HIF-1α and HIF-2α induce angiogenesis and improve muscle energy recovery.

HIF er et signalmolekyl som aktiverer angiogenese, altså produksjonen av nye blodkar. Ved lave oksygennivåer økes HIF. Dette kan komme fra trening, men også fra øvelser med å holde pusten.




Cardiovascular patients suffer from reduced blood flow leading to ischemia and impaired tissue metabolism. Unfortunately, an increasing group of elderly patients cannot be treated with current revascularization methods. Thus, new treatment strategies are urgently needed. Hypoxia inducible factors (HIFs) upregulate the expression of angiogenic mediators together with genes involved in energy metabolism and recovery of ischemic tissues. Especially, HIF-2α is a novel factor and only limited information is available about its therapeutic potential.


Gene transfers with adenoviral HIF-1α and HIF-2α were done into the mouse heart and rabbit ischemic hindlimbs. Angiogenesis was evaluated by histology. Left ventricle function was analysed with echocardiography. Perfusion in rabbit skeletal muscles and energy recovery after electrical stimulation-induced exercise were measured with ultrasound and 31 P-magnetic resonance spectroscopy (31 P-MRS), respectively.


HIF-1α and HIF-2α gene transfers increased capillary size up to 5-fold in myocardium and ischemic skeletal muscles. Perfusion in skeletal muscles was increased by 4-fold without edema. Especially AdHIF-1α enhanced the recovery of ischemic muscles from electrical stimulation-induced energy depletion. Special characteristic of HIF-2α gene transfer was a strong capillary growth in muscle connective tissue and that HIF-2α gene transfer maintained left ventricle function.


We conclude that both AdHIF-1α and AdHIF-2α gene transfers induced beneficial angiogenesis in vivo. Transient moderate increases in angiogesis improved energy recovery after exercise in ischemic muscles. This study shows for the first time that a moderate increase in angiogenesis is enough to improve tissue energy metabolism which is potentially a very useful feature for cardiovascular gene therapy.

Morning attenuation in cerebrovascular CO2 reactivity in healthy humans is associated with a lowered cerebral oxygenation and an augmented ventilatory response to CO2

Denne beskriver hvordan blodkarenes respons på CO2 er dårligere om morgenen, og det er derfor det skjer flere slag og slikt om morgenen. Den nevner mange interessante prinsipper. Bl.a. at lavere vasomotor respons (på CO2) gir mindre oksygen til hjernen. Og at i opptil 20 sekunder etter en 20 sekunder holdning av pust (etter utpust) øker fortsatt oksygenmengden og blodgjennomstrømningen i hjernen. Nevner også at siden blodkarene i hjernen reagerer dårligere på CO2 om morgenen blir det lett at pusten over- eller underkompenserer, så pustemønsteret blir uregelmessig om morgenen. Spesielt om man har underliggende faremomenter som hjerte/karsykdommer.



Furthermore, our results suggest that morning cerebral tissue oxygenation might be reduced as a result of a decreased cerebrovascular responsiveness to CO2 or other factors, leading to a higher level of desaturation.

Our data indicate that the cerebrovascular reactivity to CO2 in healthy subjects is significantly reduced in the morning and is strongly associated with an augmented ventilatory response to CO2. It is likely that this reduction in MCAV CO2 reactivity, by reducing blood flow through medullary respiratory control centers, increases both the arterial-brain tissue PCO2 difference and the H+ concentration presented to the central chemoreceptor(s) (1144). In effect, it appears the brain tissue is more susceptible in the morning to changes in arterial PCO2, which could increase the likelihood of ventilatory overshoots and undershoots.

However, as was the case with the hypercapnic challenge, subjects holding their breath in the morning experienced a significantly blunted increase in MCAV compared with evening, likely a result of a reduced cerebrovascular responsiveness to CO2.

In conclusion, our results suggest that early morning reductions in cerebrovascular CO2 reactivity strongly influence the magnitude of the ventilatory response to CO2. This may have significant implications for breathing stability, increasing the chances of periodic breathing in the morning in patients with additional risk factors. The early morning reduction in cerebral oxygenation with hypercapnic challenge, mild hypoxemia, or during apnea may be a contributing factor in the high prevalence of early morning stroke. Whether differences in the responses of CBF, oxygenation, or V̇E to CO2challenge are associated with other risk factors for stroke, such as gender or age, remains to be elucidated.

Apnea: A new training method in sport?

Veldig viktig studie om hva dykkeres trening i Apnea (å holde pusten) kan bidra med i annen idrett. Bekrefter det meste av det jeg har skrevet om, men oppklarer noe om blodverdier bl.a. Nevner EPO, nyrenes tilpasning, hypoxi, HIF-1, melkesyre, lungevolum



Breath-hold divers have shown reduced blood acidosis, oxidative stress and basal metabolic rate, and increased hematocrit, erythropoietin concentration, hemoglobin mass and lung volumes. We hypothesise that these adaptations contributed to long apnea durations and improve performance. These results suggest that apnea training may be an effective alternative to hypo- baric or normobaric hypoxia to increase aerobic and/or anaerobic performance.

Apnea durations clearly increase with training. Perhaps less well known are the findings that apnea train- ing also increases hematocrit (Hct), erythropoietin (EPO) concen- tration, hemoglobin (Hb) mass, and lung volumes [2–5]. In addition, blood acidosis and oxidative stress were shown to be re- duced after three months of apnea training [6,7]. Therefore, why not encourage apnea training for athletes?

The major determinant of aerobic performance is the capacity to deliver oxygen to the tissues [8]. An increase in the total amount of erythrocytes, as reflected by increased Hct and Hb mass, is med- iated by the glycoprotein hormone EPO, which is predominantly synthesized by the kidneys in response to chronic hypoxia [9] and to some extent (10–15% of total production) by the liver. EPO stimulates the proliferation and maturation of red blood cell precursors in bone marrow, increasing oxygen delivery to muscle and thereby enhancing sports performance [9].

