Pathophysiological changes in mitochondria of mammalian exposed to hypoxia at high altitude

Wen-xiang GAO, Gang WU, Yu-qi GAO

1. Department of Pathophysiology, 2. Institute of Plateau Special Needed Medicine and Medical Equipment, College of High Altitude Military Medicine, the Third Military Medical University, Chongqing 400038; 3. Key Laboratory of High Altitude Medicine, Ministry of Education, the Third Military Medical University, Chongqing 400038, China

Introduction

As human beings ascend to an altitude above 2 500 meters high, the human body confronts a number of environmental stimuli (hypoxia, cold et al), which may lead to injuries and sometimes diseases. Among these stimuli, hypobaric hypoxia is a primary cause of the injuries and diseases, when human body passively starts a series of physiological reactions to adapt to the high altitude environment, including intake, transportation and utility of the oxygen.

As a crucial subcellular organelle of oxygen utility,mitochondria consume high levels of oxygen to generate energy currency through oxidative phosphorylation and Krebs Cycle [1] and exert many other functions contributing to heat production, apoptosis,signaling, etc. Nevertheless, mitochondrion has been proved to be a central link of high altitude adaptative reactions responding to the hypoxic environment.そese organelles can be damaged by acute and severe hypoxia but they can gradually recover as hypoxia prolongs to contributing to the acclimatization and adaptation of human body at high altitude. On the other hand, malfunction of mitochondria have been found to be a critical factor responsible for mountain sicknesses.

In the present study, we reviewed the recent research advances in hypoxic mitochondrial responses at high altitude from aspects of acute hypoxia-induced mitochondrial impairment as well as acclimatization and adaption to acute hypoxia at high altitude including mitochondrial malfunction in mountain sicknesses.

Mitochondria impairment in acute hypoxia

Ultrastructural changes

According to the classical understandings, the number of mitochondria may be decreased upon acute hypoxia, together with ultrastructural changes. To validate this viewpoint, Cai et al [1] placed the rats into a hypobaric chamber of equivalent 4 000 meters high for 3 days and used a stereological method to analyze the ultrastructural changes of the cerebral cortex mitochondria. そey found that the mitochondria mass was increased in acute continuous threeday hypoxia group. Some mitochondrial cristae were disordered and swollen. Meanwhile, the average diameter and average area of the mitochondria were increased but specific surface was decreased afer ex-posure of the animals to acute hypoxia [2], suggesting an increase in size of the mitochondria as well as swelling, rupture and lysis of the mitochondrial cristae afer acute hypoxic insult. However, the above findings do not support a change in the number of the mitochondria. This divergence may stem from diあerent methods or study conditions, including the severity of hypoxia.

Functional changes

Oxygen consumption analysis is the most commonly used test to evaluate mitochondrial function. Luo et al[3]raised rats in a hypobaric chamber of equivalent 3 000 meters high for 3 days, and found decrease of oxygen consumption state 3 (ST3, with ADP) and elevation of oxygen consumption state 4 (ST4, ADP depleted),resulting in a low respiratory control rate (RCR=ST3/ST4) in heart mitochondria[3]. Similar results were also found in cerebral mitochondria of rats subjected to simulated 4 000 meters altitude for 3 days. ST4 is a demonstration of the coupling of oxidation and phosphorylation. In consistency with the decreased ST4,the activity of the mitochondrial uncoupling protein(UCP) was increased [4] but the phosphate-oxygen ratios (P/O ratio) was decreased [3], indicating the impaired efficiency of ATP synthesis. Nevertheless,the F0F1-ATPase activity was decreased [3] in accommodating to the decreased electron transportation, and the cellular ATP production went down [3],which caused a disturbance of energy metabolism in acute hypoxic cells and tissues.

