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Mitochondrial DNA, Early Online: 1–5! 2014 Informa UK Ltd. DOI: 10.3109/19401736.2014.915526
FULL LENGTH RESEARCH PAPER
Lower mitochondrial DNA content relates to high-altitude adaptationin Tibetans
Yue Li1,2,3*, Wei Huang4*, Qin Yu1,2,3, Yao-Ting Cheng1,2,3, and Qing-Peng Kong1,2
1State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China, 2KIZ/CUHK
Joint Laboratory of Bioresources and Molecular Research in Common Diseases, Kunming, China, 3Kunming College of Life Science, University of
Chinese Academy of Sciences, Beijing, China, and 4Department of Geriatrics, the Third People’s Hospital of Yunnan Province, Kunming, China
Abstract
Mitochondrial DNA (mtDNA) is crucial to mitochondria in energy production and otherphysiological functions. When lowlanders arrive at high altitude, the mitochondrial contenttends to decrease. However, the mtDNA content of native highlanders share the same featureas lowlanders remains unknown. It is also interesting to dissect the other changes in bloodplasma that might accompany the change of mtDNA content. To address these issues, werecruited 241 Tibetan subjects in Tibet and 220 Han subjects in Shaanxi province. RelativemtDNA copy number and blood biochemical indexes were measured. Results show thatrelative mtDNA copy number in Tibetans is significantly lower as compared to Han subjects;sex, age, blood glucose, triglyceride and total cholesterol show no influence on mtDNAcontent, but carbon dioxide combining power is negatively correlated with mtDNA content.These results indicate that an increase in CO2 combining power along with lower mtDNAcontent may provide adaptive potential.
Keywords
Adaptation, carbon dioxide combiningpower, copy number, high altitude,mitochondrial DNA
History
Received 26 February 2014Revised 10 April 2014Accepted 13 April 2014Published online 20 May 2014
Introduction
The Tibetan Plateau is the world’s highest plateau characterizedby rigorous environmental features like coldness, strong UV,windiness and hypoxia. Among these factors, hypoxia severelyimpacts the health and creates the condition for physiologicalhypoxia. However, native Tibetans have lived and successfullyreproduced at high altitude since 20 kilo-years ago (Zhao et al.,2009) in spite of the physiological challenges introduced bychronic oxygen deprivation. Previous studies have demonstratedthat Tibetans do have genetic basis enabling their successfulpeopling in the hypoxic environment (Beall et al., 2010; Bighamet al., 2010; Peng et al., 2010; Simonson et al., 2010; Xu et al.,2010; Yi et al., 2010). For instance, several positively selectedgenes in Tibetans show significantly association with decreasedhemoglobin (Hb) levels (Beall et al., 2010; Bigham et al., 2010;Peng et al., 2010; Simonson et al., 2010; Xu et al., 2010; Yi et al.,2010). Yet the arterial oxygen level of Tibetans is still lower thanthat of Andean high-altitude natives (Beall, 2007; Simonson et al.,2010; Yi et al., 2010). Hypoxia affects energy generation andconsumption in animals to a large extent. Hence adaptation ispresumably needed to maintain cellular energetics and function.As the primary energy producer, mitochondrion, therefore,becomes a valuable target for studying the adaptation to decreasedoxygen supply.