(hypoxic or ischemic conditions) results in a stabilization of the transcription factor hypoxia-inducible factor (HIF)-1a, which increases EPO secretion and the expression of EPO receptor [10].

Furthermore, any training effect vanishes rapidly (two weeks), as the newly formed red cells disappear within a mat- ter of days due to neocytolysis.

The splenic contraction effect

Apnea training may well be a future training method. Splenic contraction has been described in marine mammals as improving oxygen transport, through an increase in circulating erythrocytes. Its consequence is a prolonged dive without injuries. In humans, repeated apneas (five, in general) induce splenic contraction. This increases Hct and Hb (both between 2% and 5%) independently of hemoconcentration [19] and reduces arterial oxygen desaturation, thereby prolonging the apnea duration [3,19–22].

Repeated apneas are known to induce hypoxemia in the spleen and kidney, increas- ing respectively Hct and Hb and serum EPO concentrations [2,23].

First, the splenic contraction develops quickly after three or four apneas separated by two minutes of recovery and is associ- ated with a transient increase in Hb concentration. The amplitude of the spleen volume reduction after repeated apneas, with or without face immersion, varies widely (20–46%) depending on the rate of change in oxygenation [3,19,22,25–27]. The rapidity of the splenic contraction after simulated apneas strongly suggested a centrally-mediated feed-forward mechanism rather than the influ- ence of slower peripheral triggers [19]. These spleen and Hb re- sponses may be trainable.

Second, DeBruijns et al. [2] recently observed that repeated apneas increased EPO concentration by 24%, with the peak value reached 3 h after the last apnea and a return to baseline 2 h later.

The rapid reduction in tissue oxygen levels that oc- curs during apneas has been suggested to stimulate enhanced EPO production [25]. The decreased kidney blood flow induced by apneic vasoconstriction would result in local ischemic hypoxia, stimulating kidney EPO production. Similarly, obstructive sleep ap- nea increases the levels of EPO (􏰀1.6) and Hb (+18%) [24].

The lower SaO2 decrease found in trained divers after repeated apneas may account for the reduced oxygen delivery because of the diving response (bradycardia and vasocon- striction) and/or an increase in oxygen content [1].

Long term-effects

Another important consideration is the persistence of the per- formance gains. Most of the altitude exposure studies reported short-term effects (i.e., weeks). Repeated apneas increase Hct but this increase disappears within 10min after the last apnea [22,26].

The effects of repeated apneas on spleen and endogenous EPO may also constitute an alternative to using rhEPO or its analogues. In addition, comparison of resting Hb mass in elite BHDs and untrained subjects showed a 5% higher Hb mass in the BHDs, and the BHDs also showed a larger relative increase in Hb after three apneas (2.7%). The long-term effect of apnea training on Hb mass might be implicated in elite divers’ performances. Re- cently, it has been found that after a 3-month apnea training pro- gram, the forced expiratory volume in 1 s was higher (4.85 ± 0.78 vs. 4.94 ± 0.81 L, p < 0.05), with concomitant increases in the max- imal oxygen uptake, arterial oxygen saturation, and respiratory compensation point values during an incremental test [30].

In addition to increasing EPO and provoking splenic contraction, apnea training has been hypothesized to modify muscle glycolytic metabolism. An improvement in muscle buffer capacity [6,7,32] would reduce blood acidosis and post-apnea oxidative stress [6]. Delayed acidosis would also be advantageous for exercise perfor- mance. Finally, trained BHDs exhibit high lung volumes [15]. Ap- nea training might be interesting to improve respiratory muscle performance [15], thereby delaying the respiratory muscle fatigue during prolonged and maximal exercise.

Greater cerebral blood flow (CBF) increase was described during long apnea in elite BHDs than in controls and interpreted as a protection of the brain against the alteration of blood gas [33]. The CBF increase observed in BHDs could be the re- sult of an increased capillary density in the brain as has been de- scribed after a prolonged hypobaric hypoxia exposure [35]. These results suggest that apnea training per se provides hypoxic precon- ditioning, increasing hypoxemia and ischemia tolerance [33].

The physiological responses to apnea training exhibited by elite breath-hold divers may contribute to improving sports perfor- mance. These adaptations may be an effective alternative to hypo- baric or normobaric hypoxia to increase performance. Further experimental research of the apnea training effects on aerobic and/or anaerobic performance are needed to confirm this theory.

Intermittent Hypoxia Research in the Former Soviet Union and the Commonwealth of Independent States: History and Review of the Concept and Selected Applications

Svært viktig review om IHT – intermittend hypoxia training – som det har blitt gjort mye forskning på i russland, men først nå begynt å bli interessant i vesten. Nevner hvordan co2 reageer på hypoxi, og nervesystemet og vevet og i mitchondria, inklidert at SOD øker med 70% og at IHT normaliserer NO som lagres i blodkarene. Om at IHT kan brukes i behandling av sykdommer, inkludert strålingskader fra Chernobyl. Metoden er en «rebreathing» frem til O2% er nede på 7% – ca. 5 minutter daglig i 3 dager.


To the present day, intermittent hypoxic training (IHT) has been used extensively for altitude preacclimatization; for the treatment of a variety of clinical disorders, including chronic lung diseases, bronchial asthma, hypertension, diabetes mellitus, Parkinson’s disease, emotional disorders, and radiation toxicity, in prophylaxis of certain occupational diseases; and in sports.

The basic mechanisms underlying the beneficial effects of IHT are mainly in three areas: regulation of respiration, free-radical production, and mitochondrial respiration. It was found that IHT induces increased ventilatory sensitivity to hypoxia, as well as other hypoxia-related physiological changes, such as increased hematopoiesis, alveolar ventilation and lung diffusion capacity, and alterations in the autonomic nervous system.

