to the full capacity of the RC, leading to a maximal rate of oxygen consumption. Leak-accelerated mitochondrial respiration is known as uncoupled respiration, because it is not coupled with phosphorylation of ADP. Thus, mitochondrial leak significantly decreases ATP-production rate which limits cellular bioenergetic capacity. Mitochondrial Ca2+ handling The capacity of mitochondria to take up and accumulate Ca2+ in a Wm-dependent manner is well known [8,9]. Mitochondrial Ca2+ accumulation serves as a Ca2+ buffering system, protecting cells from the toxic effect of Ca2+, and modulates the rate of energy metabolism. Several enzymes participating in energy production in mitochondria are regulated by Ca2+. Several of the Krebs cycle dehydrogenases are very sensitive to Ca2+, (e.g. isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, pyruvate dehydrogenase) [10,11]. Ca2+ also promotes ATP generation by modulating ATP-

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ase activity via post-translational modification without alteration of the proton motive force [12]. Acceleration of mitochondrial respiration upon cellular demand by intramitochondrial Ca2+-driven activation of some of the respiratory Cx has been reported [10,12] (Fig. 1D). Ca2+ can also affect adenine nucleotide translocase, which mediates the exchange of ADP with ATP between the cytosol and mitochondrial matrix [13]. While physiological mitochondrial Ca2+ influx stimulates mitochondrial ATP generation, excessive Ca2+ load into the matrix is known to trigger an opening of mPTP. mPTP is a large-conductance pore which collapses the Wm and proton motive force, rendering mitochondria incapable of ATP production (Fig. 1D and C). Opening of mPTP is considered as an irreversible step in the cellular necrotic death pathway in ischemia–reperfusion injury [14,15]. mPTP activation is likely the mechanism linking abnormal Ca2+ handling to mitochondria cellular bioenergetics collapse caused by pathological challenge. Mitochondrial generation of ROS and self-oxidation Healthy mitochondria constantly generate superoxide due to electron escape from RC to oxygen. The previous assumption that 1–4% of oxygen consumed by mitochondria is diverted to ROS has been challenged and more realistic experimental conditions predict lower values, 0.15% [16] or 0.4–0.8% [17]. There are about ten ROS-producing sites in mitochondria, of which CxI and CxIII are considered to be the most productive [18,19]. The evidence for contribution of CxI and CxIII to mitochondrial ROS production was derived from in vitro experiments which required the use of CxI and CxIII inhibitors to interrupt electron flow, promoting electron escape to O2 and generation of ROS (Fig. 1E and G). Therefore, translational significance of in vitro-determined mechanisms of ROS release in the RC fueled with NADH-linked substrates is limited to the situations where CxI or CxIII are inhibited by pathological process. CxI can produce very large amounts of ROS by reverse electron transport (RET) [20–22]. When mitochondrial respiration is supported by succinate, a-glycerophosphate or fatty acid oxidation, under conditions with high membrane potential (e.g. resting respiration state), electrons flow back from CoQH2 into CxI, and then to NAD+, reducing it to NADH at the flavin site [18] (Fig. 1F). RET generates the highest rate of ROS production in isolated intact brain, heart, muscle and liver mitochondria [23–25]. Isolated lung mitochondria do not differ from other organs, and generate substantially greater ROS via succinate-supported RET, compared to the same mitochondria energized with NADH-linked substrates, glutamate-malate (Fig. 1G). Our discussion of the pathogenic priorities of certain mechanisms for mitochondrial ROS production in different lung diseases is preliminary. Instead, the oxidation of mitochondrial matrix structures critical for pulmonary ATP production by the ROS originating from mitochondrial RC will be discussed. In this respect, the topology of ROS release may be important, given the location of mitochondrial DNA in the matrix and its susceptibility to oxidative damage. While CxI releases ROS in the matrix, CxIII distributes ROS between matrix and mitochondrial intermembrane space [26,27]. In mitochondria excessively generating ROS, cellular bioenergetics is expected to deteriorate due to oxidative damage of the Krebs cycle enzymes (e.g. aconitase), respiratory complexes, mtDNA encoding RC units and matrix membrane [18]. MITOCHONDRIAL BIOENERGETIC FAILURE IN LUNG DISEASE Impairment in any of the above discussed mitochondrial functions is expected to alter cellular bioenergetics. As constant energy supply is essential for numerous cellular functions, it is reasonable

