altered mitochondrial biogenesis and its fusion gene expression is involved in the high-altitude...

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Respiratory Physiology & Neurobiology 192 (2014) 74–84 Contents lists available at ScienceDirect Respiratory Physiology & Neurobiology j our na l ho me pa g e: www.elsevier.com/locate/resphysiol Altered mitochondrial biogenesis and its fusion gene expression is involved in the high-altitude adaptation of rat lung Loganathan Chitra a , Rathanam Boopathy b,a Molecular Biology and Biotechnology Division, DRDO BU Center for Life Sciences, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India b Department of Biotechnology, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India a r t i c l e i n f o Article history: Accepted 10 December 2013 Keywords: Estrogen-related receptor- Intermittent hypobaric hypoxia Mitofusin-2 Mitochondrial biogenesis Oxidative phosphorylation a b s t r a c t Intermittent hypobaric hypoxia-induced preconditioning (IHH-PC) of rat favored the adaption of lungs to severe HH conditions, possibly through stabilization of mitochondrial function. This is based on the data generated on regulatory coordination of nuclear DNA-encoded mitochondrial biogenesis; dynamics, and mitochondrial DNA (mtDNA)-encoded oxidative phosphorylation (mtOXPHOS) genes expression. At 16th day after start of IHH-PC (equivalent to 5000 m, 6 h/d, 2 w of treatment), rats were exposed to severe HH stimulation at 9142 m for 6 h. The IHH-PC significantly counteracted the HH-induced effect of increased lung: water content; tissue damage; and oxidant injury. Further, IHH-PC significantly increased the mitochondrial number, mtDNA content and mtOXPHOS complex activity in the lung tissues. This observation is due to an increased expression of genes involved in mitochondrial biogenesis (PGC-1, ERR, NRF1, NRF2 and TFAM), fusion (Mfn1 and Mfn2) and mtOXPHOS. Thus, the regulatory pathway formed by PGC-1/ERR/Mfn2 axes is required for the mitochondrial adaptation provoked by IHH-PC regimen to counteract subsequent HH stress. © 2013 Elsevier B.V. All rights reserved. 1. Introduction The reduced partial pressure of oxygen (O 2 ) at high altitude has several consequences for the O 2 economy of the body. In particular, the major consequences of hypoxia are related to the limitation in cellular energy supply that it imposes through the arrest of mito- chondrial respiratory chain electrons transport and the resulting ATP depletion. So, the essence of acclimatization is the enlarge- ment of this limiting link to restore ATP production per unit of tissue mass. This is essential in restoring the behavioral and phys- ical activity of the organism. In lieu of acclimatization, several medications are available that effectively decrease susceptibility to altitude illness (Hackett and Roach, 2001). However, none of these pharmaceutical interventions (e.g., acetazolamide, nifedipine and sildenafil) except dexamethasone directly improves physical Abbreviations: ATP6/8, ATP synthetase6/8; Cyt b, cytochrome b; CO I/II/III, cytochrome c oxidase I/II/III; CS, citrate synthase; Drp-1, dynamin-related protein- 1; ERR, estrogen-related receptor-; HH, hypobaric hypoxia; IHH, intermittent hypobaric hypoxia; Mfn1/2, mitofusin1/2; mtDNA, mitochondrial DNA; ND1 to 6* 4L, NADH-dehydrogenase-ubiquinone reductase1 to 6* 4L; nDNA, nuclear DNA; NRF1/2, nuclear respiratory factor 1/2; OXPHOS, oxidative phosphorylation; PGC-1, peroxisome proliferator-activated receptor- coactivator-1; PC, precon- ditioning; Fis1, fission 1; ROS, reactive oxygen species; RNS, reactive nitrogen species; TFAM, mitochondrial transcription factor A. Corresponding author. Tel.: +91 9443654487; fax: +91 422 2425706. E-mail address: [email protected] (R. Boopathy). work performance of the individual thus, unable to prevent the exacerbating altitude induced work impairment. Hence, altitude acclimatization remains the best approach to negating the detri- mental effects of altitude on health and human performance since it promotes the development of natural mechanisms of adaptation. But, acclimatization to altitude is a relatively slow process, usually attained by staying several days or weeks at progressively higher altitudes. Hence, intermittent exposure to hypobaric hypoxia (IHH) in hypobaric chambers has been used as an alternative procedure to induce acclimation to altitude (Yoshino et al., 1990; Richalet et al., 1992; Lukyanova et al., 1995), and also to improve performance (Vallier et al., 1996; Ricart et al., 2000) in the event of real exposure to HH conditions. Transient repetitive exposure of any organism to moderate levels of stress establishes a sustained, protective response against its lethality is termed as preconditioning (PC). Two forms of PC have been described. Immediate PC occurs within minutes of stimulus initiation which are attributed to cellular changes related to the activity or functioning of tissue enzymes, response of second messengers, and alterations in ion channels. While delayed PC prin- cipally is due to the de novo protein synthesis which takes several hours to develop, and such altered physiology will persist for sev- eral days (Zhang et al., 2000; Busija et al., 2008). Several studies have demonstrated that whole body PC with IHH or intermittent hypoxia leads to increased tolerance to severe hypoxic/HH/ischemic effect on various organs like, heart, brain and lung (Lukyanova et al., 1995; Lin et al., 2011; Wang et al., 2012). However, most of 1569-9048/$ see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.resp.2013.12.007

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Page 1: Altered mitochondrial biogenesis and its fusion gene expression is involved in the high-altitude adaptation of rat lung

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Respiratory Physiology & Neurobiology 192 (2014) 74– 84