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Due to IHT, antioxidant defense mechanisms are stimulated, cellular membranes become more stable, Ca2+ elimination from the cytoplasm is increased, and 02 transport in tissues is improved. IHT induces changes within mitochondria, involving NAD-dependent metabolism, that increase the efficiency of oxygen utilization in ATP production. These effects are mediated partly by NO-dependent reactions.

Particularly at issue are the effects in humans of such transient bouts of hypoxia when repeated many times, a practice designated as intermittent hypoxia. Furthermore, when intermittent hypoxia as a specific protocol is employed to accomplish a particular aim, for example, acclimatization to high altitude, we use the term intermittent hypoxic training or IHT.

The interested reader is referred to more extensive recent scientific and historical reviews and investigative reports in Russian and Ukrainian, particularly as related to the potential thera- peutic effects of intermittent hypoxia (Berezovsky et al., 1985; Karash et al., 1988; Meerson et al., 1989; Anokhin et al., 1992; Berezovskii and Levashov, 1992; Donenko, 1992; Ehrenburg, 1992; Fesenko and Lisyana, 1992; Vinnitskaya et al., 1992; Serebrovskaya et al., 1998a,b; Volobuev, 1998; Chizhov and Bludov, 2000; Ragozin et al., 2000). Noted also is the use of IHT in the prophylaxis of professional occupational diseases (Berezovsky et al., 1985; Karash et al., 1988; Serebrovskaia et al., 1996) and in sports (Volkov et al., 1992; Radzievskii, 1997; Kolchinskaya et al., 1998, 1999). Recent reviews from Western Europe and North America are also given (Bavis et al., 2001; Clanton and Klawitter, 2001; Fletcher, 2001; Cozal and Cozal, 2001; Mitchell et al., 2001; Neubauer, 2001; Prabhakar, 2001; Prabhakar et al., 2001; Wilber, 2001).

During successive altitude exposures, compared with the initial exposure, the higher ventilation and blood arterial oxygen saturation, together with the lower blood Pco2, implied that ventilatory sensitivity to hypoxia had been increased.

In the intervening years to the present, intermittent hypoxia has been used extensively in the Soviet Union and the CIS not only for altitude pre acclimatization (Gorbachenkov et al., 1994), but also it has been proposed for treatment of a variety of clinical disorders, including chronic lung diseases and bronchial asthma in children and adults (Meerson et al., 1989; Anokhin et al., 1992; Berezovskii and Levashov, 1992; Donenko, 1992; Ehrenburg and Kordykinskaya, 1992; Fesenko and Lisyana, 1992; Redzhebova and Chizhov, 1992; Vinnitskaya et al., 1992; Lysenko et al., 1998; Serebrovskaya et al., 1998b; Chizhov and Bludov,2000; Ragozin et al.,2000, 2001), hypertension (Meerson et al., 1989; Potievskaya and Chizhov, 1992; Rezapov, 1992), emotional disorders (Gurevich et al., 1941), diabetes mellitus (Kolesnyk et al., 1999; Zakusilo et al., 2001), Parkinson’s disease (Kolesnikova and Serebrovskaya, 1998; Serebrovskaya et al., 1998a), inflammatory processes (Tkatchouk, 1994; Tsvetkova and Tkatchouk, 1999), radiation toxicity (Karash et al., 1988; Sutkovyi et al., 1995; Serebrovskaia et al., 1996; Strelkov, 1997; Strelkov and Chizhov, 1998), and certain occupational diseases (Berezovsky et al., 1985; Karash et al., 1988; Rushkevich and Lepko, 2001).

If so, then in normal humans only a few minutes of daily hypoxic exposure rapidly induces detectable increments in hypoxic ventilatory response, a hallmark of altitude acclimatization.

Hypocapnia occurs with altitude acclimatization and ventilation remains increased for days after the subjects return to sea level. By contrast, in the two above studies, eucapnia was maintained during the hypoxic exposures and neither normoxic venilation nor normoxic PC02 was altered by IHT, suggesting that only the hypoxemia and not changes in PC02 (or pH) were involved.

Changing CO2, either for the carotid body or the brain, did not induce ventilatory acclimatization to hypoxia. The work from Bisgard’s laboratory suggested that ventilatory acclimatization to hypoxia in the goat depends almost exclusively on the carotid body’s response to low oxygen. If similar mechanisms operate in humans, then ventilatory acclimatization to hypoxia, operating via the carotid body and independent of changes in pH or PCO2, can be induced by IHT. What is remarkable is that such brief periods of hypoxia can have such clearly measurable increases in the ventilatory response to hypoxia.

In addition to increasing hypoxic ventilatory sensitivity, CIS investigators have reported that IHT increases tidal volume and alveolar blood flow during exercise, improves matching of ventilation to perfusion, increases lung diffusion capacity, redistributes peripheral blood flow during exercise, decreases heart rate, increases stroke volume, and increases blood erythrocyte counts (Volkov et al., 1992; Kolchinskaya, 1993; Radzievskii 1997; Kolchinskaya et al., 1999; Maluta and Levashov, 2001). These effects have been considered to be beneficial in training athletes (Volkov et al., 1992; Radzievskii, 1997). Such findings await independent confirmation from others.

The spectral analysis suggested that after IHT there was greater parasympathetic preponderance during the hypoxic challenge than in the control group. These novel studies conducted in Ukraine suggested that IHT mimicked the usual acclimatization to high altitude, with its primarily greater parasympathetic activity (Reeves, 1993; Hughson et al., 1994). Such activation of the parasympathetic system by IHT was supported by experiments in rats (Doliba et al., 1993; Kurhalyuk et al., 2001a, b, see below).

Among these effects are that brief hypoxic stimuli of only several minutes per day for only a few days give responses that last many days, even weeks, and also that IHT apparently affects a multitude of normal functions and disease states.

