Principles

Oxygen moves from the peripheral capillaries to the mitochondria, and carbon dioxide moves in the opposite direction by the process of diffusion. Discussed in detail in Chapter 8, Fick’s law of diffusion states that the rate of transfer of a gas through a sheet of tissue is proportional to the area of the tissue and the difference in gas partial pressure between the two sides, and inversely proportional to the tissue thickness.

In the discussion of diffusion in the lung, it was noted that the alveolar-capillary barrier of the human lung is extremely thin, being only 0.2–0.3 µm in many places. By contrast, the diffusion distances in peripheral tissues are typically much greater. At rest, the diffusion distance between capillaries in muscle is of the order of 50 µm, whereas during exercise, when muscle oxygen consumption increases and capillary recruitment occurs, the diffusion distance is reduced and convective oxygen delivery is enhanced. As discussed in Chapter 8, carbon dioxide diffuses about 20 times faster than oxygen through tissues because of its much higher solubility, and therefore the elimination of carbon dioxide from muscle poses less of a problem than oxygen delivery.

Early workers believed that the movement of oxygen through tissues was by simple passive diffusion. However, it is now believed that facilitated diffusion of oxygen probably occurs in muscle cells due to the presence of myoglobin (Scholander 1960; Wittenberg 1959). This heme protein has a structure that resembles that of hemoglobin, but its dissociation curve is a hyperbola, as opposed to the s-shape of the hemoglobin-oxygen dissociation curve (Figure 14.1). Another major difference is that myoglobin has a very low P₅₀ of about 3 mmHg and, as a result, has a higher affinity for oxygen than hemoglobin. This is a necessary property if the myoglobin is to be of any use in muscle cells where the tissue PO₂ is very low.

The PO₂ in the immediate vicinity of the mitochondria is very low in some tissues, being of the order of only 1 mmHg. In fact, models of oxygen transfer in tissues often assume that the mitochondrial PO₂ is so low that it can be neglected in the context of the PO₂ of the capillary blood, which is of the order of 30–50 mmHg. In measurements of suspensions of kidney cell mitochondria in vitro, oxygen consumption has been shown to continue at the same rate until the PO₂ of the surrounding fluid falls to about 2 mmHg (Wilson et al. 1977). Measurements of PO₂ at the sites of oxygen utilization based on the spectral characteristics of cytochromes also indicate that the PO₂ is probably less than 1 mmHg (Chance 1957; Chance et al. 1962). In the quadriceps muscle of exercising humans, for example, it was shown by nuclear magnetic resonance spectroscopy that partial desaturation of myoglobin occurred at only 50% of maximal oxygen consumption, implying a PO₂ in the myoglobin of only 2–3 mmHg (Richardson et al. 1995). Given these very low PO₂ values at the sites of oxygen utilization, the much higher PO₂ of capillary blood ensures there is an adequate pressure gradient for diffusion of oxygen to the mitochondria.

Tissue partial pressures

A classical model to analyze the distribution of PO₂ values in tissue was described by August Krogh (Krogh 1919), who considered a hypothetical cylinder of tissue around a straight, thin, tubular capillary into which blood entered with a known PO₂. As oxygen diffuses away from the capillary, it is consumed by the tissue and the PO₂ falls. If simplifying assumptions are made, such as uniform consumption rate of oxygen in every part of the tissue, an equation can be written to describe the PO₂ profile (Krogh 1919; Piiper and Scheid 1986).

Another model is shown in Figure 14.2 (Hill 1928). Figure 14.2a shows a cylinder of tissue supplied with oxygen by capillaries at its periphery: in (1) the balance between oxygen consumption and delivery (determined by the capillary PO₂, the intercapillary distance R_c, and the oxygen consumption rate of the tissue) results in an adequate PO₂ throughout the cylinder; in (2) the intercapillary distance or the oxygen consumption has been increased until the PO₂ falls to zero at one point in the tissue, a phenomenon referred to as a critical situation; in (3) there is an anoxic region where aerobic (that is, oxygen-utilizing) metabolism is impossible. Under anoxic conditions, the tissue energy requirements must be met by obligatory anaerobic glycolysis with the consequent formation of lactic acid.

The situation along the tissue cylinder is shown in Figure 14.2b. It is assumed that the PO₂ in the capillaries at the periphery of the tissue cylinder falls from 100 mmHg to 20 mmHg as shown from left to right. As a result, the PO₂ in the center of the tissue cylinder falls toward the venous end of the capillary. On the basis of this model, it is clear that the most vulnerable tissue is that furthest from the capillary at its downstream end, a region referred to as the “lethal corner.” It is possible that this pattern of focal anoxia is responsible for some tissue damage at high altitude. For example, it may explain how some nerve cells of the brain might become damaged at great altitudes causing the residual impairment of central nervous system function (discussed further in Chapter 12).

Intramuscular partial pressure of oxygen

Figure 14.2 assumes that the blood in adjacent capillaries runs in the same direction, but there is evidence that this is not always the case. Instead, capillaries are organized into a network with various directions of flow and many intercommunications, a concept supported by studies emphasizing the tortuosity of capillaries around skeletal muscle cells (Mathieu-Costello 1987; Potter and Groom 1983). Although in some histological sections the capillaries of skeletal muscle appear at first sight to run chiefly parallel to the muscle fibers, this is an oversimplification. Furthermore, the density of the connections increases considerably when the muscle shortens (Mathieu-Costello 1987). Thus, a more reasonable model of oxygen delivery to muscle is a syncytium of capillaries surrounding a tubular muscle cell.