Contents lists available at ScienceDirect

Respiratory Physiology & Neurobiology

j our na l ho me pa g e: www.elsev ier .com/ locate / resphys io l

ltered mitochondrial biogenesis and its fusion gene expression isnvolved in the high-altitude adaptation of rat lung

oganathan Chitraa, Rathanam Boopathyb,∗

Molecular Biology and Biotechnology Division, DRDO – BU Center for Life Sciences, Bharathiar University, Coimbatore 641 046, Tamil Nadu, IndiaDepartment of Biotechnology, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India

r t i c l e i n f o

rticle history:ccepted 10 December 2013

eywords:strogen-related receptor-�ntermittent hypobaric hypoxia

itofusin-2

a b s t r a c t

Intermittent hypobaric hypoxia-induced preconditioning (IHH-PC) of rat favored the adaption of lungsto severe HH conditions, possibly through stabilization of mitochondrial function. This is based on thedata generated on regulatory coordination of nuclear DNA-encoded mitochondrial biogenesis; dynamics,and mitochondrial DNA (mtDNA)-encoded oxidative phosphorylation (mtOXPHOS) genes expression. At16th day after start of IHH-PC (equivalent to 5000 m, 6 h/d, 2 w of treatment), rats were exposed tosevere HH stimulation at 9142 m for 6 h. The IHH-PC significantly counteracted the HH-induced effect of

itochondrial biogenesisxidative phosphorylation

increased lung: water content; tissue damage; and oxidant injury. Further, IHH-PC significantly increasedthe mitochondrial number, mtDNA content and mtOXPHOS complex activity in the lung tissues. Thisobservation is due to an increased expression of genes involved in mitochondrial biogenesis (PGC-1�,ERR�, NRF1, NRF2 and TFAM), fusion (Mfn1 and Mfn2) and mtOXPHOS. Thus, the regulatory pathwayformed by PGC-1�/ERR�/Mfn2 axes is required for the mitochondrial adaptation provoked by IHH-PC

bsequ

regimen to counteract su

. Introduction

The reduced partial pressure of oxygen (O2) at high altitude haseveral consequences for the O2 economy of the body. In particular,he major consequences of hypoxia are related to the limitation inellular energy supply that it imposes through the arrest of mito-hondrial respiratory chain electrons transport and the resultingTP depletion. So, the essence of acclimatization is the enlarge-ent of this limiting link to restore ATP production per unit of

issue mass. This is essential in restoring the behavioral and phys-cal activity of the organism. In lieu of acclimatization, several

edications are available that effectively decrease susceptibility

o altitude illness (Hackett and Roach, 2001). However, none ofhese pharmaceutical interventions (e.g., acetazolamide, nifedipinend sildenafil) except dexamethasone directly improves physical

Abbreviations: ATP6/8, ATP synthetase6/8; Cyt b, cytochrome b; CO I/II/III,ytochrome c oxidase I/II/III; CS, citrate synthase; Drp-1, dynamin-related protein-; ERR�, estrogen-related receptor-�; HH, hypobaric hypoxia; IHH, intermittentypobaric hypoxia; Mfn1/2, mitofusin1/2; mtDNA, mitochondrial DNA; ND1 to* 4L, NADH-dehydrogenase-ubiquinone reductase1 to 6* 4L; nDNA, nuclearNA; NRF1/2, nuclear respiratory factor 1/2; OXPHOS, oxidative phosphorylation;GC-1�, peroxisome proliferator-activated receptor-� coactivator-1�; PC, precon-itioning; Fis1, fission 1; ROS, reactive oxygen species; RNS, reactive nitrogenpecies; TFAM, mitochondrial transcription factor A.∗ Corresponding author. Tel.: +91 9443654487; fax: +91 422 2425706.

E-mail address: [email protected] (R. Boopathy).

569-9048/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.resp.2013.12.007

ent HH stress.© 2013 Elsevier B.V. All rights reserved.

work performance of the individual thus, unable to prevent theexacerbating altitude induced work impairment. Hence, altitudeacclimatization remains the best approach to negating the detri-mental effects of altitude on health and human performance sinceit promotes the development of natural mechanisms of adaptation.But, acclimatization to altitude is a relatively slow process, usuallyattained by staying several days or weeks at progressively higheraltitudes. Hence, intermittent exposure to hypobaric hypoxia (IHH)in hypobaric chambers has been used as an alternative procedure toinduce acclimation to altitude (Yoshino et al., 1990; Richalet et al.,1992; Lukyanova et al., 1995), and also to improve performance(Vallier et al., 1996; Ricart et al., 2000) in the event of real exposureto HH conditions.

Transient repetitive exposure of any organism to moderatelevels of stress establishes a sustained, protective response againstits lethality is termed as preconditioning (PC). Two forms of PChave been described. Immediate PC occurs within minutes ofstimulus initiation which are attributed to cellular changes relatedto the activity or functioning of tissue enzymes, response of secondmessengers, and alterations in ion channels. While delayed PC prin-cipally is due to the de novo protein synthesis which takes severalhours to develop, and such altered physiology will persist for sev-eral days (Zhang et al., 2000; Busija et al., 2008). Several studies have

demonstrated that whole body PC with IHH or intermittent hypoxialeads to increased tolerance to severe hypoxic/HH/ischemic effecton various organs like, heart, brain and lung (Lukyanova et al.,1995; Lin et al., 2011; Wang et al., 2012). However, most of
Page 2: Altered mitochondrial biogenesis and its fusion gene expression is involved in the high-altitude adaptation of rat lung

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uch studies with PC of lungs have focused on the restoration ofystemic adaptive physiology like, enhanced hypoxic ventilatoryesponse, improved gas-exchange function, increase in blood O2ransport activity through erythropoiesis, contractile response ofracheal smooth muscle, arterial O2 saturation and decreased pul-

onary vascular permeability (Rodriguez et al., 1999; Zhang et al.,004; Chakrabarty and Fahim, 2005; Guner et al., 2007). But suchdaptations in lungs are not only the result of increased strength ofxternal respiratory systems which transport O2 from the environ-ent to the tissue mitochondria. Other important factors in such

daptation are changes that follow at the cellular level, which ishe increased ability of tissue to utilize the low O2 content of bloodnd increased efficiency of mitochondrial system to maintainellular energy homeostasis for workload of hypoxic ventilation.n turn, mitochondrial function is influenced by the coordinatedxpression of genes involved in mitochondrial biogenesis as wells its dynamics and mitochondrial oxidative phosphorylationmtOXPHOS).