This work, as well as more recent studies (Lukyanova, 1997; Sazontova et al., 1997; Kondrashova et al., 1997; Temnov et al., 1997; Lebkova et al., 1999), indicated that the basic molecular response to any type of hypoxic challenge involves the mitochondrial enzyme complexes (MchEC). There is evidence that energy metabolism under acute hypoxia is affected even before a significant decrease in oxygen consumption becomes measurable and before cytochrome c oxidase (CO) activity is significantly reduced.

In the cascade of hypoxia-induced metabolic alterations, MchEC I is most sensitive to intracellular oxygen shortage. The reversible inhibition of MchEC I leads to both the suppression of reduced equivalent flux through the NAD-dependent site of the respiratory chain and the emergency activation of compensatory metabolic pathways, primarily the succinate oxidase pathway. The switch of energy metabolism to this pathway is the most efficient energy-producing pathway available in response to a lack of O2 (Lukyanova et al., 1982).

Skulachev (1996) proposed a two-stage mechanism that allows mitochondria to regulate O2 concentrations and to protect against oxidative stress: (1) «soft» decoupling of oxidative phosphorylation for «fine-tuning» and (2) decrease of the reduction level of respiratory chain components by opening nonspecific mitochondrial inner membrane pores as a mechanism to cope with massive O2 excess.

However, parallel nonenzymatic processes result in O2 formation. This is especially the case when 02 concentrations reach the capacity of the respiratory chain enzymes. Reduction in O2 concentrations leads to an exponential decrease in radical formation. Skulachev (1995) hypothesizes that mitochondria have a mechanism for «soft» decoupling in stage 4 of the respiratory chain. This mechanism prevents the complete inhibition of respiration, the complete reduction of respiratory carriers, and the accumulation of reactive compounds such as ubisemiquinone (CoQ·-). Such a mechanism will be activated, for example, if the capacity of the respiratory chain is decreased due to a reduced availability of ADP.

In contrast to the constriction of capillaries, which prevents undesirably high O2 concentrations in tissue, «soft» decoupling allows fine-regulation on an intracellular level.

Adaptation to hypoxia by an intermittent hypoxic challenge is associated with the expression of, and a shift toward, enzyme isoforms that can efficiently function in a mitochondrial environment with high concentrations of reduced equivalents as generated during hypoxia. This prevents inactivation of the MchEC and may constitute one of the adaptation mechanisms triggered by intermittent hypoxia.

One of the most significant peculiarities of adaptation to intermittent hypoxia is free-radical processes.

The periods of reoxygenation could lead to oxygen radical formation, which might be analogous to that occurring with normoxic reperfusion of transiently hypoxic or ischemic tissues (Belykh et al., 1992; Meerson et al., 1992a). If periods of hypoxia followed by normoxia led to formation of oxygen radicals, but if the hypoxia were much briefer than the periods of normoxia, and if the exposure sequence were repeated over days, then one might expect that antioxidant defenses could be enhanced much more effectively than in sustained hypoxia.

In a recent study rats were given shorter hypoxic exposures: 15 min five times daily for 14 d (Kurhalyuk and Serebrovskaya, 2001). When they were subsequently challenged by exposure to 7% oxygen, blood catalase and glutathione reductase activity were increased and malon dialdehyde concentration was half that of non-IHT controls. The findings were consistent with the concept that intermittent hypoxia stimulates increased antioxidant defenses.

While there was considerable individual variability in the findings (Table 2), our indexes of oxidant stress before IHT were higher in the Chernobyl workers. We used a program of IHT: isocapnic, progressive, hypoxic rebreathing lasting for 5 to 6 min until inspired air O2 reached 8% to 7%, with three sessions, separated by 5 min, of normoxia, per day for 14 consecutive days. The use of IHT was accompanied by a decrease of spontaneous and hydrogen peroxide-initiated blood chemiluminescene, as well as considerable reduction of MDA content (Table 2). Of interest, more recent study on patients with bronchial asthma, who also were characterized by indexes suggestive of oxidative stress, have shown that similar IHT produced the increase of superoxide dismutase (SOD) activity by nearly 70%. This increase correlated with a decrease in MDA content (r = -0.61, P< 0.05) (Safronova et al., 1999; Serebrovskaya et al., 1999c).

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Thus studies in humans and in tissues have shown that adaptation to intermittent hypoxia induces increased antioxidant defenses, acceleration of electron transport in the respiratory chain, stabilization of cellular membranes, and Ca2+ elimination from the cytoplasm. These data served as the basis for IHT in the treatment of various diseases in which free-radical production might be anticipated, for example, bronchial asthma and post-Chernobyl syndromes.

Recent studies have shown the following principal results: (1) adaptation to intermittent hypobaric hypoxia stimulates NO production in the organism; (2) excessive NO synthesized in the course of adaptation is stored in the vascular wall; (3) adaptation to hypoxia prevents both NO over-production and NO deficiency, resulting in an improvement in blood pressure; and (4) effects of intermittent hypoxia on mitochondrial respiration are mediated mainly by NO-dependent reactions (Manukhina et al., 1999, 2000a,b; Malyshev et al., 2000; Ikkert et al., 2000; Smirin et al., 2000; Kurhalyuk and Serebrovskaya, 2001; Kurhalyuk et al., 2001b; Serebrovskaya et al., 2001).

A somewhat different hypothesis has also been suggested, that the electron transport function of the myocardial respiratory chain in the NAD-cytochrome-b area is limited to a greater extent in animals poorly tolerant to hypoxia than in those that are not. In intolerant animals, even mild hypoxia leads to diminution of the oxidative capacity of the respiratory chain and of ATP production and, as a consequence, to a suppression of the energy-dependent contractile function of the myocardium. In animals more tolerant of hypoxia, this process is less manifest and develops very slowly, which confirms the lesser role of NAD-dependent oxidation of substrates in the metabolism of the myocardium of these animals (Korneev et al., 1990).