Studies by Honig et al. (1991) indicate that the PO₂ profiles shown in Figure 14.2 may be misleading in skeletal muscle. After rapidly freezing working muscles of experimental animals the investigators measured the degree of oxygen saturation of the intracellular myoglobin using a spectrometer with a narrow light beam and inferred the intracellular PO₂ from the myoglobin oxygen saturation. These data and theoretical work by the same group suggest that the major resistance to oxygen diffusion from capillary to muscle fiber mitochondria is at the capillary-to-fiber interface, i.e., the thin carrier-free region including plasma, endothelium, and interstitium. This, in turn, necessitates a large driving pressure (PO₂ difference) at that site to deliver oxygen to the muscle fibers. Some of the results of these studies are shown in Figure 14.3, where it can be seen that most of the fall of PO₂ occurs in the immediate vicinity of the peripheral capillary and that, throughout the muscle cell, the PO₂ is remarkably uniform and very low (∼1–3 mmHg). This pattern results in part from the presence of myoglobin, which, as noted above, facilitates the diffusion of oxygen within the muscle fibers.

From the limited available data, it appears that environmental hypoxia exerts only a minor effect on the intramuscular PO₂ (reviewed in Favier et al. 2015). Using various methodological approaches in humans (Jung et al. 1999; Richardson et al. 2006; Richardson et al. 1995), dogs (Hutter et al. 1999), and rats (Johnson et al. 2005), the resting intramuscular PO₂ has been estimated to be around 27 mmHg at sea level. One minute of exposure to very severe hypoxia (F_IO₂ 0.07, equivalent to 8300 m) was shown to decrease the PO₂ only to about 10 mmHg in rat cremaster skeletal muscle (Johnson et al. 2005) (Figure 14.4). At a less extreme simulated altitude (F_IO₂ 0.10, equivalent to 5800 m), the PO₂ was found close to 23 mmHg in skeletal muscles (Richardson et al. 2006).

These intramuscular PO₂ values contrast drastically with the values seen during exercise where the skeletal muscle PO₂ is low and remains constant with increasing work. To understand this, we can begin by examining data on exercise in normoxia from Richardson (2000) who studied muscle oxygenation during single knee extensor exercise. Using magnetic resonance spectroscopy of myoglobin as a measure of tissue oxygenation and exploiting the fact that the P₅₀ of myoglobin is about 3.2 mmHg, they found that although the calculated PO₂ was relatively high up to a maximal work rate of 60% of V˙O2max, above that the intracellular PO₂ fell to a relatively uniform and constant value of about 3.8 mmHg in all subjects. When the hypoxic stress of exercise is then combined with environmental hypoxia, the intramuscular PO₂ decreases a bit further to as low as 2 mmHg (Masschelein et al. 2014; Richardson et al. 1995), just above the minimal PO₂ at which mitochondrial metabolism is inhibited (∼1.25 mmHg) (Richmond et al. 1997). As argued by Favier et al. (2015), these results suggest that the hypoxic stress of physical exercise is drastically more severe than that resulting from ambient hypoxia alone and is further exacerbated when performed in a hypoxic environment.

Capillary geometry

Some early studies suggested that the tortuosity of existing muscle capillaries was increased in response to hypoxia, effectively contributing to improve capillary surface area and enhance gas diffusion (Appell 1978). However, this result has not been confirmed by other investigators (Mathieu-Costello 1989; Poole and Mathieu-Costello 1990), who showed that muscle capillary tortuosity does not increase with chronic exposure to hypoxia when sarcomere length is taken into account. These later investigators argued that the early results may be explained by failure to control the state of contraction of the muscle, as it is known that the capillary length and tortuosity change during muscle shortening (Mathieu-Costello 1987). Similar to hypoxia, endurance training does not seem to have any major impact on capillary tortuosity (Poole et al. 1989).

Capillary recruitment

In his pioneering work, Krogh (1919) suggested that capillary recruitment could increase capillary blood flow and oxygen delivery to active skeletal myofibers during exercise. His model was essentially based on the existence of precapillary sphincters at the proximal end of each capillary that could regulate the blood flow accordingly to the metabolic needs of the myofibers. During exercise for example, more capillaries would “open” to improve oxygen delivery to the muscle tissue. Although appealing at first glance, this model has not been consistently supported by experimental models (Angleys and Ostergaard 2020; Poole et al. 2011), and the anatomical and functional existence of precapillary sphincters that could fully restrict blood flow has not been demonstrated (Golub and Pittman 2013).

Another model to explain these blood flow adjustments is the concept of longitudinal capillary recruitment (Ellis et al. 1994), which relies on the unique architecture of the skeletal muscle capillary network (i.e., a branching pattern and orthogonal anastomoses). In this model, blood can be redirected from capillaries with high flow (mainly longitudinal capillaries) to capillaries exhibiting lower blood flow (capillary bifurcations and orthogonal anastomoses), thereby increasing the homogeneity of red blood cell transit times within the capillary network and improving oxygen supply to active myofibers. This redistribution of blood flow between capillary segments is thought to enhance myocyte oxygen extraction by increasing the surface area and time available for blood-myocyte oxygen exchange (Ellis et al. 1994; Poole et al. 2011). Regardless of the mechanism, evidence supports the concept that capillary blood flow increases with exercise. Bourdillon et al. (2009), for example, estimated capillary recruitment in exercising human skeletal muscle in both sedentary and endurance-trained subjects at simulated altitudes of 1000 m, 2500 m, and 4500 m, and demonstrated that capillary recruitment significantly increased at 4500 m, with greater recruitment seen in trained versus sedentary subjects (Bourdillon et al. 2009).