The role of nDNA-encoded “transcription co-regulator” callederoxisome proliferator-activated receptor-� coactivator-1�PGC-1�) along with nuclear receptor, like, estrogen-relatedeceptor-� (ERR�) is important in regulating cellular energyomeostasis and mitochondrial biogenesis (Wu et al., 1999;ootha et al., 2004; Schreiber et al., 2004). Nuclear respiratory

actors (NRF1/2) are shown to act downstream of “PGC-1�-RR�” axes in driving the transcription and replication ofhe mitochondrial genome through activating mitochondrialranscription factor A (TFAM) (Wu et al., 1999; Ekstrand et al.,004). Hence, through these regulatory factors nuclear DNAnDNA) is involved in controlling the expression of mitochondrialNA (mtDNA)-encoded genes, like, seven subunits of NADH-ehydrogenase-ubiquinone reductase (ND1–ND6 and ND4L),ytochrome b (Cyt b) of ubiquinol-cytochrome c reductase, threeubunits of cytochrome c oxidase (CO I, CO II and CO III) and twoubunits of ATP synthetase (ATP6 and 8) involved in mtOXPHOSFernandez-Silva et al., 2003). In addition to mitochondrial bio-enesis, the coordination of mitochondrial function is dependentn dynamics nature of mitochondria, which is controlled by twopposing processes, mitochondrial fission and fusion. In mammals,itofusin 1 and 2 (Mfn1 and Mfn2) control the mitochondrial

usion process, whereas, mitochondrial fission 1 (Fis1) and theynamin-related protein 1 (Drp1) are involved in fission ofitochondria (Hales and Fuller, 1997; Smirnova et al., 1998; James

t al., 2003).In our previous study, we have shown that exposure of rats

o severe HH conditions decreased the expression of ERR�, whichpparently resulted in the decreased level of mitochondrial num-er, mtDNA content and mtOXPHOS in the lung tissue (Chitra andoopathy, 2013). Along with these results, the emerging paradigmsuggest that, during high altitude acclimatization, multiple adap-ive pathways through the regulation of nDNA-encoded regulatorsike ERR� are required to sustain tissue mitochondrial respira-ory function. However, at present there is paucity of informationith respect to functional significance and molecular mechanismsnderlying such changes in lung under IHH exposure-induced PCIHH-PC) conditions. It is a well known fact that these adaptivehanges are accompanied by the activation of nucleic acid androtein synthesis in various tissues. Hence, we hypothesized that

HH-PC co-ordinately up-regulates nDNA-encoded mitochondrialegulatory genes and even the mitochondrial number in lung, asart of an adaptive response to sustain normal physiology. There-ore, the aim of the present study was to examine the effects of HH

exposure to 9142 m for 6 h) on alveolar edema, cellular toxicitynd oxidative stress in rat lung subjected with or without IHH-C regimen (exposure to 5000 m for 6 h/day for 14 days). Also, toetermine whether the beneficial effect of IHH-PC is accompanied

gy & Neurobiology 192 (2014) 74– 84 75

by alteration in transcript levels of nDNA-encoded regulators ofmitochondrial biogenesis, dynamics and mtDNA-encoded mtOX-PHOS genes. Such an understanding would link the efficiency ofmtOXPHOS and respiratory function to sustained lung function inresponse to an increased HH stress.

2. Materials and methods

2.1. Animals

Male Sprague-Dawley rats (160–180 g) were used in the study.The animals were maintained in the Animal house of the instituteat 28 ◦C ± 2 ◦C and subjected to a 12–12 h light–dark cycle withfree access to food and water. The experiments were carried outin accordance with the guidelines of the ethics committee of theinstitute Bharathiar University, Coimbatore, India.

2.2. Hypobaric hypoxic exposure

The rats were randomly assigned to four experimental groups(n = 6): normobaric normoxia (NN); HH; IHH-PC regimen; andIHH-PC regimen followed by HH exposure. IHH-PC regimen wasimposed by exposure of rats to simulated altitude of 5000 m for6 h/day lasting for 14 days. After the next day, beginning on the16th day of IHH-PC regimen, rats were continued in NN or exposedto severe simulated altitude of 9142 m in animal decompressionchamber at 28 ◦C for 6 h. The airflow in the chamber was 2 L/minwith relative humidity maintained at 50–55%. Rats were providedwith food and water ad libitum during the experimental protocol.All the animals survived the high-altitude exposure. After expo-sure to HH, the animals were sacrificed by cervical dislocationand lungs were dissected out. One portion of fresh lung tissuefrom the various experimental groups was used for extraction oftotal RNA and isolation of mitochondria. The second portion wassnap frozen and stored at −80 ◦C for biochemical and enzymeassay.

2.3. Analysis of edema formation in lung

Lung water content was used as an index of edema formation.The water content of the lung tissue was calculated as the differencebetween wet weight and dry weight and expressed as mg of waterper mg of dry tissue (Shukla et al., 2011).

2.4. Preparation of lung tissue

All the lung tissue obtained for biochemical measurements washomogenized as described previously (Chitra and Boopathy, 2013).The protein concentrations of the tissue homogenates were deter-mined using BSA as standard (Lowry et al., 1951).

2.5. Assay of lactate dehydrogenase activity

Lactate dehydrogenase (LDH) activity in the lung homogenatewas quantified using reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye (Abe and Matsuki, 2000).