Practical mitochondrial function is based on gene expression and protein synthesis. Mitochondrion is a semi-autonomous organelle, i.e., mitochondrial proteins are coded by nuclear DNA (nDNA) and mitochondrial genome. Human mitochondrial DNA(mtDNA) comprises 13 genes encoding subunits of the electron transport chain, 2 rRNA genes and a number of tRNA genes. そe transcription and translation efficiency of mtDNA were both impaired by acute hypoxia, according to an in vitro study with isolated rat brain mitochondria [3]. Other nDNA-encoded proteins were also found low in mRNA or protein levels after acute hypoxia exposure, such as F0F1-ATPase, VDAC, and so on [5].

The biogenesis of mitochondrial genome may also be involved in hypoxic acclimatization at high altitude. As the Han Chinese from plains ascends to high altitude up to 5 300 meters, the sperm mtDNA copy number was increased and reached its peak after 1 month [6]. In another study, the liver mtDNA was depleted in the rats raised in equivalent 5 000 meters high hypobaric chamber for 30 days [6]. Further studies needs to be carried out to unvail the association of mitochondrial genetic features and high altitude acclimatization.

Mitochondria are separated by outer and inner membranes, which are composed of phospholipid bilayers and proteins, from other sub-cellular structures [7]. Mitochondria-produced ATP needs to be transported out to the cytoplasm to support cell metabolism and functions, while ADP and other substrates into the mitochondria. Transmembrane transportation of ATP depends on a mitochondrial membrane complex containing ATP/ADP carrier (AAC) and mitochondrial membrane potential(MMP). An experimental study showed that the mitochondrial ATP/ADP transportation was decreased after acute hypoxic insulting, the mechanisms of which may involve low AAC activity and decreased MMP [8].

Mitochondrial acclimatization in chronic hypoxia

With prolonged resident time at high altitude, the mitochondria gradually acclimatize to the hypoxic environment, as part of the acclimatization of human body.

A stereological study showed no observable change in mitochondrial morphology and numerical density during chronic hypoxia. Compared with acute hypoxia group, the average diameter and average area were deceased[2], suggesting that the mitochondrial number and ultrastructure recover to the normal status.

As for the oxygen consumption in heart (4 000 meters for 30 days) and brain (5 000 meters for 30 days) of the mitochondria in chronic hypoxic rats,though ST3 and RCR levels are higher than the levels of acute hypoxia rats, the above two parameters still stayed lower than the normal, except for ST4 returning to the normal level, suggesting that mitochondria in chronic hypoxia rats regained a good coupling of oxidation and phyorarylation. Other mitochondrial functional parameters, such as F0F1-ATPase activity,P/O ratio, ATP synthesis efficiency, AAC activity,MMP as well as mitochondria transcription and protein synthesis in vitro were partially restored but still remained lower than the normal levels [8-10].

Mitochondrial adaptation in native high altitude residents

Animals and human beings living at high altitude over a number of generations can acquire abilities to adapt to high altitude hypoxic environment. Generally, there are structural, functional and genetic adaptation responses.

Structural mitochondrial adaptation

Native Tibetan in Qinghai-Tibet Plateau is widely accepted as the best adaptive human race at high altitude. To understand the mitochondrial adaptive responses in native Tibetans, we recruited the immigrated Han Chinese as unadapted group and the Tibetans as adapted group to investigate the mitochondrial ultratructure and function and found that the placental mitochondria showed distinct swelling in Hans but partial vacuolization in Tibetans [11].

Functional mitochondrial adaptation

Similar to the structural observations, the Tibetans show better mitochondrial oxidative phosphorylation functions than Hans when exposed to similar hypoxic environment at high altitude. For example, the placental mitochondrial ST3, RCR, ATP synthesis,and oxidative phosphorylation (OPR), accompanying with the activity of mitochondrial electron transportation chain complexes I, II, III were found higher in Tibetans than in Hans. ST4 was not markedly diあerent, in consistency with the fact that this parameter could return to normal after acclimatization [12].そe mitochondrial ATP/ADP ratio and placental tissue energy charge were also higher in the Tibetans,as a comprehensive result of that AAC activity and MMP were higher in placental mitochondria of the Tibetans [3].