Capillary density

Increasing tissue capillarization contributes to improved oxygen delivery to mitochondria. Early studies on the effects of high altitude exposure on capillary density suggested an increased level of capillarization in the brain, retina, skeletal muscle, and liver of experimental animals exposed to low barometric pressures over several weeks (Cassin et al. 1971; Mercker and Schneider 1949; Opitz 1951; Valdivia 1958).

Two useful histological parameters can be determined from transverse cross-sections of skeletal muscle: the capillary density (CD, capillaries mm⁻²) and the capillary-to-fiber ratio (C/F). Whereas the CD provides information about the abundance of capillaries in a given tissue, C/F ratio changes reflect the true formation or loss of capillaries. Increasing muscle CD can indeed result from the formation of new capillaries from pre-existing ones. Referred to as angiogenesis, this has been well described in response to prolonged endurance training in human and rodent muscles. An increase in CD can also result from tissue remodeling, in which there is a decrease in myofiber size in the absence of angiogenesis. The former is associated with an increase in the C/F ratio, while the latter does not change this parameter.

Early studies performed in simulated or terrestrial high altitude have shown that hypoxia increases muscle capillarization, although the specific role of angiogenesis in these observed changes has been the subject of debate (Banchero 1987; Breen et al. 2008). It is now believed that the increase in CD observed in human and animal skeletal muscles following prolonged hypoxic exposure results from a reduction in the size of skeletal myofibers rather than angiogenesis (i.e., no change in the C/F ratio). This has been demonstrated by studies in guinea pigs native to the Andes at sea level, 1610 m, 3900 m, and at a simulated altitude of 5100 m (Banchero 1982) (Figure 14.5). The same pattern has been described in studies of acclimatized humans where muscle samples were obtained by biopsy. For example, Cerretelli et al. obtained muscle biopsies on climbers immediately after several weeks attempting to climb Lhotse Shar (8398 m) in Nepal and showed that, although the capillary density was somewhat increased, the change could be wholly accounted for by a reduction of muscle fiber size (Boutellier et al. 1983; Cerretelli et al. 1984). Following 75 days of exposure to 5250 m or higher, Mizuno et al. (2008) also reported reduced mean muscle fiber area and increased CD (from 320 to 405 capillaries mm⁻²). It is important to recognize, however, that some studies present different results. For example, during Operation Everest II, where six volunteers were gradually decompressed to the simulated altitude of Mount Everest over a period of 40 days, needle biopsies from the vastus lateralis revealed a statistically significant 25% decrease in the mean fiber cross-sectional area (FCSA) of type I fibers, and a nonstatistically significant 26% decrease in FCSA of type II fibers. The C/F ratio was unchanged, while there was only a nonsignificant trend toward an increase in the CD (Green et al. 1989; MacDougall et al. 1991). More recently, Lundby et al. (2004) showed that there was no change in CD in skeletal muscles of lowlanders after acclimatization to an altitude of 4100 m.

In animal models, Sillau and Banchero (1977) measured the FCSA, C/F ratio, and CD in different skeletal muscles from rats exposed for six to seven weeks to ambient hypoxia (F_IO₂ 0.125) and their body weight-matched controls maintained in normoxic conditions and found that an increase in CD only occurred at high altitude if skeletal myofibers atrophied (Sillau and Banchero 1977). This suggests that while skeletal muscle atrophy during prolonged exposure to high altitude is often presented as a detrimental effect of high altitude, from a vascular standpoint it could represent an adaptive response to increase the CD without the cost of true capillary synthesis.

Skeletal muscle angiogenesis

As discussed above, the general consensus is that an increase in CD occurs in human or animal skeletal muscle due to atrophy of existing myofibers during prolonged hypoxic exposure in the absence of angiogenesis. Early studies suggesting true capillary formation at altitude might have been the result of methodological issues involving sample collection and handling, study design (e.g., differences in body weight between hypoxic and normoxic groups), and the histologic technique (e.g., enzymatic versus nonenzymatic marker).

Nevertheless, true capillary formation, as evidenced by a significant increase in the C/F ratio, has been observed in a few animal species living at high altitude. For example, an increased C/F ratio has been reported in flight and leg muscles from finches living at high altitude (Hepple et al. 1998). Similarly, deer mice raised from highland populations at 4300 m show 20–30% greater C/F ratio compared to lowland mice (Lui et al. 2015). This muscle phenotype was not apparent in lowland mice even after six to eight weeks of acclimatization to 4300 m. These investigators argued that whether increased capillary numbers (and mitochondrial density) occur at high altitude depends on the level of metabolic stress on the muscle that can also be distinct from initial acclimatization (Lui et al. 2015). Similar considerations occur with the issue of training at altitude, which are discussed later in this chapter.