2.6. Measurement of reactive nitrogen species (RNS)

The total of nitric oxide (NO) present in the supernatant of lunghomogenate was assayed using Griess reagent (Sigma) in accor-dance with the manufacturer’s instructions (Boyaci et al., 2006).

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.7. Measurement of reactive oxygen species (ROS)

ROS production was measured spectrophotometrically by theuantitative nitro blue tretazolium (NBT) reduction assay aseported previously (Mookerjee et al., 2006).

.8. Gene expression analysis using real time-PCR

About 1 �g of total RNA extracted from lung tissue usingotal RNA extraction kit-animal tissues (RBC Real Genomics, Realiotech Corporation, Taiwan) was reverse-transcribed with therst Strand cDNA Synthesis Kit (Fermentas, Thermo Scientific,SA) in accordance with the manufacturer’s instructions. RNA

solated from each two different animals per group was pooledroportionately (i.e. to make three biological replicates per group).uantitative Real Time PCR (qRT-PCR) amplification reactions wereerformed in 20 �l of final volume via SYBR Green chemistryRoche Diagnostics India Pvt., Ltd., India) according to the manufac-urer’s instructions on LightCycler 480 system (Roche, Singapore).

he beta-actin (�-actin) gene amplification acted as an internalontrol. The details of the primer sequences (Schlecht et al., 2004;ikula et al., 2005; Cassano et al., 2006; de Cavanagh et al., 2008;alena et al., 2009; Ding et al., 2010) for various genes studied are

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ig. 1. Effect of IHH-PC on HH-induced pulmonary edema (A) and LDH activity (B)n rat lung. The pulmonary edema (i.e. increase in water content) and tissue damage

i.e. LDH activity) in lung of different groups of animals exposed to, NN ( );

HH-PC plus NN ( ); HH ( ); and IHH-PC plus HH ( ) conditions wereetermined. Results are expressed in % variation in comparison to NN animals asean ± SD (n = 6 per group). *P < 0.05 vs. NN; #P < 0.05 vs. HH. The 100% value in

bsolute quantity of water content and LDH activity, respectively, corresponds to.13 ± 0.037 mg/mg dry tissue and 0.55 ± 0.016 U/mg of protein. Determination ofdema and LDH activity are described in Section 2.

gy & Neurobiology 192 (2014) 74– 84

given in table S1 (Supplementary material). Amplification speci-ficity was controlled by melting curve analysis. The “REST-2009”application was used to understand the relative expression profile(Pfaffl, 2009).

2.9. Determination of mitochondrial DNA integrity(mtDNA/nDNA ratio)

MtDNA/nDNA ratio was quantified as described previously (deCavanagh et al., 2008). The mtDNA/nDNA ratio was reported as2−�Cp.

2.10. Determination of citrate synthase activity

Citrate synthase (CS), a mitochondrial matrix enzyme, is com-monly used as a marker for the contents of intact mitochondria.Enzyme activity was assayed following the acetylation of 5,5′-dithiobis-(2-nitrobenzoic acid) at 412 nm (Craig, 1973).

2.11. Assay of mtOXPHOS complex activity

Mitochondria of the lung tissue were isolated using MITOISO1kit (Sigma Chemical Company, USA) following the manufacturer’sinstructions. The activity of Complex I was measured by monitoring

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Fig. 2. Protective effect of IHH-PC on HH-induced ROS (A) and RNS (B) production.The ROS (i.e. increase in NBT reduction) and RNS (i.e. increase in NO) generationin lung tissues of different groups (as described in Fig. 1 legend) of animals weredetermined as detailed in Section 2. Results are expressed in % variation in com-parison to NN animals as mean ± SD (n = 6 per group). *P < 0.05 vs. NN; #P < 0.05 vs.HH. The 100% value in absolute quantity of ROS and NO, respectively, correspondsto 0.422 ± 0.010 OD at 630 nm/mg tissues and 829 ± 26.26 nmoles/mg of protein.

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L. Chitra, R. Boopathy / Respiratory Physiology & Neurobiology 192 (2014) 74– 84 77

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ig. 3. Protective effect of IHH-PC on HH-induced mitochondrial complexes dysfuomplex V (D) of lung tissues of different groups (as described in Fig. 1 legend) ofn comparison to NN animals as mean ± SD (n = 6 per group). *P < 0.05 vs. NN; #P <

orresponds to 172 ± 10.89, 1483 ± 62.19, 1359 ± 149.37 and 264.64 ± 50.72 nmole

he reduction of 2,6-dichlorophenol indophenol in the presence of00 mM NADH at 600 nm (Janssen et al., 2007). The activity of com-lex III was measured as an increase in absorbance because of theeduction of cytochrome c at 550 nm (Krahenbuhl et al., 1994). Thectivity of complex IV was measured as a decrease in absorbanceecause of the oxidation of cytochrome c at 550 nm (Capaldi et al.,995). Complex V activity was measured as oligomycin-sensitiveg2+-ATPase activity by measuring the increasing oxidation ofADH at 340 nm in the presence of LDH, pyruvate kinase and ATP

Zheng and Ramirez, 2000). All the assays were performed at 30 ◦C.

.12. Statistical analysis

For relative expression studies REST 2009 uses randomizationnd bootstrapping methods to test the statistical significance ofene expression ratios and calculates 95% confidence intervals forelative fold changes. The hypothesis test P(H1) obtained repre-ents the probability of the alternate hypothesis that the differenceetween the sample and control groups is due only to chance. Theignificance level was set at P(H1) < 0.05 (Pfaffl, 2009). The genexpression ratio was reported in the form of a box and whisker plothowing median expression (horizontal line), surrounded by therst and third quartiles of those values (box) and the extreme valueswhiskers). Results of the biochemical experiments were reporteds mean values ± SD. Data was analyzed with one-way analysisf variance (ANOVA). When a significant effect was detected, theonferroni post hoc test was used to compare between groups aspplicable. The significance level was set at P < 0.05.