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to propose that poor cellular bioenergetics may contribute to the mechanisms of pulmonary cell malfunction defining a disease state of the entire organ. Mitochondrial bioenergetic dysfunction in ALI Acute lung injury is characterized by acute onset of respiratory failure associated with diffuse interstitial pulmonary edema in the absence of left ventricular failure. A classic example of ALI is ARDS which often manifests as a part of multiple organ failure syndrome in critically ill patients. One of the pathogenic features in ARDS is abnormalities of surfactant system [28]. Reduced secretion of surfactant is commonly seen in ARDS and is associated with worse outcomes [29,30]. Type II pneumocytes contain about threefold greater mitochondrial mass compared to type I cells [31]. A potential role for intact mitochondria in surfactant production and secretion may be supported by studies reporting that intramitochondrial but not cytosolic delivery of glutathione in the ethanol-fed rats significantly preserved surfactant-producing and secreting function of type II cells. [32]. Mechanistic importance of bioenergetics is supported by decreased pulmonary contents of ADP, ATP, guanosine 50 -diphosphate, guanosine 50 -triphosphate, nicotinamide-adenine-dinucleotide and creatine phosphate in an LPS-induced ARDS model [33]. Robust depletion of the lung ATP content has been observed following intra-tracheal LPS challenge in mice and neutrophils + LPS exposure in MLE12 cells [34]. Our preliminary data demonstrated significantly poorer ADPphosphorylating respiration in mitochondria isolated from murine lungs injured by intra-tracheal administration to LPS (Fig. 2A-C). These data suggest that one of the mechanisms of LPS-induced injury and energy reserve depletion in the lungs could be inhibition of mitochondrial RC. Of note, significant pulmonary protection against murine LPS-induced ALI was attributed to mitochondrial transfer from bone-marrow-derived stromal cells to alveoli [35], with elevated ATP content in the vicinity of transferred mitochondria. Mitochondrial transfer from mesenchymal stem cells to lung macrophages has also been shown to enhance phagocytosis in an animal model of E. coli pneumonia [36]. Again, this mitochondrial transfer improved mitochondrial respiration and ATP turnover in macrophages in vitro and enhanced macrophage phagocytic capacity both in vitro and in vivo. These reports highlighted a therapeutic role of improved mitochondrial bioenergetics in alleviation of ALI. In the rat model of acute pneumococcal pneumonia, increased ATP availability and turnover in the lungs attenuated lung damage induced by hypothermic exposure [37]. Given that generalized and pulmonary inflammation is considered as a pathogenic prerequisite of ARDS, direct inhibition of hepatocyte CxIV by TNFa [38] and IL-1b-induced depression of mitochondrial respiration in macrophages [39] may have mechanistic relevance to ALI. Thus, there is a body of evidence suggesting that bioenergetic dysfunction of pulmonary mitochondria may be considered as a component of the ARDS development. It is still unclear what causes inhibition of RC in the lungs affected by ARDS. One of the potential causes could be oxidative damage of the mitochondrial matrix. Although ROS originating in mitochondria have been implicated in the mechanisms of cell injury in ARDS [40], LPS, TNF-a, or IL-1b exposure did not elevate ROS production or inactivate aconitase in mitochondria but did inhibit mitochondrial respiration in human A549 or murine L929 cells [41]. Another potential cause of pulmonary bioenergetic dysfunction is mitochondrial uncoupling. Upregulation of uncoupling protein 2 (UCP2) exacerbated lung injury and increased animals’ mortality in association with significantly decreased ATP content and moderately reduced Wm in the lung tissue [42], but in contrast to the well-documented mitochondria-uncoupling action of UCP1, no

experimental evidence for mitochondrial uncoupling directly induced by UCP2 has been reported. With respect to mitochondrial Ca2+ handling, Kiefmann et al. [29] reported increased mitochondrial Ca2+ uptake in type II cultured pneumocytes in response to hypocapnia (simulation of alveolar hypocapnia in ARDS). This mitochondrial Ca2+ effect was governed by the activity of Krebs cycle isocitrate dehydrogenase, and lead to activation of apoptotic cellular death [29]. It is yet to be determined whether intramitochondrial Ca2+ flux contributes to ARDS evolution and stimulates or inhibits pulmonary bioenergetics. In the models of ALI associated with pulmonary ischemia– reperfusion, mitochondrial Ca2+ overload-driven mPTP opening has been implicated in the mechanism of pulmonary epithelial cell death [43,44]. Thus, in spite of considerable attention to potential pathogenic roles for mitochondria in pulmonary injury during ARDS, existing data are sketchy with respect to mechanistic input and the cause of mitochondrial bioenergetic dysfunction. Nevertheless, data demonstrating alleviation of experimental ARDS severity afforded by a transfer of intact mitochondria from other cells [35,36] strongly support contribution of mitochondrial bioenergetic dysfunction to ALI.