Taken together these studies suggest that IHT induces increased ventilatory sensitivity to hypoxia in the absence of Pco2 or pH changes; that it induces other hypoxia-related physiological changes such as increased hematopoiesis and decreased plasma volume and increase in alveolar ventilation and lung diffusion capacity; and that it may be useful in the management of certain disease states. The effects appear to be mediated, at least in part, through release of reactive oxygen species, which then induce an increase of antioxidant defenses. In addition, IHT appears to induce changes within mitochondria, possibly involving NAD-dependent metabolism, that increase the efficiency of oxygen utilization in ATP production.

Response of skeletal muscle mitochondria to hypoxia

Alt om hvordan mitochondrier reagerer på hypoxi, inkludert under trening. Nevner ROS, kapillærer, oxygencascade, HIF-1,tibetanere og høydetreningmen ingen ting om CO2. Nevner også hypoxi inntrer 20s etter trening starter – ref HIIT.


In contrast to earlier assumptions it is now established that permanent or long-term exposure to severe environmental hypoxia decreases the mitochondrial content of muscle fibres. Oxidative muscle metabolism is shifted towards a higher reliance on carbohydrates as a fuel, and intramyocellular lipid substrate stores are reduced. Moreover, in muscle cells of mountaineers returning from the Himalayas, we find accumulations of lipofuscin, believed to be a mitochondrial degradation product. Low mitochondrial contents are also observed in high-altitude natives such as Sherpas.

In these subjects high-altitude performance seems to be improved by better coupling between ATP demand and supply pathways as well as better metabolite homeostasis.

The hypoxia-inducible factor 1 (HIF-1) has been identified as a master regulator for the expression of genes involved in the hypoxia response, such as genes coding for glucose transporters, glycolytic enzymes and vascular endothelial growth factor (VEGF). HIF-1 achieves this by binding to hypoxia response elements in the promoter regions of these genes, whereby the increase of HIF-1 in hypoxia is the consequence of a reduced degradation of its dominant subunit HIF-1a.

A further mechanism that seems implicated in the hypoxia response of muscle mitochondria is related to the formation of reactive oxygen species (ROS) in mitochondria during oxidative phosphorylation. How exactly ROS interfere with HIF-1a as well as MAP kinase and other signalling pathways is debated.

The current evidence suggests that mitochondria themselves could be important players in oxygen sensing.

The focus on mitochondria neglects the fact that for the dominant function of mitochondria, aerobic ATP re-synthesis, the mitochondrial organelle cannot function independently. It is embedded, at least in animal phyla, in a systems physiological context. Mitochondria house the final biochemical steps in the production of reducing equivalents that react at the terminal oxidases of the respiratory chain with molecular oxygen provided by the respiratory system from the environment.

In mitochondria, O2 finally disappears and oxygen partial pressure goes to zero (Fig. 1). Mitochondria thus are an effective oxygen sink and this allows organisms to use all of the available oxygen partial pressure of the actual environment to drive the respiratory cascade from lungs through circulation to the mitochondria (Taylor & Weibel, 1981).

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Figure 1

The pathway of oxygen. Simplified model of the oxygen transport pathway showing the principal structures and the corresponding partial pressure of oxygen (PJ) in these compartments, during maximal exercise, while breathing normoxic or hypoxic room air. mb, myoglobin. The data are derived from Richardson et al. (1995).

What is known is that isolated mitochondria do not need more than a few torr of O2 partial pressure for full function (Gayeski & Honig, 1997). Similarly, it is generally acknowledged that assessing mitochondrial function in hypoxia in cultured cells requires the lowering of the oxygen pressure at the cell surface to values between 0.5 and 5 Torr, values completely incompatible with the survival of all but a few highly specialized species (Boutilier & St Pierre, 2002).

In the context of this review we mostly neglect the hypoxic response of the microvasculature as well as the cardio- vascular and pulmonary system, which all conspire to maintain mitochondrial function under hypoxic conditions (Hochachka, 1998).

With ‘short- term’ hypoxia we refer to continuous bouts of hypoxia exposure lasting from minutes to hours (such as during a physical exercise bout), whereby during most of the day subjects are subjected to normoxic conditions.

With the term ‘intermittent’ hypoxia we designate experimental protocols whereby short periods of hypoxia (minutes) are interspersed with similarly short periods of normoxia. The physiological effects of inter- mittent hypoxia interventions have not yet been well defined. In particular, these types of interventions have not to our knowledge been analysed with regard to changes of the mitochondrial compartment. This literature will thus not be reviewed here (see Serebrovskaya, 2002).

One of the first notions of a change of muscle oxidative capacity with hypoxia is found in Reynafarje’s (1962) landmark paper on muscle adaptations in Peruvian miners. He found cytochrome c reductase activity (+78 %) and myoglobin content (+16%) to be significantly increased in biopsies of sartorius muscle of permanent high-altitude residents as compared to low-landers. This paper, together with data of larger muscle capillary densities in guinea pigs native to the Andeas as compared to data from low-land control animals (Valdivia, 1958), had a strong impact on physiologists’ thinking for at least two decades.

When it became clear towards the end of the sixties, that endurance-type exercise was also a potent stimulator of muscle mitochondrial enzyme activity (Holloszy, 1967), the connection was made that local tissue hypoxia provoked by exercise might be an important signal for mitochondrial proliferation under heavy exercise conditions.

The capillary density (i.e. the number of capillaries counted per unit area of muscle fibre) was indeed found to be increased in post-expedition samples. However, this was not due to capillary neo-formation since the capillary to muscle fibre ratio was unchanged. What had occurred over the time of the expedition was a significant loss of muscle fibre cross- sectional area. Hence, the same number of capillaries supplied a smaller tissue space. In addition, we found a decrease of mitochondrial volume density, which led to a total loss of muscle mitochondrial volume of close to 30 %.