. Results

.1. Protective effect of IHH-PC on HH-induced lung injury

There was no significant difference in lung edema (as deter-ined in terms of lung water content) on IHH-PC exposed animals

. The integrity of mitochondrial complex I (A), complex III (B), complex IV (C) andals were determined as detailed in Section 2. Results are expressed in % variations. HH. The 100% value in absolute quantity of complex I, III, IV and V, respectively,mg of protein.

in comparison to control (non-IHH animals kept in NN condi-tion). Contrary to it, in animals directly exposed to HH, therewas a significant increase in edema (∼119%; P < 0.01) in com-parison to control animals (Fig. 1A). However, the increase inedema observed in HH condition was not observed and in factmaintained (P < 0.01) to NN level in animals subjected to IHH-PCregimen.

The increased cellular injury was measured in terms of LDHactivity. It was significantly lower (∼79%; P < 0.05) in animalsexposed to IHH-PC but kept under NN conditions in compar-ison to control animals. On the other hand, animals exposedto HH alone recorded a significant increase (∼149%; P < 0.05) intheir lung tissue LDH activity. However, IHH-PC regimen signif-icantly reduced the HH-induced increase in LDH activity to NNlevel (Fig. 1B).

3.2. Protective effect of IHH-PC on HH-induced oxidative stress

The oxidative stress markers, ROS (∼194%) and NO (∼142%)were significantly increased (P < 0.01) in animals subjected onlyto HH conditions in comparison to control animals (Fig. 2).Surprisingly, IHH-PC has also triggered a moderate increase(P < 0.05) in ROS (∼125%) and NO (∼114%) generation in lungtissues. However, it is interesting to note that, the increasein ROS (∼129%) and NO (∼119%) generation due to IHH-PC regimen were not further augmented when exposed toHH conditions.

3.3. Protective effect of IHH-PC on mitochondrial complexesunder HH conditions

The exposure of animals to HH, significantly reduced (∼45–65%;P < 0.01) the specific enzyme activities of different mitochondrialcomplexes (I, III, IV and V) in lung tissues in comparison with control

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nimals (Fig. 3). Surprisingly, the IHH-PC of animals has signifi-antly recorded an increase (∼170–190%; P < 0.01) in their specificctivities of all complexes. Thus on expected line the IHH-PC reg-men exposed animals, subsequently subjected to HH conditionsecorded a stabilized functional activity of all mitochondrial com-lexes but at elevated levels. Hence, it is clear that IHH-PC regimenffers functional protection of mitochondrial complexes.

.4. Protective effect of IHH-PC on mitochondrial number andtDNA content under HH conditions

The mitochondrial number and the mtDNA content were deter-ined, respectively, by the CS activity (Fig. 4A) and the ratio oftDNA to nDNA (Fig. 4B). The lung tissues from IHH-PC regi-en exposed animals showed an increased CS activity (∼190%;

< 0.01) and also mtDNA/nDNA ratio (∼160%; P < 0.01). Contraryo it, in animals exposed to HH alone, a drastic fall in CS activity isbserved concomitant to the decreased (∼50–55%; P < 0.01) contentf mtDNA relative to nDNA. Furthermore, the increased level of CS

ctivity (∼190%; P < 0.01) and mtDNA content (∼160%; P < 0.01) inHH-PC regimen animals were consistently maintained even afterhe subsequent HH exposure.

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ig. 4. Protective effect of IHH-PC on HH-induced mitochondrial disintegrity. Theitochondrial number (i.e. CS activity) and mtDNA content (i.e. mtDNA/nDNA ratio)

f lung tissues of different groups (as described in Fig. 1 legend) of animals wereetermined as detailed in Section 2. Results are expressed in % variation in com-arison to NN animals as mean ± SD (n = 6 per group). *P < 0.05 vs. NN; #P < 0.05s. HH. The 100% value in absolute quantity of CS activity and mtDNA/nDNA ratio2−�Cp), respectively, corresponds to 1.70 ± 0.07 �moles/min/mg of protein and71.46 ± 45.77. Determination of CS activity and mtDNA/nDNA ratio is described

n Section 2.

gy & Neurobiology 192 (2014) 74– 84

3.5. Independent effects of IHH-PC and HH, on expression ofgenes involved in mitochondrial biogenesis

To investigate the molecular mechanisms on the increased lev-els of mitochondrial number during IHH-PC regimen, the transcriptlevels of nDNA-encoded transcription factors involved in mito-chondrial biogenesis was examined using qRT-PCR (Fig. 5A; TableS2). In comparison to control animals, the IHH-PC regimen exposedanimals have significantly increased levels of PGC-1� [∼12-fold;P(H1) < 0.05] and ERR� (∼3-fold) transcripts. Further, IHH-PC reg-imen also increased [∼4-fold; P(H1) < 0.05] the mRNA level ofseveral genes involved in the downstream regulation of mitochon-drial biogenesis, like, NRF1, NRF2 and TFAM. On the other hand,under HH conditions alone, PGC-1� mRNA was increased [∼2-fold; P(H1) < 0.05] together with the selective repression of ERR�(Fig. 5B; Table S3). Despite such up-regulation of PGC-1� mRNA, theexpression of downstream regulatory genes involved in mitochon-drial biogenesis like NRF1, NRF2 and TFAM has not changed underHH condition in comparison to control animals. Further, the expres-sion of genes in animals exposed to HH after IHH-PC was shown inFig. 5C. The increased levels of PGC-1� [∼8-fold; P(H1) < 0.05] andERR� (∼3-fold) due to IHH-PC were maintained even after the sub-sequent exposure to HH conditions. The transcript levels of NRF1,NRF2 and TFAM were also maintained (∼3–3.2-fold) to the IHH-PClevel (Table S4).