More recently, Deveci et al. (2001, 2002) examined sedentary rats exposed to hypoxia (F_IO₂ 0.12, equivalent to 4400 m) for three and six weeks, and analyzed the CD, C/F, and FCSA in different muscles including the diaphragm, the soleus (a postural and mainly slow-twitch [type I fibers] oxidative muscle), the tibialis anterior, and the extensor digitorum longus (mainly fast-twitch [type IIa/IIb] and predominantly glycolytic muscles). At 3 weeks, they found a significant increase in both CD and C/F in the diaphragm and soleus muscle without any myofiber atrophy. More interestingly, regional tissue differences were apparent, as an increase in the C/F ratio was found in muscle regions predominantly composed of larger fibers (richer in type IIa/IIb fibers and less type I fibers) (Figure 14.6). For example, in the tibialis anterior muscle, where no increase in C/F was detected at the whole muscle level, angiogenesis occurred specifically in the cortex region predominantly composed of large and glycolytic fibers rather than in the core region, which has a higher proportion of smaller and oxidative type I fibers. Since many previous studies have analyzed muscle capillarization over a longer duration of hypoxia/altitude exposure, Deveci et al. (2002) repeated the experiment at six weeks and found a significant increase in the C/F ratio in all muscles, thereby suggesting a progressive angiogenic response with prolonged hypoxic exposure.

Regardless of whether the observed changes are due to tissue remodeling from myofiber atrophy or angiogenesis, increased capillarization can be seen in a resting muscle at high altitude in response to ambient hypoxia. It is important to keep in mind, however, that mountaineering and trekking at high altitude combine several proangiogenic stimuli that can affect muscle and its capillarization (Figure 14.7). For example, high volumes of exercise, such as hiking, running, and climbing, are powerful proangiogenic stimuli in skeletal muscle. As discussed above, exercise alone induces severe hypoxic stress at the tissue level, which can be exacerbated by ambient hypoxia. In a study from Olfert et al. (2001), for example, no change was observed in the C/F ratio of gastrocnemius muscles from rats trained for eight weeks in normoxia, whereas a significant increase was reported when training in hypoxia (F_IO₂ 0.12). All animals were trained at the same absolute intensity, which could also explain the impact of hypoxic training (Olfert et al. 2001). Mechanical stress on muscle, as well as increases in blood flow and shear stress, can also serve as proangiogenic stimuli, as can exposure to cold (Deveci and Egginton 2002; Deveci et al. 2002).

Vascular endothelial growth factor and hypoxia

Vascular endothelial growth factor-A (VEGF-A) has been extensively studied for its role in promoting physiological and pathological angiogenesis (Ferrara 2001; Folkman and Shing 1992). Given its role in stimulating endothelial cell proliferation, migration, and increased vascular permeability (Semenza 2001), as well evidence that hypoxia is a well-established stimulus for HIF-1-mediated VEGF-A expression, there has been considerable interest in the role of VEGF-A in the response to high altitude.

Exercise and the subsequent hypoxic stress at the tissue/cellular level are strong stimuli for VEGF-A mRNA expression in human (Gustafsson et al. 1999; Hoppeler 1999) and rodent (Breen et al. 1996) skeletal muscles. In line with work from Deveci at al. (Deveci and Egginton 2002; Deveci et al. 2001, 2002) reporting an increased C/F ratio in muscle regions predominantly composed of large fibers, Birot et al. (2003) quantified VEGF-A mRNA expression in isolated myofibers from the rat plantaris at rest and in response to exercise and observed similar basal expression level among I, IIa, IIx, and IIb fiber types. In response to exercise, however, VEGF-A mRNA expression was specifically increased in type IIb fibers, predominantly the largest and less vascularized fibers (Birot et al. 2003). At the protein level, Aiken at al. (2016) have reported a very similar pattern of expression between human and rodent skeletal muscles in response to a single bout of intense running exercise, including a strong increase in VEGF-A protein levels immediately postexercise and attenuation over a few hours of recovery (Aiken et al. 2016). Endurance training attenuates the responsiveness of VEGF-A mRNA to a single bout of exercise (Gavin and Wagner 2001; Richardson 2000), but whether chronic environmental hypoxia has a similar effect remains unclear. Indeed, whereas acute hypoxia can increase VEGF mRNA expression in animal and human skeletal muscles, some studies suggest that chronic hypoxia might reduce the levels to those seen in acute hypoxia or even lower (Lundby et al. 2004; Olfert et al. 2001). Some discrepancies between these studies could result from different methodological approaches and use of different time-points for analysis.

The frequently cited mechanism for increased VEGF-A expression under hypoxia is the increased transcriptional activity of hypoxia-inducible factor (HIF), resulting in increased levels of VEGF-A mRNA (Hoppeler et al. 2003). However, this simplified interpretation does not reflect the complexity of the proangiogenic activity of VEGF-A in response to hypoxia. As illustrated in Figure 14.8, hypoxia can indeed play a role on VEGF-A mRNA expression, but also on mRNA stabilization, translation into native protein, the subsequent maturation and secretion of the VEGF-A protein, and the expression of receptors required for VEGF-A proangiogenic action. For example, hypoxia can stimulate the expression of the human antigen R protein (HuR) that in turn stabilizes VEGF-A mRNA and facilitates their translation into protein (Amadio et al. 2008; Morfoisse et al. 2015; Osera et al. 2015). This mechanism has been shown in rat ischemic gastrocnemius muscle (Tang et al. 2002).