Mitochondrion is the major organelle where more than 95% ofcellular energy is produced, thus called the energy-producingfactory of the organism. Changes in mitochondrial function play acrucial role in homeostasis in ATP production/consumption(Wallace et al., 2003). Mitochondria contain their own mitochon-drial-DNA (mtDNA). These small molecules encode 2 ribosomalRNAs, 22 tRNAs and 13 proteins that are essential for oxidativephosphorylation and ATP production. Each cell contains hundredsto thousands of copies of mtDNA, which are important for manymtDNA-mediated pathological processes (Diez-Sanchez et al.,2003; Liu et al., 2006), mitochondrial biogenesis and normalcellular functions (Clay Montier et al., 2009). Besides, alterationsof mtDNA copy number are considered to be important hallmarksin a variety of cancers (Feng et al., 2011; Kim et al., 2004;Lin et al., 2010; Wang et al., 2006, Xing et al., 2008; Yamadaet al., 2006; Yu et al., 2007). Particularly, certain mitochondrialhaplotypes defined by specific variants such as nt3010G-nt3970Cshow implications of high-altitude adaptation in Tibetans(Luo et al., 2011b). Studies also showed that certain mitochon-drial haplogroups are predisposed to high-altitude diseases whileothers can protect against mountain sickness (Li et al., 2011, Luoet al., 2011a, 2012).
Mitochondrial content alters during acclimatization to highaltitude. It is reported that lowlanders returning from high altitudehad decreased muscle mitochondrial densities and lipofuscin, amitochondrial degradation product, was found in muscle cells ofthose subjects (Hoppeler et al., 2003). Moreover, a previous studyindicated that sperm mtDNA copy number initially increasedwhen the donors arrived at higher altitude, then graduallydecreased after one year’s acclimatization (Luo et al., 2011c).Hence, it is necessary to find out how mtDNA copy number
*These authors have contributed equally to this work.
Correspondence: Qing-Peng Kong, State Key Laboratory of GeneticResources and Evolution, Kunming Institute of Zoology, ChineseAcademy of Sciences, Kunming 650223, China. Tel/Fax: +8687165197967. E-mail: [email protected]
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changes and to what extent it contributes to high-altitudeadaptation in native Tibetans.
Materials and methods
Sample collection and DNA extraction
Since we have previously observed a significant associationbetween mtDNA content and temperature, Han population fromShaanxi, a place having similar latitude with Qinghai-Tibetplateau, was then chosen as control to reduce the potentialinfluence of the temperature in this study. After a 12 h fastingperiod, blood samples were collected from 241 unrelatedindividuals in Tibet (above 3500 m), and 220 unrelatedindividuals in Shaanxi province (400 m). Blood glucose, totalcholesterol, triglycerides and carbon dioxide combining powerwere measured by standard procedures using fasting bloodobtained by venipuncture in the early morning. Each participantwas informed about the study and signed an informed consent.All clinical investigations were conducted according to theprinciples expressed in the Declaration of Helsinki. Thestudy project was approved by the Ethics Committee atKunming Institute of Zoology, Chinese Academy of Sciences.This ethics committee has the authority to approve studiesinvolving human participants and/or human material. Totalgenomic DNA was extracted from peripheral blood usingthe AxyPrep� Blood Genomic DNA Miniprep Kit (Axygen,Union City, CA).
Measurement of mtDNA copy number
The procedure for quantifying relative mtDNA content hasbeen described in our previous study (He et al., 2014) andreplicated in another study in our lab (Cheng et al., 2013).Specifically, relative mtDNA copy number was measuredby the fluorescence-based quantitative real-time PCR. Primerpair L375: 50-CACCAGCCTAACCAGATTTC-30 and H475:50-GGGTTGTATTGATGAGATTAGT-30 (Shanghai SangonBiological Engineering Technology & Services Co., Ltd.,Shanghai, China) were used to quantify the mtDNA. Primerpairs HBG1: 50-GCTTCTGACACAACTGTGTTCACTAGC-30
and HBG2: 50-CACCAACTTCATCCACGTTCACC-30
(Shanghai Sangon Biological Engineering Technology &Services Co., Ltd., Shanghai, China) were used for normalization.The quantitative real-time PCR was performed with SYBR�
Premix Ex Taq� II kit (TaKaRa Biotechnology Co. Ltd., Dalian,China) on the CFX Connect� Real-Time system (BioRadLaboratories, Hercules, CA) according to the manufacturer’sinstructions. Each sample was run in triplicates for bothmitochondrial gene and nuclear gene amplification and only thereactions within a standard deviation of 0.2 were included forfurther analysis.