3.6. Independent effects of IHH-PC and HH on expression ofmtDNA-encoded mtOXPHOS genes

The mtDNA-encoded mtOXPHOS genes that include ND1, ND2,ND3, ND4, ND4L, ND5, ND6, Cytb, COI, COII, COIII, ATP6 and ATP8were significantly increased (∼2.5–12-fold) by IHH-PC regimen(Fig. 6A; Table S2). Contrary to this, exposure of animals to HH con-ditions alone has significantly decreased mtOXPHOS genes (Fig. 6B;Table S3) in comparison to control animals. Such, HH-induceddown-regulation of genes involved in mtOXPHOS are prevented(Fig. 6C; Table S4) and in fact their transcripts levels were increased(∼3.2–11-fold) relative to NN level by IHH-PC regimen.

3.7. Independent effects of IHH-PC and HH on expression of genesinvolved in mitochondrial dynamics

The expression of mitochondrial fusion genes, namely, Mfn1 and2 was increased (∼3–4-fold), while the transcript level of fissiongenes, Fis1 and Drp1 was maintained at the same level relative toNN under IHH-PC regimen (Fig. 7A; Table S2). In contrast, under HHconditions alone, among the two genes involved in mitochondrialfusion, only Mfn2 expression was significantly decreased and theexpression of Mfn1 gene was unaltered. On the other hand, mito-chondrial fission genes, Fis1 and Drp1 was significantly increased[∼2–3-fold; P(H1) < 0.05] after the exposure of animals to HH condi-tions (Fig. 7B; Table S3). It is interesting to note that IHH-PC regimenexposure in animals has prevented a loss of Mfn2 level, as well asthe increased level of mitochondrial fission genes observed underHH conditions (Fig. 7C; Table S4).

4. Discussion

IHH-PC regimen is a fast-acting mechanism, a prudent shortcuteffective induction of resistance to HH sickness. The observationsmade in the present study constitute the description of intrinsicprotective mechanisms induced by IHH-PC regimen in unaccli-

matized rat through functional alteration of the mitochondrialsystem. Such an alteration in turn would increase the ability oftissue to maintain energy homeostasis for workload challengesunder hypoxic conditions. In particular, improvement in both
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iogenesis and fusion of mitochondria play a significant rolen inducing adaptation to HH insult by prior IHH-PC regimen.omplementing this, the role of nDNA gene ERR� in influencinghe mitochondrial function cannot be ignored.

.1. IHH-PC regimen-induced HH adaptation is dependent onitochondrial function

IHH-PC regimen has significantly reduced the pulmonarydema (Fig. 1A) and cytotoxic effect (Fig. 1B) in lung caused dueo exposure of rats to HH. Further, IHH-PC has significantly inhib-ted the HH-induced ROS and NO generation altogether. IHH-PCegimen itself has triggered a moderate increase in ROS and NO

evel (Fig. 2) due to its immediate but possibly direct effect on

itochondrial system. IHH-PC induces normal physiology adaptiveechanism to changing environment to escape from non-lethal

onditions and one of the peculiar adaptations is free-radical

ig. 5. Effect of IHH-PC regimen followed by NN (A), HH (B) and IHH-PC regimen followedxpression of genes in rat lung tissue was measured using qRT-PCR as detailed under Secf different genes denoted in the X-axis. The gene expression ratio of rat lung tissue exposing �-actin gene expression for normalization. Note that mRNA transcript levels of all thowever, under HH exposure, PGC-1� mRNA transcript level was increased (∼2-fold), wh

he loss of ERR� transcript and maintained the mRNA transcript of other mitochondrial ber group. The name of the genes as abbreviated in X-axis is given in expansion under ab

gy & Neurobiology 192 (2014) 74– 84 79

processing. In IHH-PC, the long period of NN interspersed betweenbrief periods of HH, which is repeated over days are expected toincrease ROS and NO generation, which subsequently will activatethe pro-survival signaling mechanism. In particular, presence ofsuch low concentrations of oxidants under IHH-PC would haveenhanced the antioxidants production more effectively (Goncharand Mankovska, 2010), thus, suppressed the lethal effect ofoxidants under HH conditions (Fig. 2).

In addition, the basic molecular response to any type of hypoxicchallenge will involve mtOXPHOS complexes. On this expectedline, IHH-PC has shown beneficial effect on mtOXPHOS complexesby increasing its activity, whereas, HH exposure has deleteriouslyaffected its activity (Fig. 3). Intensification of oxidant production

due to HH-related decrease in O2 availability as an electron accep-tor would elicit the mtOXPHOS complex impairment. The effects ofexcessive NO on the deregulation of the mtOXPHOS complex alsowill lead to increased mitochondrial superoxide anion production

by HH(C) on the relative expression of mitochondrial biogenesis genes. The relativetion 2. The Box and whisker plot reports the mRNA expression ratio on the Y-axissed to various conditions was calculated relative to the control group (fixed at 1),e mitochondrial biogenesis genes were increased (∼3–12-fold) by IHH-PC regimen.ile a selective repression of ERR� (∼0.045) was observed. But, IHH-PC has preventediogenesis genes to IHH-PC level. *P(HI) < 0.05 in comparison to control group; n = 3breviations section.

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Borutaite and Brown, 1996; Poderoso et al., 1996; Mohanraj et al.,998). Consequently, the ability of mitochondria to synthesize ATPecomes vulnerable under HH conditions. In contrast, IHH-PC isssociated with accelerated mtOXPHOS complex activity, which isurther maintained even after exposure to HH condition in the lung.hus, the data from the present study clearly show that IHH-PC reg-men favored enhanced cellular bioenergetics, which plays a criticalole in lung adaptation to HH insult.