Mitochondrial bioenergetic dysfunction in hyperoxia and BPD BPD or chronic lung disease is characterized by postnatal alveolar developmental arrest leading to pulmonary insufficiency in premature infants. Among numerous postnatal stresses strongly associated with BPD, exposure to high concentrations of oxygen has been considered as one of the major triggers. Accumulation of dysmorphic, swollen mitochondria with abnormal cristae in pulmonary cells of animals exposed to chronic hyperoxia has been noted for a long time [45,46]. It is now known that disrupted mitochondrial function in pulmonary tissue is one of the toxic effects of prolonged oxygen therapy. For example, hyperoxia significantly inhibited basal and maximal respiration in MLE12 cells, and this was associated with reduced CxI and CxII-dependent respiration rates in mitochondria isolated from mice subjected to hyperoxic stress [47]. In pulmonary A549 cells, 96 hours of hyperoxic exposure significantly depleted ATP content, dissipated mitochondrial membrane potential and inhibited mitochondrial respiration [48]. These signs of mitochondrial dysfunction were associated with arrested pulmonary endothelial cell proliferation and death [49]. Earlier, Ratner et al. showed that partial pharmacological inhibition of CxI in normoxic neonatal mice fully reproduced the phenotype of arrested alveolar development induced by hyperoxia in their littermates [50]. In this model, hyperoxia did not cause a massive cellular demise as described earlier in the neonates affected by the ‘‘old BPD” form; this injury was defined by the extent of their developmental arrest, a hallmark of the current form of the disease, the ‘‘new BPD”. Some authors, using in vivo or in vitro hyperoxic exposure in neonatal mice or cultured pulmonary endothelial cells reported that hyperoxia primarily affected CxI activity, causing an inhibition of NADH-linked mitochondrial respiration [49,50]. Others, using MLE12 cells exposed to hyperoxia and lung mitochondria isolated from the mice exposed to hyperoxia have shown inhibition of CxI and CxIIdependent respirations with CxIV activity being spared [47]. Of note, in fibroblasts (N12) and alveolar epithelial cells (A549), hypercapnia, a common functional outcome of BPD, also significantly decreased mitochondrial ATP production and impaired cellular proliferation, independent of acidosis and hypoxia [51]. Given that postnatal pulmonary cellular proliferation and growth highly depends on adequate energy supply and nutrition [52– 55], hyperoxia and hypercapnia-driven inhibition of mitochondrial

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Fig. 2. H and E stained sections of the lungs obtained from sham (normal saline) or LPS-treated p10 mice (A). LPS (10 lg) was administered intra-tracheally at p5. Scale bar = 50l. B and C show tracings of mitochondrial respiration recorded using organelles isolated from LPS or Sham treated mice (B). Substrate, glutamate-malate (10 mM and 5 mM), ADP (100 nM), DNP (70 lM). State 3 (phosphorylating respiration) rates in lung mitochondria isolated from sham and LPS (n = 3) treated mice (C). Fig. D and E, actual tracing of H2O2 fluorescence changes in the lung mitochondria incubated in normoxic (RA) or hyperoxic (O2) buffer (D). Ca2+ buffering capacity in mitochondria incubated (5 min) in the normoxic (RA), compared to the same mitochondria tested in hyperoxic (O2) buffer with or without cyclosporine A (CsA) (E). Hyperoxic buffer was prepared by pre-bubbling with 100% O2 for two minutes (pO2  500 mmHg). F and G, the density of A549 cells grown in the galactose media (ATP generation is only of mitochondrial origin) following 48 hours of incubation in normoxia (naïve), hyperoxia (O2 + vehicle) or hyperoxia with supplemented succinate (O2 + succinate) (F). Hyperoxia (95% O2 and 5% CO2), Normoxia (21% O2 and 5% CO2), methyl-succinate 10 mM. Red is ethidium homodimer, green is CalceinAM stainings. Cellular proliferation of A549 cells exposed to pyridaben (CxI inhibitor, 1 lM) or pyridaben + succinate or pyridaben + 3-nitroproionate (CxII inhibitor, 5 mM) + succinate, n = 6 in each group (G). A549 pulmonary epithelial cells were seeded (150,000 cells per flask) in the galactose media. At 24 hrs before experimental exposure, cells stained with CFSE-dye using Cell traceTM Cellproliferation kit (Promega Inc). CFSE-dye is initially colorless and non-fluorescent. Once CFSE is passively diffused into cells, it is cleaved by esterase and becomes highly fluorescent. The dye is retained by cells throughout proliferation and can be used for tracing using FACS. The label is inherited by daughter cells after cell division. Thus, final intensity of CFSE fluorescence inversely corresponds to the number of divisions of original cells. The proliferation index is the ratio of the median value of CFSE fluorescence and the number of cells at the baseline (before any experimental exposure) divided by the same ratio after the exposure. The proliferation index is expressed as the % of naïve cells.