This adaptation was suggested to represent an anti-apoptotic mechanism allowing protection against the lack of oxygen in oxidative muscles (Riva et al. 2001).

We would assume that the increased transcription of genes involved in the metabolic pathways favouring glucose metabolism in muscle cells is responsible for the observed shift towards glucose metabolism in organisms at altitude. The functional advantage of this adaptation would be a larger ATP yield per molecule of oxygen (Wilmore & Costill, 1994). It has recently been shown that protein expression of the enzymes and transporters of lactate, lactate dehydrogenase (LDH) and monocarboxylate transporters (MCT1, 2 and 4), is affected in a tissue-specific manner by long-term exposure to hypobaric hypoxia (McClelland & Brooks, 2002). These findings are broadly compatible with the idea that hypoxia induces mechanisms favouring glucose utilization in muscle cells.

Despite the low muscle mitochondrial oxidative capacity, physical performance capacity of native high-land populations at altitude is excellent. A number of physiological mechanisms have been invoked to explain the ‘paradoxical’ finding of superior aerobic performance in hypoxia despite modest muscle oxidative capacities (Hochachka et al. 2002). Hochachka et al. (1998) describe systemic mechanisms, among them being a blunted hypoxia ventilatory and pulmonary vasoconstrictor response as well as an up-regulation of erythropoietin expression, which help native high-land populations to excel at altitude. At the level of muscle tissue it is suggested that the control contributions from cellular ATP demand and ATP supply pathways are up-regulated whereas the contribution of control steps in O2 delivery are down-regulated (Hochachka et al. 2002). This leads to an improved coupling between ATP demand and supply pathways and better metabolite (i.e. lactate, adenylates) homeostasis.

We thus decided to explore hypoxia protocols under which hypoxia was present only during the exercise session but not during recovery (e.g living low–training high). Analysis of pre- and post- training biopsies of m. vastus lateralis revealed that total mitochondria increased significantly in all groups; in contrast, subsarcolemmal mitochondria, i.e. those located near capillaries, increased significantly only in those groups training under hypoxic conditions, irrespective of training intensity. Noticeably, the group which trained at high intensity in hypoxia showed the highest increase in total mitochondrial volume density (+59 %) and capillary length density was increased significantly in this group only (+17.2 %) (Geiser et al. 2001; Vogt et al. 2001).

Several independent lines of evidence suggest the occurrence of ‘local hypoxia’, i.e. a drop in intramyocellular oxygen pressure with exercise.

This finding of a low muscle oxygen tension during exercise is supported by more recent data using 1H-nuclear magnetic resonance spectroscopy which demonstrates that myoglobin desaturation occurs within 20 s of onset of exercise in human quadriceps muscle (Richardson et al. 1995).

Mitochondria, the oxygen sink, are more prevalent in oxidative fibres by a factor of at least three, and even within fibres they are clustered in the fibre periphery close to the capillaries (Howald et al. 1985).

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Figure 3
HIF-1a-mediated oxygen sensor and hypoxia-inducible gene expression. Oxygen stabilizes hypoxia- inducible factor 1a (HIF-1a) through Fe2+-dependent proline hydroxylases and asparaginyl hydroxylase(s) which hydroxylate HIF-1a within its oxygen-dependent degradation domain (ODDD) and C-terminal portion, respectively. Such modification causes recruitment of the von Hippel-Lindau tumour- suppressor protein (pVHL), which targets HIF-1a for proteosomal degradation thereby silencing HIF-1a activity. Conversely, hypoxia causes a stabilization of HIF-1a and activates the MAPK pathway which enhances the transcriptional activity of HIF-1a through phosphorylation. Both mechanisms contribute to transcriptional activation of downstream angiogenic factor (VEGF), erythropoietin (EPO), glucose transporters (glut 1 and 3) and glycolytic genes (PFK) via HIF-1a/HIF-1b dimers (HIF-1). Moreover, lack of oxygen (anoxia) causes HIF-1a stabilization, possibly by reducing the activity of hydoxylases by depleting their substrate O2.

In vitro experiments show that an activation of HIF-1 leads to an up-regulation of anaerobic enzymes of the glycolytic pathway (Wenger, 2002).

To date there is no clear-cut explanation for the paradoxical finding of increased reactive oxygen species (ROS) production in hypoxia as there is controversy about whether hypoxia, in fact, causes an increase or a fall in ROS production (Archer & Michelakis, 2002). Also the mechanisms by which ROS and cytosolic H2O2 levels are involved in stabilization/activation and eventual degradation/silencing of HIF-1a and other redox-sensitive transcription factors are controversially discussed (Chandel et al. 2000; Kietzmann et al. 2000).

It is reported that ~2% of the O2 used by the mitochondrial electron transport chain creates ROS and in particular the superoxide radical, due to its incomplete reduction (see Kietzmann et al. 2000; Archer & Michelakis, 2002; reviewed in Abele et al. 2002). The superoxide anion (O2•_) radical is very unstable and is rapidly converted either spontaneously or after its export into the cytoplasm by mitochondrial and cytoplasmic superoxide dismutases, MnSOD and Cu/ZnSOD, to the more stable H2O2.

Recent findings in hepatoma cells indicate a small (approximately 2.5-fold) and transient increase in ROS after 40–50 min of hypoxia onset (0.5 % O2; Vanden Hoek et al. 1998). These and subsequent in vitro experiments in the same system point to mitochondria, and to respiratory complex III of the electron transport chain in particular, as the source of ROS production in hypoxia (Chandel et al. 2000).