To further understand the role of mitochondria as “centralegulator” involved in HH adaptation, we intend to study theifferential effect of IHH-PC regimen and HH exposure either

ndependently or together on mitochondrial number, mtDNAeplication/transcription, mitochondrial biogenesis and dynam-cs. HH condition has significantly decreased the mitochondrial

ig. 6. Effect of IHH-PC regimen followed by NN (A), HH (B) and IHH-PC regimen followelative expression of genes in rat lung tissue was measured using qRT-PCR as detailed un-axis of different genes denoted in the X-axis. The gene expression ratio of rat lung tissue), using �-actin gene expression for normalization. Note that mRNA transcript levels of

egimen. Also, IHH-PC regimen has prevented the loss of mRNA transcripts of mtOXPHOSroup.

gy & Neurobiology 192 (2014) 74– 84

number and mtDNA content (Fig. 4). Decrease in mitochondrialnumber would increase the workload of available mitochondria,leading to the higher membrane potential and increased ROS pro-duction (Maassen et al., 2002). This in turn would affect mtDNA,which lacks protective histones, and further its proximity tothe mtOXPHOS complexes. Such an unopposed mtDNA oxida-tion would interfere with mitochondrial transcription and thusthe mtOXPHOS protein synthesis, which not only impairs respira-tory capacity but also would exacerbate ROS production (Wallace,1999). So, an increased mitochondrial number and mtDNA content

essentially an important adaptive mechanism. Such an adaptationby IHH-PC regimen has probably inhibited the injury on mitochon-dria and mtDNA upon exposure to HH condition.

ed by HH (C) on the relative expression of mtDNA-encoded mtOXPHOS genes. Theder Section 2. The Box and Whisker plot reports the mRNA expression ratio on the

exposed to various conditions was calculated relative to the control group (fixed atall the mtDNA-encoded mtOXPHOS genes were increased (∼3–10-fold) by IHH-PC

genes under HH conditions. *P(HI) < 0.05 in comparison to control group; n = 3 per

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.2. IHH-PC regimen-induced HH adaptation accompaniesitochondrial biogenesis

The increase in mitochondrial number and mtDNA contentnder IHH-PC indicates an increased role of nDNA and mtDNA,hich can critically activate the synthesis of mtOXPHOS complex

ubunits. In fact, IHH-PC regimen in rat has significantly increasedhe nDNA-encoded expression of genes (Fig. 5A) involved in mito-hondrial biogenesis (PGC-1�, ERR�, NRF1, NRF2 and TFAM), andtDNA-encoded 13 subunits of mtOXPHOS complex (Fig. 6A).lthough, the nDNA-encoded subunits of mtOXPHOS were noteasured in the present study, the increased NRF1 and NRF2

ould mediate the expression of several nDNA-encoded mtOX-

HOS genes (Au and Scheffler, 1998; Virbasius et al., 1993a,b).onversely, the decreased expression of ERR� and mtDNA-ncoded mtOXPHOS gene under HH condition would compromise

ig. 7. Effect of IHH-PC regimen followed by NN (A), HH (B) and IHH-PC regimen followedxpression of genes in rat lung tissue was measured using qRT-PCR as detailed under Secf different genes denoted in the X-axis. The gene expression ratio of rat lung tissue exposing �-actin gene expression for normalization. Note that IHH-PC regimen has increased

Fis1 and Drp1) were not altered. Nevertheless, HH exposure has decreased the fusion geegimen prior to HH exposure has prevented the increase in fission genes under HH cond

gy & Neurobiology 192 (2014) 74– 84 81

mitochondrial function through impairing mitochondrial biogen-esis and mtOXPHOS complexes activity. This is because, theinhibition of ERR� diminishes the effect of PGC-1� in inducingmitochondrial biogenesis and cellular respiration (Mootha et al.,2004; Schreiber et al., 2004). It is important to note that, IHH-PC reg-imen has inhibited the loss of mRNA transcripts involved in mito-chondrial biogenesis and mtOXPHOS complex under HH condition,wherein, the transcript level of ERR� is preserved (Fig. 5C and 6C).

4.3. IHH-PC regimen-induced HH adaptation acceleratesmitochondrial fusion

The expression of key factors involved in mitochondrial dynam-ics is regulated at transcriptional level and their activities aremuch more needed to the bioenergetics state of mitochondria(Honda and Hirose, 2003; Jendrach et al., 2008; Westermann,

by HH (C) on the relative expression of mitochondrial dynamics genes. The relativetion 2. The Box and whisker plot reports the mRNA expression ratio on the Y-axissed to various conditions was calculated relative to the control group (fixed at 1),

the mRNA transcript levels of fusion genes (Mfn1 and Mfn2) while the fission genesne: Mfn2 while the fission genes (Fis1 and Drp1) were increased. However, IHH-PCitions. *P(HI) < 0.05 in comparison to control group; n = 3 per group.

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012). Hence it is conceivable that, an increased transcript level ofenes involved in fusion (Mfn1 and Mfn2) is yet another adaptiveesponse gained by mitochondria from IHH-PC regimen (Fig. 7A).lso, the decreased expression of fusion gene (Mfn2) and increasedxpression of fission genes (Fis1 and Drp1) under HH conditionurther suggest the mutual importance of mitochondrial morphol-gy and bioenergetics status in adaptation to HH (Meeusen et al.,004; Youle and Karbowski, 2005; Benard et al., 2007; Sauvanett al., 2010). Furthermore, IHH-PC regimen has inhibited both, theoss of transcripts involved in fusion and increase of transcriptsnvolved in fission under HH exposure (Fig. 7C). This suggestshat, increased mitochondrial fusion by IHH-PC can dramaticallyampen the deleterious effects through maintenance of mitochon-rial bioenergetics under metabolic stress conditions like HH. This

s supported by the emerging concept from various other studieshich shows, interconnected mitochondria are energetically active

nd associated with pro-survival mechanism against apoptotictress (Tondera et al., 2009); fragmented mitochondria are inactivend always associated with pathological conditions like, diabetes,

besity, ischemia-reperfusion, and neurodegeneration (Cho et al.,009; Zorzano et al., 2009; Ong et al., 2010; Galloway et al., 2012).