RC in lungs may represent a fundamental mechanism for alveolar developmental failure in premature infants. In addition to hyperoxia, normoxic mechanical ventilation, especially with excessive tidal volume, also significantly depressed pulmonary mitochondrial ADP-phosphorylating activity and arrested alveolarization in neonatal mice. Importantly, pharmacological uncoupling of mitochondria in non-ventilated, normoxic control neonatal mice reproduced the phenotype of alveolar developmental arrest [56]. This suggests that, whether by inhibition of RC or mitochondrial leak, decreased energy production contributes to the development of the BPD-like phenotype. In vitro, direct shear

stress mimicking volutrauma of ventilation suppressed mitochondrial RC, and superimposed hyperoxia exacerbated mitochondrial dysfunction in pulmonary endothelial cells. Both these effects were associated with increased production of superoxide and peroxynitrite in mitochondria [57,58], suggesting self-oxidative nature of mitochondrial RC dysfunction. It is not yet clear whether hyperoxia-induced inhibition of mitochondrial RC in lungs is mediated by the ROS derived from mitochondria. What is known, however, is that the BPDphenotype induced in postnatal day 4 (P4) mice after 72 h exposure to 75% oxygen could be attenuated by simultaneous exposure

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to MitoTEMPO, mitochondria-specific antioxidant [59]. One of the targets for MitoTEMPO is mitochondrial ROS-mediated upregulation NADPH oxidase-2. Our preliminary data highlighted another target for mitochondria-derived ROS, mitochondrial Ca2+ buffering capacity. In response to hyperoxia, lung mitochondria isolated from normal P5 mice, dramatically elevated their H2O2 production, associated with decline of cyclosporine A-sensitive Ca2+ buffering capacity (Fig. 2D and E). This suggests that mitochondria-originating ROS may indirectly (e.g. reduction of Ca2+ buffering capacity) deteriorate ATP production. While a mechanistic role of mitochondrial ROS has been proposed in hyperoxia-induced lung injury in premature infants [60], and in primary rat alveolar epithelial cellular apoptosis [61], some of pulmonary responses to hyperoxia were not dependent on elevated mitochondria-derived ROS formation [62,63]. A primary role for mitochondrial free radicals on cellular bioenergetics in the inhibition of developing lung still needs to be well documented. Regarding mitochondrial leak in hyperoxia and BPD, early induction of UCP2 has been reported in pulmonary macrophages during hyperoxia-induced lung injury [64], but direct induction of mitochondrial leak by this protein was not shown. In addition, we have shown that neonatal mice treated with the mitochondrial uncoupler 2,4-dinitrophenol fully replicated the phenotype of arrested alveolar development induced in their littermates by the exposure to normoxic mechanical ventilation for 8 hours [56]. This supports a deleterious effect of mitochondrial leak for normal alveolar development. Mitochondrial bioenergetic dysfunction in reactive airway disease With regard to bronchial asthma, data on the role of mitochondrial RC dysfunction in the evolution of this disease are conflicting. In the model of ovalbumin-sensitized mouse bronchial asthma, a significant depletion of pulmonary ATP content and deactivation of CxIV was mechanistically linked to airway hyper-reactivity [65,66]. Furthermore, this group demonstrated decreased ATP levels, CxIV activity, the loss of mitochondrial cristae and mitochondrial swelling in the lungs of the mice sensitized and challenged with ovalbumin [67]. In contrast, Huttemann et al. have demonstrated that mice with genetic ablation of the lung-specific sub-unit of CxIV, cytochrome c oxidase (sub-unit 4, isoform 2), exhibited 30% depletion of pulmonary ATP content associated with significantly reduced airway hyper-responsiveness and resistance upon challenge with methacholine. The authors proposed that, because energy is required for airway constriction, reduction in cellular energy supply could lead to smooth muscle relaxation and asthma relief [68]. The same group found that therapeutic doses of theophylline results in Tyr304 phosphorylation of the cytochrome c catalytic sub-unit I in the liver [69] and, in similar fashion, inhibits cytochrome c oxidase in the lungs, supporting their notion that airway hyper-reactivity is sensitive to ATP production. Of note, significant overexpression of CxV, ATP synthase, H+ transporting and mitochondrial F1 complex, b polypeptide (ATP5b, involved in ATP synthase) was detected in the asthmaaffected lungs in mice and attributed to airway smooth muscle thickening [70]. Thus, with respect to acute exacerbation of asthma, restricted mitochondrial ATP production may be beneficial. Interestingly, mitochondrial mass and oxygen consumption were greater in the airway smooth muscle cells from asthmatic patients than from COPD patients and controls. This was associated with increased cell proliferation and enhanced mitochondrial biogenesis mediated by abnormal Ca2+ influx in response to acetylcholine [71]. In this study, accelerated proliferation of airway smooth muscle cells in asthma directly depended upon elevation of mitochondrial mass. Thus, it seems that enhanced mitochondrial