At the translation level, whereas the global and classical cap-dependent protein translation process can be silenced under hypoxia, an alternative mechanism exists for mRNA-possessing internal ribosome entry sites (IRESs) that permit the initiation of translation; that is, IRES sites have been described for VEGF-A (Boutellier et al. 1983; Huez et al. 1998). Following translation, the oxygen regulated protein-150 (ORP-150), an endoplasmic reticulum chaperone, assists VEGF-A protein post-translational modification and secretion (Ozawa et al. 2001a; Ozawa et al. 2001b). Birot at al. (2003) showed concomitant increased expression of ORP-150 and VEGF-A mRNA in rat plantaris muscles following an acute bout of running exercise. Finally, the secreted VEGF-A protein exerts its proangiogenic action by binding to receptors. Expression of VEGF-A receptors 1 (Flt-1) and 2 (Flk-1) is influenced by hypoxia or ischemia, including a HIF-1-dependent regulation for Flt-1 (Gerber et al. 1997). Interestingly, Birot et al. (2004) have measured both VEGF-A mRNA and protein time-course expression for in the right cardiac ventricle of rats exposed for up to 25 days to hypobaric hypoxia (5500 m). In this tissue, which hypertrophies with concomitant angiogenesis during chronic hypoxia, mRNA changes were not mirrored at the protein level (Birot et al. 2004). Therefore, VEGF-A mRNA quantification might represent an easy and practical measurement of a HIF-1 target; however, it certainly has some limitation in the interpretation of the full and complex proangiogenic activity of VEGF-A. It is also important to keep in mind that the angiogenic process is highly complex and tightly orchestrated by a plethora of pro- and antiangiogenic factors (Olfert and Birot 2011). The contribution of antiangiogenic factors in regulating physiological muscle vascular adaptation remains understudied, particularly in the context of environmental hypoxia.

Muscle mass

As indicated above, one way to increase capillary density and thus reduce diffusion distance within skeletal muscle is to reduce muscle fiber size. A decrease in the whole muscle mass and in myofiber size during prolonged exposure to hypoxia has been well described (Boutellier et al. 1983; Cerretelli et al. 1984; Hoppeler et al. 1990b; MacDougall et al. 1991; Mizuno et al. 2008). This is illustrated in Figure 14.9, which shows a significant reduction in muscle volume as measured by computed tomography in the thigh and upper arm regions of the subjects of Operation Everest II (MacDougall et al. 1991).

Some studies, however, have not observed such a decrease in muscle cross-sectional area with exposure to hypoxia (D’Hulst et al. 2016; Green et al. 2000; Jacobs et al. 2016; Levett et al. 2012; Lundby et al. 2004). D’Hulst and Deldicque (2017) proposed that this discrepancy could be explained by the interaction between the severity and duration of hypoxic exposure between studies and ultimately concluded there is a critical altitude threshold of about 5000 m, above which skeletal muscle wasting occurs (Figure 14.10). While this general assumption of a dose-response relationship is likely correct, it has been extensively debated (D’Hulst & Deldicque 2017; Millet et al. 2017), and a number of important issues must be taken into account, including (1) the most appropriate method for determining the dose of hypoxia (i.e., altitude hours versus time spent at a certain level of oxygen saturation [Millet et al. 2016]); and (2) the difficulty distinguishing between the severity and duration of hypoxia using data from previous studies. Indeed, studies of severe hypoxia typically involve longer durations due to the need for acclimatization and the overarching mountaineering goals of the participants (Millet et al. 2017).

To what extent high altitude might differentially affect oxidative versus predominantly glycolytic muscles is still unclear. On one hand, by being more oxygen-dependent, oxidative muscles might be more hypoxia-sensitive. On the other hand, glycolytic muscles possess a greater proportion of fast twitch glycolytic fibers (type II), which are larger and less vascularized than oxidative fibers and therefore potentially more sensitive to hypoxic stress. For example, de Theije et al. (2015) showed increased atrophy of the glycolytic extensor digitorum longus muscle compared to the oxidative soleus in mice exposed to three weeks of hypoxia, suggesting that type II fibers would be greatly affected by hypoxia. This observation is in line with the previous section about muscle capillarization and angiogenic activity, which considered (1) the possibility of muscle angiogenesis in response to environmental hypoxia in muscle regions predominantly composed of the large type II fibers (Deveci et al. 2001, 2002) and (2) the increased expression of VEGF-A mRNA in response to exercise, specifically in type IIb fibers (Birot et al. 2003). These studies, however, were conducted in rodents rather than humans, and in hypoxic chambers rather than at terrestrial high altitude. However, during Operation Everest II, MacDougall (1991) reported similar levels of atrophy between thighs and arms (respectively –13% and –15% muscle area) as well as between type I and type II fibers (respectively –25% and –26% mean fiber cross-sectional area) (MacDougall et al. 1991). Reviewing animal and human studies, Favier et al. (2015, 2010) suggested that both oxidative and glycolytic muscles and fibers can atrophy with exposure to extreme altitude and that the impact of muscle typology on hypoxia sensitivity still remains unclear.