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Figure 4

Targets of mitochondrial ROS production. Incomplete reduction of O2 during normal metabolic conversion of pyruvate in mitochondria gives rise to a low level of superoxide anion (O2•_). Exposure of cells to hypoxia increases the aberrant production of O2•_ at the mitochondrial electron chain complex III. Catalysed or spontaneous dismutation of the unstable superanion and export via anion channels enhances the concentration of cytosolic H2O2 and other reactive oxygen species (ROS). Increased ROS may increase the level of oxidatively damaged lipids (lipofuscin) or activate downstream redox-sensitive signal transduction events. For example ROS stabilize HIF-1a possibly by interfering with the activity of Fe2+-dependent proline hydroxylases. ROS may also activate the MAPK or PI-3K/Akt pathway, which enhance the transcriptional activity of HIF-1a through its phosphorylation. Lastly, mechanical stress may also increase ROS production which may influence HIF-1a activation.

Clearly there is evidence pointing to a role of mitochondria as oxygen sensors by modulation of ROS production.

Recently, evidence for increased oxidant production in rat skeletal muscle during prolonged exercise has been provided, with both the mitochondrial respiratory chain and the NADPH oxidase as potential sources for oxidants (Bejma & Ji, 1999). This argues for ROS production in skeletal muscle due to local tissue hypoxia.

Two routes to functional adaptation: Tibetan and Andean high-altitude natives

Om hvordan pusten endres når man bor i høyland. Nevner mye om oksygentilgjengelighet, oksygenkaskade, hypoxi, kapillærer, mitokondrier, m.m., og om hvorfor vi har lite oksygen i mitokondriene. Studier undersøker spesifikt om at tibetanere har endrede gener siden de med gener som er tilpasset lave oksygennivåer har mindre fysiologisk stress og dermed større sjangse for barn som overlever, og hvilke forkjeller i hypoxi-tilpasning som har sjedd i de to befolkningene.

Ny, som må gjennomgåes: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1876443/


These reveal generally more genetic variance in the Tibetan population and more potential for natural selection. There is evidence that natural selection is ongoing in the Tibetan population, where women estimated to have genotypes for high oxygen saturation of hemoglobin (and less physiological stress) have higher offspring survival.

At 4,000-m elevation, every breath of air contains only ≈60% of the oxygen molecules in the same breath at sea level. This is a constant feature of the ambient environment to which every person at a given altitude is inexorably exposed. Less oxygen in inspired air results in less oxygen to diffuse into the bloodstream to be carried to the cells for oxygen-requiring energy-producing metabolism in the mitochondria.

Humans do not store oxygen, because it reacts so rapidly and destructively with other molecules. Therefore, oxygen must be supplied, without interruption, to the mitochondria and to the ≥1,000 oxygen-requiring enzymatic reactions in various cells and tissues (4).

Fig. 1. Ambient oxygen levels, measured by the partial pressure of oxygen (solid line) or as a percent of sea-level values (dashed line), decrease with increasing altitude, a situation called high-altitude or hypobaric hypoxia. The atmosphere contains ≈21% oxygen at all altitudes.

The oxygen level is near zero in human mitochondria at all altitudes (5). This condition is described as “primitive,” because it has changed little for the past 2.5 billion years despite wide swings in the amount of atmospheric oxygen (at times it has been 10,000-fold lower; refs. 6 and 7) and “protective” in the sense that it circumvents potentially damaging reactions of oxygen with other molecules (8).

Fig. 2. The oxygen transport cascade at sea level (solid line) and at the high altitude of 4,540 m (dashed line) illustrates the oxygen levels at the major stages of oxygen delivery and suggests potential points of functional adaptation (data from ref. 60).

Potential and Actual Points of Adaptation to Hypoxia

Energy Production.

Lowlanders traveling to high altitude display homeostatic responses to the acute severe hypoxia. The responses are energetically costly, as indicated by an increase in basal metabolic rate (BMR; the minimum amount of energy needed to maintain life with processes such as regulating body temperature, heart rate, and breathing). BMR is increased by ≈17–27% for the first few weeks upon exposure to high altitude and gradually returns toward sea-level baseline (9). In other words, for acutely exposed lowlanders, the fundamental physiological processes required to sustain life at high altitude require more oxygen despite lower oxygen availability.

In contrast to acutely exposed lowlanders and despite the equally low level of oxygen pressure in the air and lungs, both Andean and Tibetan highlanders display the standard low-altitude range of oxygen delivery from minimal to maximal. Both populations have the normal basal metabolic rate expected for their age, sex, and body weight (1416), implying that their functional adaptations do not entail increased basal oxygen requirements. Furthermore, Andean and Tibetan highlanders have maximal oxygen uptake expected for their level of physical training (12, 13, 17).


One potential point of adaptation in oxygen delivery is ventilation, which, if raised, could move a larger overall volume of air and achieve a higher level of oxygen in the alveolar air (Fig. 2) and diffusion of more oxygen. An immediate increase in ventilation is perhaps the most important response of lowlanders acutely exposed to high altitude, although it is not sustained indefinitely and is not found among members of low-altitude populations born and raised at high altitude, such as Europeans or Chinese (3, 18).

For example, a comparative analysis summarizing the results of 28 samples of Tibetan and Andean high-altitude natives at an average altitude of ≈3,900 m reported an estimated resting ventilation of 15.0 liters/min among the Tibetan samples as compared with 10.5 liters/min among the Andean samples (19).

Fig. 3 illustrates the higher resting ventilation of Tibetans as compared with Andean highlanders evaluated using the same protocol at ≈4,000 m. The mean resting ventilation for Tibetans was >1 SD higher than the mean of the Andean highlanders (20).

Oxygen in the Bloodstream.