bioenergetics promotes pathological pulmonary phenotype in asthma. Indeed, Aguilera-Aguire et al. found a mechanistic contribution of oxidatively damaged ubiquinol-cytochrome c reductase (CxIII) core II protein to exacerbation of airway inflammation in asthma as it was linked to elevated ROS release from CxIII rather than to interruption of electron carrier capacity of this complex [72]. Mitochondrial bioenergetics dysfunction in PH PH could be defined as pulmonary insufficiency caused by pathological constriction and remodeling of pulmonary arteries, leading to increased resistance in these vessels which causes hypertrophy and eventual failure of the right ventricle. Pulmonary arterial remodeling is a key process determining the extent of pulmonary artery resistance. It is characterized by structural modifications leading to a hyperproliferative and apoptosis-resistant phenotype of pulmonary vascular cells: pulmonary arterial smooth muscle cells (PASMC), endothelial cells and fibroblasts [73]. PH is, perhaps, the most investigated lung disease in terms of mechanistic contribution of different types of mitochondrial dysfunction. In the pulmonary vasculature, mitochondria are the physiological source of ROS acting as oxygen sensors in coordination of hypoxic pulmonary vasoconstriction [74]. Two hypotheses, both focused on a critical role of mitochondrial ROS, have been proposed: a) the redox hypothesis in which decreased generation of mitochondrial ROS and reduced redox state initiates hypoxic vasospasm [75] and b) the ROS hypothesis which proposes that hypoxic vasoconstriction is mediated by increased generation of ROS [76]. Vascular remodeling in PH has been closely associated with a metabolic shift from mitochondrial oxidative phosphorylation to anaerobic glycolysis in actively proliferating PASMCs, endothelial cells and fibroblasts [77–79], called the Warburg effect. The Warburg effect confers bioenergetic and biosynthetic advantages to proliferating cells by increasing non-oxidative ATP production [80]. This shift has been attributed to stabilization and activation of HIF1a [81]. Active HIF1a, in turn, activates pyruvate dehydrogenase kinase, an inhibitor of pyruvate dehydrogenase (PDH), which then suppresses mitochondrial oxidative phosphorylation due to the inability to produce acetyl-CoA for the Krebs cycle [82]. This may account for hyperpolarized mitochondria in PH-affected smooth muscle cells and right ventricular myocytes [79]. Currently, PDH inhibition is viewed as the primary mechanism for suppression of mitochondrial oxidative phosphorylation, pulmonary vasoconstriction and remodeling in PH [83]. This view explains the anti-pulmonary hypertension effect of dichloroacetate, which enhances mitochondrial oxidative phosphorylation by increasing the levels of intramitochondrial pyruvate and acetylCoA [84,85]. Another mechanism in PH could be related to direct suppression of mitochondrial RC complexes. Rafikova et al. reported that chronic inhibition of CxIII with antimycin A initiated sustained pulmonary vasoconstriction, increased vascular edema, and most importantly, a glycolytic metabolic shift in pulmonary arterial endothelial and PASMC from healthy female rats [86]. The same group found a tight link between CxI damage, glycolytic switch in PASMCs and vascular remodeling in monocrotaline-induced PH model in rats [87]. Pulmonary artery endothelial cells from patients with idiopathic pulmonary hypertension have demonstrated decreased CxIV activity [77]. These data suggest that primary inhibition of mitochondrial RC may also account for glycolytic shift and PH-phenotype in the lungs. It is intriguing that UCP2 knock-out murine PASMCs exhibited decreased PDH activity, intramitochondrial Ca2+ content, elevated Wm, resistance to apoptosis and spontaneously developed pulmonary vascular remodeling and PH [88,89]. The mechanism