Mechanism of muscle atrophy

The mechanism for muscle atrophy at high altitude is also not well understood. Muscle volume is essentially determined by muscle cells and myofibrillar proteins. Therefore, the regulation of muscle mass very likely results from a balance between muscle protein synthesis and breakdown. In response to acute exposure to hypoxia (∼48 hours), no difference in resting urinary nitrogen (whole-body) or 3-methylhistidine (myofibrillar) markers of protein degradation have been observed (Imoberdorf et al. 2006). Moreover, during longer periods of acclimatization (seven to nine days at 4559 m), sarcoplasmic protein synthesis remains unchanged, and the rate of myofibrillar contractile protein synthesis actually increases (Holm et al. 2010). Thus, one assumption from these data to explain muscle atrophy during prolonged exposure to hypoxia is that myofibrillar protein breakdown must have been elevated to an even greater extent than the increase in myofibrillar contractile protein synthesis. In support of this argument, whole body protein breakdown tended to be elevated (Holm et al. 2010). Muscle protein breakdown is mainly triggered by the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP). Some studies have reported increased expression of UPS and ALP molecular factors. For example, an increase in mRNA expression for the E3-ubiquitin ligases MAFbx and MurF1 has been described in response to early exposure to hypoxia (de Theije et al. 2015), but not in response to prolonged hypoxia exposure (Chaillou et al. 2014). The timing of these signaling pathways therefore seems to be important. Both UPS and ALP processes are controlled by the Akt/mTOR signaling, and mTOR expression was also shown to be reduced under hypoxic conditions (Vigano et al. 2008). Interestingly, Chaillou et al. (2014), who reported greater atrophy in the glycolytic extensor digitorum longus muscle of mice exposed to hypoxia versus their soleus muscles, also observed in the same samples a hypoxia-based stimulation of several UPS and ALP markers (Chaillou et al. 2014).

The Akt/mTOR pathway also affects muscle mass by regulating protein synthesis. A down regulation of this pathway has been observed by Favier et al. (2010) in rats exposed to a simulated altitude of 6300 m in a hypobaric chamber. This was associated with an increased expression of REDD1, a direct target of HIF-1 and negative regulator of muscle mass in response to hypoxia. REDD1 could reduce protein synthesis by inhibiting the mTOR signaling pathway. Finally, myostatin is a muscle-secreted molecule that could also contribute to muscle atrophy by inhibiting the mTOR pathway. It has been shown that myostatin expression was increased under hypoxic conditions (Chaillou et al. 2014).

It has been hypothesized that hypophagia at altitude could be a contributing factor in muscle atrophy. However, it is important to note that hypoxia per se can regulate molecular factors and pathways involved in muscle protein synthesis or breakdown (de Theije et al. 2015; Favier et al. 2010). As outlined in Chapter 15, it has been reported that consuming a controlled higher protein hypocaloric diet relative to a controlled standard-protein hypocaloric diet did not protect fat-free mass during weight loss over 22 days at 4300 m (Berryman et al. 2017). The likely mechanism to explain the lack of influence of the high protein diet in preventing weight loss was due to an enhanced protein oxidation. Unfortunately, there seems to be little data on the rate of protein synthesis and metabolism and breakdown at higher altitudes (>5000 m) and over many months where the loss of muscle mass is most prevalent. Finally, if protein synthesis rates remain constant at high altitude, an alternative or complementary mechanism may be a partial resistance of the skeletal muscle to normal anabolic stimuli such as exercise. Indeed, decreased myofibrillar protein synthesis (Etheridge et al. 2011) and muscle cross-sectional area (Kayser et al. 1996) has been observed when resistance training is conducted under hypoxic conditions.

Fiber type distribution

With regard to the muscle contractile phenotype, it does not seem that prolonged hypoxia significantly affects the fiber type distribution in human skeletal muscles (Chaillou 2018; Hoppeler et al. 1990a; Hoppeler et al. 1990b; Kayser et al. 1991). No significant difference was observed after analyzing vastus lateralis muscle biopsies following Operation Everest II (40 days in a hypoxic chamber simulating the Mount Everest altitude, 8848 m) (Green et al. 1989; MacDougall et al. 1991), results that were supported by field experiments at different altitudes (Juel et al. 2003). Conversely, Doria et al. (2011) have observed an increase in slow twitch type I fiber and a decrease in fast twitch type IIa fibers in the vastus lateralis muscle following a Himalayan expedition (43 days including 23 days above 5000 m) (Doria et al. 2011). Again, it is very difficult in this type of field study to distinguish the effect of ambient hypoxia per se from other confounding factors (cold, high volume of physical activity, nutritional intake). Finally, skeletal muscles from high altitude natives (Himalayas, South American Andes, or Ethiopian Highlands) seem to have smaller fibers but a fiber type distribution close to that of lowlanders despite a tendency for a higher proportion in type I fibers (Chaillou 2018; Kayser et al. 1991; Kayser et al. 1996).

Mitochondria volume density

The mitochondria are the primary sites of oxygen utilization by the body and thus constitute the final link of the oxygen cascade. In general, mitochondrial volume density (number of mitochondria per unit volume of tissue) in skeletal muscle is related to maximal oxygen uptake (Lundby and Jacobs 2016). For example, mitochondrial volume density is greater in highly aerobic animals, such as the horse, compared with less active animals, such as the cow (Taylor et al. 1987), and increases with exercise training (Holloszy and Coyle 1984; Meinild Lundby et al. 2018). It has been proposed that mitochondria half-life is about two weeks (Menzies and Gold 1971) and the turnover in tissues depends on mitochondria biogenesis and degradation (autophagy/mitophagy) (Horscroft et al. 2017). The impact of high altitude on mitochondrial volume density changes in animals and humans is considered below.