Statistical analysis
Statistical calculations were performed using the statisticalpackage SPSS version 13.0 (Beijing Stats Data Mining Co. Ltd.,Beijing, China). The mtDNA content differences between Han andTibetan subjects and between male and female subjects wereanalyzed using the Mann–Whitney test. Quantitative data wasexpressed as means ± SEM (Standard Error of Mean). Correlationbetween the mtDNA copy number and age, blood glucose,triglyceride, total cholesterol or carbon dioxide combining powerwere analyzed using linear regression. Figures were drawn byPrism 5 (GraphPad Software, Inc., La Jolla, CA). p50.05 wasconsidered to be statistically significant; p50.01 was consideredstatistically very significant.
Results
mtDNA copy number is significantly lower in highlanders
As shown in Figure 1, mtDNA copy number in Tibetans(Mean ± SEM, 136.2 ± 8.60) was significantly lower comparedto Han subjects (Mean ± SEM, 237.9 ± 20.65) (p¼ 0.0017;Figure 1). In fact, our previous research also confirmed thatmtDNA content in Tibetans was significantly lower than other 27Chinese ethnic populations residing in different geographicregions across China (unpublished data).
To exclude bias caused by gender and age, relative mtDNAcopy-number in two genders were compared by Mann–Whitneytest, and the correlation between mtDNA copy-number and agewas analyzed by linear regression. Our results showed that themean mtDNA copy-number in female were comparable to that ofmale in both Shaanxi (p¼ 0.063; Figure 2A) and Tibet(p¼ 0.273; Figure 2B). Furthermore, no correlation betweenmtDNA copy number and age was observed in either Shaanxi(r2¼ 0.002, p¼ 0.073; Figure 3A) or Tibetan subjects (r2¼ 0.003,p¼ 0.409; Figure 3B), suggesting that the observed distributionpattern for mtDNA copy-number was unlikely influenced bygender or age.
MtDNA copy-number is negatively associated with carbondioxide combining power
Changes of mtDNA content and energy metabolism seem to beclosely related (Meierhofer et al., 2004). To determine thepotential impact associated with mtDNA content in Tibetans, wetook an analysis of some metabolic parameters in blood plasmaincluding blood glucose, triglyceride, total cholesterol and carbondioxide combining power in Tibetan subjects. The resultsindicated that CO2 combining power was inversely associatedwith mtDNA copy number (r2¼ 0.425, p¼ 0.012; Figure 4D), butnone of the rest indexes showed a correlation with mtDNA copynumber (r2¼ 0.035, p¼ 0.312; r2¼ 0.061, p¼ 0.338; r2¼ 0.097,p¼ 0.209, respectively; Figure 4A–C).
Discussion
Mitochondria are major ATP producers in mammalian cells.Mitochondrial function can be affected by mtDNA polymorph-isms (Kazuno et al., 2006) and mtDNA content changes(Clay Montier et al., 2009). Besides, mitochondria are susceptibleto hypoxia as well. Ultrastructural and other analyses haveshown that both acclimatized lowlanders (Evett et al., 2012) andadapted highland natives (Kayser et al., 1991) had decreased
Figure 1. Comparison of the relative mtDNA copy number betweenShaanxi subjects (n¼ 220) and Tibetan subjects (n¼ 241).