The higher ventilation levels among Tibetans that move more oxygen through the lungs, along with the higher HVRs that respond more vigorously to fluctuations in oxygen levels, might be expected to result in more oxygen in the bloodstream. However, the level of oxygen in the arterial blood (Fig. 2) of a sample of Tibetans at ≈3,700 m was lower than that of a sample of Andean high-altitude natives at the same altitude (54 as compared with 57 mmHg; 1 mmHg = 133 Pa) (24, 25). In addition, hemoglobin, the oxygen-carrying molecule in blood, is less saturated with oxygen among Tibetans than among their Andean counterparts (26, 27). Fig. 3 illustrates the lower percent of oxygen saturation of hemoglobin in a sample of Tibetans at ≈4,000 m. The increased breathing of Tibetans does not deliver more oxygen to the hemoglobin in the arteries.

Fig. 3 illustrates the markedly lower hemoglobin concentrations in a sample of Tibetan men and women as compared with their Andean counterparts at ≈4,000 m. [The average hemoglobin concentrations were 15.6 and 19.2 g/dl for Tibetan and Andean men, respectively, and 14.2 and 17.8 g/dl for women (28).] Hemoglobin concentration is influenced by many factors, including erythropoietin, a protein that causes differentiation of the precursors that will become hemoglobin-containing red blood cells. Tibetans have slightly lower erythropoietin concentrations than Andean highlanders at the same altitude (25). When matched for volume of red blood cells, a procedure that would effectively compare the highest Tibetan and the lowest Andean values, Andean highlanders have much higher erythropoietin levels, which implies that some sensor is responding as if the stress were more severe, even though the samples were collected at the same altitude of ≈3,700 m.

Andean highlanders have overcompensated for ambient hypoxia according to this measure, whereas Tibetan highlanders have undercompensated. Indeed, Tibetans are profoundly hypoxic and must be engaging other mechanisms or adapting at different points in the oxygen transport cascade to sustain normal aerobic metabolism.

Fig. 4. The calculated arterial oxygen content of Tibetan men and women is profoundly lower than their Andean counterparts measured at ≈4,000 m (data from ref. 62), whereas the exhaled NO concentration is markedly higher (recalculated from data reported in ref. 34).

Blood Flow and Oxygen Diffusion.

Other potential points of functional adaptation include the rate of flow of oxygen-carrying blood to tissues and the rate of oxygen diffusion from the bloodstream into cells.

Because blood flow is a function of the diameter of blood vessels, dilating factors could, in principle, improve the rate of oxygen delivery. Sea-level populations respond to high-altitude hypoxia by narrowing the blood vessels in their lungs, the first point of contact with the circulation. Known as hypoxic pulmonary vasoconstriction, that reflex evolved at sea level to direct blood away from temporarily poorly oxygenated toward better oxygenated parts of the lung. High-altitude hypoxia causes poor oxygenation of the entire lung and general constriction of blood vessels to the degree that it raises pulmonary blood pressure, often to hypertensive levels (3, 29).

In contrast, most Tibetans do not have hypoxic pulmonary vasoconstriction or pulmonary hypertension. This is indicated by essentially normal pulmonary blood flow, as measured by normal or only minimally elevated pulmonary artery pressure (29, 30).

a mean pulmonary artery pressure of 31 mmHg for the Tibetan 28% lower than the mean of 43 mmHg for the Andean (35 mmHg is often considered the upper end of the normal sea-level range) (30, 31). Andean highlanders are consistently reported to have pulmonary hypertension (29). Thus, pulmonary blood flow is another element of oxygen delivery for which Tibetans differ from Andean highlanders in the direction of greater departure from the ancestral response to acute hypoxia.

A probable reason for the normal pulmonary artery pressure among Tibetans is high levels of the vasodilator nitric oxide (NO) gas synthesized in the lining of the blood vessels. Low-altitude populations acutely exposed to high-altitude down-regulate NO synthesis, a response thought to contribute to hypoxic pulmonary vasoconstriction (32, 33). In contrast, NO is substantially elevated in the lungs of Tibetan as compared with Andean highlanders and lowlanders at sea level (Fig. 4) (34). Among Tibetans, higher exhaled NO is associated with higher blood flow through the lungs (30).

Several other lines of evidence highlight the importance of high blood flow for Tibetans. These include greater increase in blood flow after temporary occlusion (35) and higher blood flow to the brain during exercise (36) as compared with lowlanders.

Generally, Tibetans appear to have relatively high blood flow that may contribute significantly to offsetting their low arterial oxygen content.

A denser capillary network could potentially improve perfusion and oxygen delivery, because each capillary would supply a smaller area of tissue, and oxygen would diffuse a shorter distance. Tibetans (the study sample were Sherpas, an ethnic group that emigrated from Tibet to Nepal ≈500 years ago) who are born and raised at high altitude have higher capillary density in muscles as compared with Andean high-altitude natives, Tibetans born and raised at low altitude, or lowlanders (Fig. 5) (40).

Fig. 5. High-altitude native Tibetans have higher capillary density than their Andean counterparts or populations at low altitude; Tibetan and Andean highlanders both have lower mitochondrial volume than low-altitude populations (data from refs. 40, 44, 63, and64).

The last potential point of adaptation is at the level of the mitochondrion itself. Acutely exposed lowlanders lose mitochondria in leg muscles during the first 3 weeks at altitude. Similarly, both Tibetan (Sherpas) and Andean high-altitude natives have a lower mitochondrial volume in leg muscle tissue than sea-level natives at sea level (Fig. 5) (40).

Among Tibetans, a smaller mitochondrial volume somehow supports a relatively larger oxygen consumption, perhaps by higher metabolic efficiency (12, 43, 44).

Another candidate gene is the transcription factor hypoxia-inducible factor 1 (HIF1) often called the “master regulator” of oxygen homeostasis, because it induces >70 genes that respond to hypoxia (5658). An investigation of polymorphisms in the HIF1A gene of Tibetans (Sherpas) found a dinucleotide repeat in 20 Tibetans that was not found in 30 Japanese lowlander controls (59).