Results from animal studies remain equivocal. In an investigation of the mitochondrial density of the myocardium of rabbits and guinea pigs from Cerro de Pasco, Peru (4330 m), it was found that the values were the same as those at sea level (Kearney 1973). However, it was shown that the number of mitochondria in samples of myocardium was 40% greater in cattle born and raised at 4250 m compared with cattle raised at sea level (Ou and Tenney 1970). The size of individual mitochondria was found to be the same and it was argued that the increase in mitochondrial number was advantageous because it reduced the diffusion distance of the intracellular oxygen. Gamboa and Andrade (2010) compared mitochondrial volume density in the diaphragm and gastrocnemius muscles of mice exposed to hypoxia (F_IO₂ 0.10) for four weeks and found a decrease in the diaphragm only (about 20% reduction). Interestingly, they also reported in this tissue decreased mRNA and protein levels of PGC1-α and PPAR-γ, biomarkers of mitochondria biogenesis, whereas mRNA and protein levels of BNIP3, used as an autophagy biomarker, were increased.

From human studies, it is now known that the mitochondrial volume density in human skeletal muscle decreases with exposure to very high altitude (Hoppeler et al. 1990a; Levett et al. 2012). In a study on muscle biopsies of climbers returning from two Swiss expeditions to the Himalayas, mitochondrial volume density decreased by 20%. Given the associated 10% decrease in muscle mass, this corresponded to a nearly 30% decrease in absolute mitochondrial volume (Hoppeler 1990). A feature of the electron micrographs of muscle biopsies was the presence of poorly defined material known as lipofuscin, a substance thought to be the consequence of lipid peroxidation related to loss of mitochondria (Howald and Hoppeler 2003). Conversely, there was no decrease in mitochondrial volume density in biopsies of vastus lateralis muscles from subjects of Operation Everest II (MacDougall et al. 1991). Jacobs et al. (2016) even observed an increase in mitochondrial volume density in muscle biopsies obtained after 28 days at 3454 m when subjects attempted to maintain their sea-level physical activity (a technically challenging task). In contrast, Levett et al. (2012) reported that ascent to Everest base camp at 5300 m over 19 days was not associated with loss of mitochondria; after 66 days at altitude including ascent above 6400 m, mitochondrial density fell by 21% including a 73% decrease in subsarcolemmal mitochondria. In addition, levels of transcriptional coactivator PGC-1α fell by 35%, suggesting downregulation of mitochondrial biogenesis. Sustained hypoxia also decreased expression of electron transport chain complexes I and IV and UCP3 levels. This is consistent with the fact that during subacute hypoxia, mitochondria are protected from oxidative stress. However, following sustained exposure, mitochondrial biogenesis is impaired and uncoupling is downregulated, perhaps to improve the efficiency of ATP production (Levett et al. 2012). This is relevant to the discussion of high altitude acclimatization seen at moderate altitudes and deterioration seen at extremely high altitudes (Chapter 7).

In line with the idea that mitochondria volume density would be dependent on the duration of exposure and altitude reached, Horscroft and Murray (2014) have reviewed related changes in expression of biomarkers of mitochondria volume density in human vastus lateralis muscle. On the basis of 23 research studies, a decrease was reported in 31% at high altitude, 43% at very high altitude, and 57% at extreme altitude. For example, 19 days at 5300 m or 40 days at an equivalent of 8000 m did not reveal any changes, whereas 55 days at 5000 m or 66 days above 6600 m showed decreased biomarkers of mitochondria volume density (Horscroft and Murray 2014). This could also explain some discrepancy between the studies cited previously. For example, Jacobs et al. (2016) found an increase in the mitochondrial volume density of about 7–8% after 28 days of exposure at 3454 m, an increase attributed to intermyofibrillar mitochondria (+11%). However, both exposure duration and altitude were, in fact, much lower compared to other studies reporting opposite changes, i.e., a decrease in mitochondria volume density preferentially affecting the subsarcolemmal mitochondria population (Hoppeler 1990; Levett et al. 2012; Kayser et al. 1996). Interestingly, although statistically nonsignificant, the Jacobs at al. (2016) study showed a trend toward decreased subsarcolemmal mitochondria volume density (–12%) as well. Further information on the response of skeletal muscle mitochondria to hypoxia is available elsewhere (Hoppeler et al. 2003; Horscroft and Murray 2014; Murray and Horscroft 2016) and is summarized in Figure 14.11.

Skeletal muscle changes in long-term residents

Earlier in this chapter, we discussed the idea that muscle atrophy, often seen as a sign of deterioration, might in fact be part of the beginning of an adaptive process since native populations at high altitude also present smaller myofibers. It is therefore very interesting to note that Tibetans and Quechuas also have reduced mitochondrial volume density in skeletal muscle, including second-generation of lowland-dwelling Tibetans, suggesting a long-term adaptive process rather than deterioration (Hoppeler et al. 2003; Horscroft et al. 2017; Kayser et al. 1991; Kayser et al. 1996). These findings are summarized above and illustrated in Figure 14.11.

Myoglobin concentration

Myoglobin is a monomeric globin protein that facilitates diffusion of oxygen into and storage within skeletal and heart muscle, where it is normally expressed at high levels in most vertebrates (Wittenberg and Wittenberg 2003). Because it has a higher affinity for oxygen than hemoglobin, myoglobin efficiently extracts oxygen from the blood by creating a steep pressure gradient at the sarcoplasmic membrane that increases the rate of diffusion, while also delivering it to the mitochondria through its role as an intracellular oxygen carrier (Wittenberg and Wittenberg 2003). When expressed at high concentrations, myoglobin also functions as an oxygen store, making oxygen available for respiration during muscle contractions when gas exchange with the peripheral capillaries is impaired (Fago 2017).