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mitochondrial density. This phenomenon was regarded as anadaptive response aiming to minimize the production of harmfulreactive oxygen species (ROS) (Hoppeler et al., 2003). Althoughthe mtDNA content in lowlander semen samples increasedsignificantly when initially exposed to high altitude, it tended todecline when the sojourn extended to more than one year (Luoet al., 2011c), likely reflecting a gradual adaptation to highaltitude over time. Evidence from heart exposed to in vivohypoxia also demonstrated a decrease in mitochondrial respir-ation, which was believed to be a protective phenotype withreduced ROS generation and an outcome of mitochondriaresisting to hypoxic stress (Heather et al., 2012). Consistently,our result showed that mtDNA copy-number was significantlylower in high-altitude Tibetans than in Han subjects from loweraltitude. These foresaid phenotypes (decreased mitochondrial andmtDNA content and impaired respiration function) suggest anadaptive mechanism of protecting cells from the damage of ROS.Furthermore, this phenomenon may be not restricted to onlyTibetans but also high-altitude residents of other ethnic groupssince our previous study revealed that populations from the samegeographic region, even belonging to different ethnic groups, tendto share similar level of mtDNA content (Cheng et al., 2013).Further studies are still needed to determine whether low mtDNAcontent is a general phenotype in all high-altitude populations.
Meanwhile, according to Farlex Partner Medical Dictionary(carbon dioxide combining power. (n.d.) Farlex PartnerMedical Dictionary. (2012). Retrieved February 2 2014 from
http://medical-dictionary.thefreedictionary.com/carbon+dioxide+combining+power), carbon dioxide combining power (CO2 CP) isthe ability of blood plasma to combine with carbon dioxide,indicating the alkali reserve and a measure of the acid-basebalance in the blood. Intriguingly, our data suggested a negativecorrelation between mtDNA copy number and carbon dioxidecombining power, indicating those who have lower mtDNAcontent tend to have higher carbon dioxide combining power. It isthen possible that Tibetans may have higher CO2 CP since theirmtDNA copy number was significantly lower than that of thelowlanders. Although Tibetans have been through thousands ofyears developing adaptive phenotypes, the fact is that they stillhave lower level of oxygen in the arterial blood (Beall, 2007).Considering that the intensive metabolic demands could not besatisfied by lower mtDNA content and impaired oxidativephosphorylation, other energy producing pathways, such asglycolysis, become tempting solutions to the problem of main-taining energy homeostasis under hypoxia (Murray, 2009).Indeed, a strong association of increased lactate concentrationand an EPAS1 (endothelial PAS domain protein 1) haplotype thatshows a signal of positive selection was revealed (Ge et al., 2012).However, increased glycolysis can result in an accumulation oflactate within the body, which, if failed to be expelled, could leadto metabolic acidosis. As a representative of bicarbonate in blood,CO2 combining power can neutralize acid. After swimmingtraining, CO2 combining power was negatively correlated withblood lactate concentration in athletes (Zhang et al., 2009).
Figure 2. Relative mtDNA copy number in different gender groups in Shaanxi (A) and in Tibet (B).
Figure 3. Relationships between age and the mtDNA content in Shaanxi (A) and Tibetan (B) subjects.
DOI: 10.3109/19401736.2014.915526 mtDNA reveals high-altitude adaptation 3
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A study revealed that Ochotona curzoniae, a better adaptedTibetan Plateau alpine meadow species, had higher level ofcarbon dioxide combining power than Microtus oeconomus at analtitude of 8000 m (Bai, 2010). Thus it is possible that a higherlevel of CO2 CP presents an adaptive mechanism to neutralize theproducts of increased anaerobic metabolism.
Our findings revealed that Tibetan natives at high altitude hadsignificantly lower mtDNA copy-number than lowlanders.Correlation analyses showed mtDNA content was not associatedwith metabolic substances such as glucose and triglyceride, butwas reversely associated with CO2 combining power, a represen-tative of alkali reserve. Overall, it seems that an increase in CO2
CP accompanied with lower mtDNA content could provideadaptive potential by producing less ROS and compensatingenergy generation without the risk of acidosis under hypoxia.
Acknowledgements
We thank Dr. He Y-H for helpful suggestions.
Declaration of interest
This work was supported by non-profit grants from National BasicResearch Program of China (2012CB518205), Yunnan Province(2011FA024), Natural Science Foundation of China (31123005,31322029), and the Chinese Academy of Sciences. The authors declareno conflicts of interest. The authors alone are responsible for the contentand writing of this article.
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