Mfn2 gain-of-function under IHH-PC regimen and loss-of-unction under HH exposure can be related to its role in

ig. 8. Schematic of IHH-PC regimen-induced biogenesis and fusion of mitochondria. IHHamely, PGC-1� and ERR�, which interact and co-ordinately increased the expression ofuently, NRFs enhances the TFAM expression, which translocates into mitochondria and di3 subunits along with nDNA-encoded subunits are increased to form multi-subunit mtOXlso increased the expression of genes involved in mitochondrial fusion, namely, Mfn1 anre coordinated to induce biogenesis and fusion of mitochondria required for the mitochegulation of mitochondrial biogenesis by moderately increased ROS and NO; regulationnd function of hyperfused mitochondria is warranted to wholly understand the role plaH tolerance in the lung tissue.

gy & Neurobiology 192 (2014) 74– 84

mitochondrial metabolism, which is not second to its involvementin regulation of mitochondrial dynamics. In this regard, severallines of evidence indicate that two mutually non-exclusive mecha-nisms were likely to be played by Mfn2 to maintain mitochondrialmetabolism: first, Mfn2 is involved in expression of nDNA-encodedmtOXPHOS subunits (Chen et al., 2005; Pich et al., 2005; Sorianoet al., 2006); and second, Mfn2 is a component of intracellu-lar signaling machinery that regulate mitochondrial energizationthrough mitochondrial membrane potential (Soriano et al., 2006).The important fact to be noted is regulation of Mfn2 expression byPGC-1� is dependent on ERR� (Cartoni et al., 2005). Hence, lossof ERR�, which eventually led to the Mfn2 deficiency is the majorgenomic deregulation under HH condition, which has been espe-cially prevented by IHH-PC regimen to induce adaptation in thelung.

4.4. Molecular mechanisms yet to be understood in IHH-PCregimen-induced HH adaptation

The phenomenon of stabilized mitochondrial bioenergeticsadaptation through enhanced mitochondrial biogenesis and itsfusion, is in agreement with the several previous studies, wherein,various other PC like responses, viz., low-dose of cobalt chloride

-PC regimen increased the expression of nDNA-encoded transcription regulators, downstream regulators of mitochondrial biogenesis, like, NRF1 and NRF2. Subse-rectly activates the transcription and replication of mtDNA. In turn, mtDNA-encoded

PHOS complexes required for efficient O2 consumption and ATP synthesis. IHH-PCd Mfn2, which may result in formation of hyperfused mitochondria. These events

ondrial adaptation to HH exposure. Further, molecular insight into the events, like, of mitochondrial metabolism by Mfn2; and characterization of both morphologyyed by mitochondria under IHH-PC regimen for enhanced aerobic respiration and

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upplementation (Saxena et al., 2012); ischemia (McLeod et al.,004); and carbon monoxide inhalation (Suliman et al., 2007),alorie restriction (Civitarese et al., 2007) and endurance exer-ise (Gomez-Cabrera et al., 2008) have been imposed in rodentsr humans. Also, all these studies have concentrated in metabol-cally active tissues like, heart, liver, skeletal muscle and brain.owever, with respect to IHH-PC regimen-induced adaptation on

ung mitochondrial bioenergetics, our evidence is the first but isnly preliminary which shows the molecular event of accelerateditochondrial biogenesis and fusion as the “central regulator” ofitochondrial adaptation under HH conditions (Fig. 8). An elab-

rate study linking the molecular events that has been shown inhe present study will give an insight into the IHH-PC regimen-nduced mitochondrial bioenergetics. Especially, two aspects oftudy are of importance, first, the role of moderately increasedxidant production under IHH-PC regimen on mitochondrial alter-tion has to be characterized. This is because, moderate dose ofOS is a must for increased mitochondrial biogenesis under vari-us PC regimen (McLeod et al., 2004; Civitarese et al., 2007; Sulimant al., 2007; Gomez-Cabrera et al., 2008; Saxena et al., 2012),hich has been shown to be abolished by antioxidant (Vitamin) intake (Gomez-Cabrera et al., 2008). Also, moderate increase

n NO stimulates mitochondrial biogenesis mainly through activa-ion of regulatory proteins such as PGC-1�, NRF1, and TFAM (Nisolind Carruba, 2006). Second, further morphological and functionalharacterization of hyperfused mitochondria in the lung tissueections formed by IHH-PC regimen is desirable. Such study willmprove the understanding of mitochondrial morphology requiredor enhanced utilization of available low concentration of O2 underH condition. Such a question cannot be addressed with isolateditochondria, which will inevitably fragment during processing.

. Conclusion

IHH-PC regimen has evoked multiple signaling processes espe-ially in lung cellular biology in protecting mtOXPHOS function.ore importantly, mitochondria have adopted themselves a long-

asting resistance against lethal stress like, HH exposure. Furtherrom the outcome of the present study one can envision a scenariohere the presence of a regulatory pathway formed by PGC-

�/ERR�/Mfn2 axes is required for the mitochondrial adaptationrovoked by IHH-PC regimen in order to counteract subsequent HHtress. In conclusion, the present study delineates the existence ofntergenomic control of mitochondrial function in generating adap-ation in lung by IHH-PC regimen to resist against an eventual HHxposure. The practical importance of this discovery lies in the facthat, IHH-PC regimen is a simple intervention and less intrusive.f these results are translated for clinical benefits, then the IHH-PCegimen may have important implications toward enhancing theerformances of athletic and military personnel under high altitudeypoxic conditions.

cknowledgments

This work was supported by grants from DRDO-BU Centeror Life Sciences, Bharathiar University, Coimbatore, Tamil Nadu,ndia. One of the authors Ms. L. Chitra acknowledges DRDO-BUenter for Life Sciences for the award of Senior Research Fellow-hip.

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