Early studies by Hurtado et al. (1937) showed increased concentrations of myoglobin in the diaphragm, adductor muscles of the leg, pectoral muscles of the chest, and the myocardium of dogs born and raised in Morococha, Peru (4550 m), when compared to dogs at sea level in Lima. Other studies that have shown an increase in myoglobin as a result of acclimatization to hypoxia include those of hamster heart muscle (Clark 1952), rat heart muscle and diaphragm (Vaughan and Pace 1956), and various guinea pig tissues (Tappan and Reynafarje 1957). In many other species, elevations in myoglobin expression have been reported at high altitude (reviewed in [Fago 2017]), albeit with marked variation between different organs

In contrast to the animal studies, results from human analyses remain equivocal. Reynafarje (1962) measured myoglobin concentrations in the sartorius muscle of healthy humans native to Cerro de Pasco (4400 m) and in other Peruvians native to sea level and found higher concentrations of myoglobin in the high altitude natives tissue (7.03 mg g⁻¹) than in the sea-level controls (6.07 mg g⁻¹). These results occurred in the absence of changes in lean body mass and body water content and, as a result, were likely not an artifact due to dehydration. Likewise, in humans who exercise in normobaric hypoxia, it has been reported that circulating erythropoietin (Guadalupe-Grau et al. 2015) and HIF-1α stabilization (Vogt et al. 2001) enhance myoglobin expression in exercising skeletal muscle, and possibly in other species as well (reviewed in [Fago 2017]). In contrast to these reports of elevated myoglobin concentration, others have shown that myoglobin expression in human is unaltered after return from the summit of Mount Everest (Levett et al. 2012) or decreased following a seven-to-nine-day stay at 4559 m (Robach et al. 2007). Therefore, apart from one earlier study at 4440 m (Reynafarje 1962) and one that involved exercise training in normobaric hypoxia (Vogt et al. 2001), there is little consistent evidence in humans for elevations in myoglobin expression at high altitude.

Exercise training and detraining

All of the changes in peripheral tissue outlined in this chapter are intimately influenced by the extent of exercise training or detraining while at high altitude. For example, exercise training at sea level increases muscle capillarity including both the capillary/fiber ratio and number of capillaries per square millimeter within several weeks (Andersen and Henriksson 1977; Brodal et al. 1977; Ingjer and Brodal 1978). Furthermore, the increase in number of capillaries is found in all fiber types that are recruited during training (Andersen and Henriksson 1977; Nygaard and Nielsen 1978), and the increased capillary supply is proportional to the increased maximum oxygen uptake (Andersen and Henriksson 1977). Endurance exercise training also increases mitochondrial volume (Meinild Lundby et al. 2018) and respiratory capacity through enzymes involved in β-oxidation, the Krebs cycle, and the electron transport chain (Holloszy and Coyle 1984; Lundby and Jacobs 2016). Myoglobin concentrations may also be affected by training status. In experimental animals, myoglobin content increases with exercise (Lawrie 1953; Pattengale and Holloszy 1967), with animals, such as seals who have large amounts of myoglobin, exhibiting high oxygen uptake in conditions of reduced oxygen availability (Castellini and Somero 1981). However, a study comparing trained and untrained human subjects (Jansson et al. 1982) and another study of short-term training in humans (Svedenhag et al. 1983) both failed to show any effect of training on muscle myoglobin concentration. Interestingly, when exercise training was performed under hypoxic conditions, muscle myoglobin concentrations were increased (Terrados et al. 1990).

Detraining or inactivity, a common phenomenon during high altitude expeditions, can also lead to profound deconditioning within the peripheral tissues. For example, bed rest typically results in a loss of skeletal muscle mass, capillary density, skeletal muscle oxidative function, mitochondrial biogenesis, and remodeling (Buso et al. 2019; Standley et al. 2020; Trevino et al. 2019). Interestingly, the addition of hypoxia seems to aggravate inactivity-induced muscle wasting, but has little effect on mitochondrial and oxidative function (Debevec et al. 2018; Salvadego et al. 2016; Salvadego et al. 2018). While these data highlight that exercise or lack thereof may augment or counteract adaptations to hypoxia, changes in physical activity are unlikely to be the whole story as evidenced by the experience of the investigators on the 1960–61 Himalayan Scientific and Mountaineering Expedition. During several months at 5800 m, the level of physical activity was well maintained with opportunities for daily skiing, yet the expedition members suffered a relentless and progressive loss of weight that averaged 0.5–1.5 kg per week (Pugh 1964) (discussed further in Chapter 15). Moreover, one study directly compared active versus passive ascent to 5250 m and found that the loss in muscle fiber area was independent of the method of ascent (Mizuno et al. 2008). Nevertheless, the influence of well-controlled and comparable (e.g., intensity and duration) exercise training studies at both sea level and high altitude on peripheral tissue adaptations have not been conducted. It must be stressed, however, that it is difficult to maintain the same level of physical activity during exposure to chronic hypoxia and difficult to match sea level residents with residents at altitude with respect to physical activity Future research that accounts for the effects of exercise and inactivity may yield further insights into the peripheral tissue adaptations to hypoxia. Table 14.1 compares some of the tissue changes caused by training with those resulting from exposure to high altitude.

Stay updated, free articles. Join our Telegram channel

Join