Genetic mitochondrial adaptation

Plateau pika (Ochotona curzoniae) is a native animal that has been residing at over 3 000 meters high altitude for over ten thousand years. During these endless years, the plateau pika has evolved to adapt to the hypoxic environment of high altitude. So far, the mitochondrial genome of the plateau pika has been sequenced and detected the coding information of mtDNA in the mitochondrial adaptation. By comparing mitochondrial genome of the plateau pika with other high altitude native animals and low altitude resident pikas, Luo et al [13] found 15 specific amino acid species in the pika, with two α-helix amino acid replacements in cyclooxygenase-1 (COX1), and a hydrophilic to hypdrophobic amino acid substitution.There was another amino acid substitution at locus 47 located in the matrix region of COX1, a subunit of mitochondrial ecletron transport chain complex IV,these amino acid substitutions may aあect the activity of cytochrome c oxidase and therefore NO generation.

The mtDNA sequence has also been studied in a number of other high altitude animals, including yak[14], Tibetan antelope[15], Tibetan wild donkey[16], Tibetan chicken[17], etc. There found 16 amino acid substitutions in mtDNA encoding proteins by comparing Tibetan wild donkey and Equusasinus and Equuscaballus. Among these above results, there were amino acid substitutions in NADH dehydrogenase subunit 4 (ND4) and ND5 [18], indicating a functional diあerence of mitochondria.

Tibetan mtDNA was also analyzed, which showed that the nt3010G-nt3970C haplotype was positively correlated with high altitude adaptation in the Tibetans, while the D4 haplotype was negatively correlated[19].

Native Tibetans and immigrant Han Chinese showed diあerence in mitochondrial gene and protein expression. By using cDNA array and two dimensional electrophoresis, a set of different expression genes and proteins have been pinpointed and classified. These genes and proteins were associated with energy metabolism, signaling, cell proliferation and adhesion, electron transport, nucleotide-excision repair, etc[20]. Different mitochondrial protein expression could also been found between native and immigrant animals at high altitude, represented by the plateau pika (5 000 meters) and rats (5 000 meters, 30 days), according to a 2 DE electrophoresis analysis[21].

Mitochondrial DNA changes in mountain sicknesses

Recent studies found association of mtDNA variations with mountain sicknesses, which may provide new risk factors for prevention of high altitude pulmonary edema (HAPE) and high altitude polycythemia (HAPC). For example, the genotypes of mtDNA,3397G and 3552A, were found to increase the risk of HAPE [22]. そe newly results of our study team indicated that a mtDNA genotype could help decrease the risk of HAPE in the immigrant Han Chinese at high altitude (unpublished data). そe genetic features of mtDNA seem to be considerably involved in the pathogenesis of both acute HAPE and chronic HAPC mountain sickness, the specific mechanism for which still needs to be clarified.

Summary

Studies focusing on hypoxic mitochondrial responses at high altitude have been lasted for over half a century. Experimental studies on structural, functional and genetic changes of the mitochondria suggest a central link of mitochondria in the processes of hypoxic injuries, acclimatization and adaptation at high altitude, and in the pathophysiological processes of mountain sicknesses. Mitochondrial genome and functions should be a good target for prevention medicines and acclimation promoting measurements against mountain sickness.

In conclusion, acute hypoxia causes structural and functional mitochondrial injuries, which partially recovers in chronic hypoxia. And native high altitude animals and populations show a genetic adaptation in mitochondrial genomes, together with intact mitochondrial structure and elevated mitochondrial function. On the other hand, variations in mtDNA may be associated with mountain sicknesses.

Acknowledgements

そis work was supported by the Grants from National Natural Science Foundation of China (81071610,81471814).

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