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behind this important observation remains unclear. One group proposed that genetic ablation of UCP2 mimics hypoxia-induced changes in smooth muscle cell mitochondria, eventuating into activation of HIF1a [88]. Other authors suggest that mitochondrial hyperpolarization acts as a novel mechanism for spontaneous vascular remodeling in UCP2 deficiency [89]. These authors also suggested that the mechanism for hyperproliferation, downstream to mitochondrial hyperpolarization is elevated ROS release in the hyperpolarized mitochondria, as ROS scavenging reversed the hyperproliferative phenotype. Thus, mitochondrial bioenergetic dysfunction observed in the cells participating in pulmonary vascular remodeling in PH is characterized by metabolic shift toward glycolysis secondary to inhibition of PDH and decreased enzymatic activities of CxI, III and IV. DIRECTIONS FOR FUTURE RESEARCH Mitochondrial bioenergetic dysfunction has been detected in different pulmonary cell types and in a variety of respiratory diseases. However, its pathogenic contribution to mechanisms of the lung injury depends upon the nature and the stage of the disease. Inhibition of mitochondrial respiratory complexes and oxidative metabolism has been strongly associated with arrested alveolar development in the models of neonatal BPD. In mature subjects, the same inhibition of RC is associated with vascular cellular hyperproliferation, overgrowth and PH. While stimulation of oxidative metabolism attenuates severity of PH, in asthma it has been linked to exacerbation of the airway hyper-reactivity and remodeling. Thus, therapeutic strategies targeting mitochondrial bioenergetics are expected to be different depending on the nature of the disease. For example, strategies enhancing oxidative metabolism in pulmonary epithelial and endothelial cells, reactivation of PDH with dichloroacetate, protection of RC and matrix membrane integrity during mechanical ventilation and hyperoxia, or transfer of intact mitochondria to already injured cells, could be protective against ARDS, BPD and vascular remodeling in PH. In contrast, controlled inhibition of oxidative phosphorylation or induction of mitochondrial uncoupling may alleviate exacerbation of asthma. Mitochondria-targeted anti-oxidants (MitoTEMPO, MitoQ) have been reported to be beneficial in experimental models mimicking all discussed diseases. It has to be noted, however, that the intramitochondrial carrier of anti-oxidative agents, triphenylphosphonium cation, uses the electrochemical gradient of Wm. Therefore, the compound will saturate mitochondria as long as the mitochondrial membrane charge exceeds that of the cytosol, potentially leading to uncontrolled intramitochondrial delivery of the drug and mitochondrial depolarization [90]. Finally, detailed understanding of the mechanisms for mitochondrial bioenergetics dysfunction will guide future therapeutic strategies. For example, hyperoxia-induced alveolar developmental arrest has been shown to inhibit CxI but not CxII-dependent mitochondrial respiration [50]. This implies that during hyperoxia, supplementation of succinate which does not require CxI activity to support mitochondrial respiration may preserve cellular proliferation in the developing lungs. Our initial data demonstrated significant preservation of cellular proliferation in hyperoxia-exposed A549 cells in the presence of succinate (Fig. 2F). Similarly, succinate rescued cellular proliferation when CxI was inhibited with pyridaben. However, when both CxI and CxII were inhibited, no benefit of succinate supplementation was detected (Fig. 2G). Acknowledgement This work was partially supported by the NIH, United States grant 5K08HL96953 (V.R).

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