am j physiol heart circ physiol 296: h363-h374, 2009

Region-specific adaptations in determinants of rat skeletal muscle oxygenationto chronic hypoxia

R. C. I. Wust,1,2 R. T. Jaspers,2 A. F. van Heijst,3 M. T. E. Hopman,4 L. J. C. Hoofd,4

W. J. van der Laarse,5 and H. Degens1

1Institute for Biomedical Research into Human Movement and Health, Manchester Metropolitan University, Manchester;2Research Institute MOVE, Faculty of Human Movement Sciences, Vrije Universiteit Amsterdam, Amsterdam; 3Department ofPediatrics and 4Department of Physiology, Radboud University Nijmegen Medical Centre, Nijmegen; and 5Department ofPhysiology, Institute for Cardiovascular Research, VU University Medical Centre, Amsterdam, The Netherlands

Submitted 19 March 2009; accepted in final form 28 April 2009

Wust RC, Jaspers RT, van Heijst AF, Hopman MT, HoofdLJ, van der Laarse WJ, Degens H. Region-specific adaptations indeterminants of rat skeletal muscle oxygenation to chronic hyp-oxia. Am J Physiol Heart Circ Physiol 297: H364 –H374, 2009.First published May 8, 2009; doi:10.1152/ajpheart.00272.2009.—Chronic exposure to hypoxia is associated with muscle atrophy (i.e.,a reduction in muscle fiber cross-sectional area), reduced oxidativecapacity, and capillary growth. It is controversial whether thesechanges are muscle and fiber type specific. We hypothesized thatdifferent regions of the same muscle would also respond differently tochronic hypoxia. To investigate this, we compared the deep (oxida-tive) and superficial (glycolytic) region of the plantaris muscle ofeight male rats exposed to 4 wk of hypobaric hypoxia (410 mmHg,PO2: 11.5 kPa) with those of nine normoxic rats. Hematocrit washigher in chronic hypoxic than control rats (59% vs. 50%, P ! 0.001).Using histochemistry, we observed 10% fiber atrophy (P ! 0.05) inboth regions of the muscle but no shift in the fiber type compositionand myoglobin concentration of the fibers. In hypoxic rats, succinatedehydrogenase (SDH) activity was elevated in fibers of each type inthe superficial region (25%, P ! 0.05) but not in the deep region,whereas in the deep region but not the superficial region the numberof capillaries supplying a fiber was elevated (14%, P ! 0.05). Modelcalculations showed that the region-specific alterations in fiber size,SDH activity, and capillary supply to a fiber prevented the occurrenceof anoxic areas in the deep region but not in the superficial region.Inclusion of reported acclimatization-induced increases in mean cap-illary oxygen pressure attenuated the development of anoxic tissueareas in the superficial region of the muscle. We conclude that thedeterminants of tissue oxygenation show region-specific adaptations,resulting in a marked differential effect on tissue PO2.

tissue oxygenation; capillarization; oxidative capacity

CHRONIC AND/OR INTERMITTENT HYPOXIA have been suggested tobe factors that contribute to changes in muscle structure andfunction in patients suffering from several cardiorespiratorydiseases such as chronic obstructive pulmonary disease, severepneumonia, and heart failure (10, 29, 63, 64, 78). Oxygen iscrucial for aerobic energy metabolism, and even a brief reduc-tion in oxygen supply due to hypoxia leads to acutely dimin-ished muscle performance (9, 17, 34). During long-term expo-sure to hypoxia, skeletal muscle adapts to the reduced PO2 bymarked changes in skeletal muscle fiber size and cellularmetabolism (25, 51).

An increase in capillary density (CD) in rat muscle after6 wk of exposure to 12% oxygen (21, 22) may serve tofacilitate oxygen supply to muscle cells by reducing thediffusion distances of and exchange area for oxygen. Thisresponse is realized by angiogenesis (21, 22) and/or adecrease in fiber cross-sectional area (FCSA) (3, 25). Afurther improvement of the oxygen supply to the mitochon-dria might be realized by an increase in myoglobin (Mb)concentration enhancing the facilitation of intracellular dif-fusion of oxygen (60). These results, however, are notconsistent as unaltered FCSA (21, 67), capillarization (1,65), and/or Mb concentration in response to chronic hypoxiahave also been reported (41, 66).

Besides adaptations in capillarization, chronic hypoxiamight also affect energy metabolism. In both human and ratmuscles, a decreased oxidative capacity has been reported andis accompanied by an increased glycolytic capacity (1, 12, 30,38, 39, 48). Others, however, have reported that the oxidativecapacity is unchanged (47) or even increased after exposure tohypoxia (26, 33).

The observation that adaptations to hypoxia are musclespecific and even specific to the region within a musclecould explain these discrepancies (22, 24, 55, 66, 67).Indeed, it has been reported that slow and fast musclesrespond differently to chronic exposure of hypoxia (22, 24).Even within one muscle with a nonuniform fiber typedistribution, the capillary adaptation to hypoxia seems todepend on the region of the muscle, where the hypoxia-induced angiogenesis was greater in regions within a musclewith a lower oxidative capacity (22).

To investigate whether adaptations to chronic hypoxia arefiber type and/or region specific, rats were exposed for 4 wk tochronic hypobaric hypoxia (410 mmHg). We hypothesized that1) overall, there is atrophy and a decrease in oxidative capac-ity; 2) in the superficial region of the muscle, angiogenesis ismore pronounced than in the deep region; and 3) these adap-tations prevent the occurrence of anoxic areas in both regionsof the muscle during hypoxia.

We used an integrative approach to assess the skeletalmuscle fiber adaptations to chronic hypoxia by determiningchanges in fiber type, size, capillary supply, oxidative ca-pacity, and Mb concentration in muscle fibers of distinctoxidative and glycolytic regions in serial histological sec-tions of the plantaris muscle of the rat. The functionalconsequences of these adaptations were calculated using aKrogh model (36, 46) and a Hill model (19, 32).

Address for reprint requests and other correspondence: R. Wust, Institute ofMembrane and Systems Biology, Faculty of Biological Sciences, Univ. ofLeeds, Worsley Bldg., Leeds LS2 9JT, UK (e-mail: [email protected]).

Am J Physiol Heart Circ Physiol 297: H364–H374, 2009.First published May 8, 2009; doi:10.1152/ajpheart.00272.2009.

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METHODS

Animals

Seventeen male Wistar rats, aged 8 wk at the start of the study,were used. Experimental rats (n " 8) were housed in a hypobarichypoxic chamber at a barometric pressure of 410 mmHg and 20.9%O2 [equivalent to a PO2 of 11.5 kPa (86 mmHg) and that experiencedat #5,000 m of altitude]. Normobaric rats (n " 9) lived in the sameroom but at ambient pressure (#760 mmHg). All rats were kept atroom temperature and a 12:12-h light-dark cycle. Food and waterwere given ad libitum, and the cage was opened for 30 min maximallyonce a week for cleaning. After 4 wk of exposure to hypobarichypoxia or normobaric conditions, rats were anesthetized by anintraperitoneal injection of pentobarbital sodium (70 mg/kg), and theright plantaris muscle was dissected from the surrounded tissue,blotted dry, and weighed. The muscle was then pinned on cork andslightly stretched above its slack length to minimize any bias in thedetermination of FCSA and capillary parameters, related to differ-ences in muscle length, quickly frozen in liquid nitrogen, and storedat $80°C until further analysis. All protocols and procedures wereapproved by the University of Nijmegen Institutional Animal Careand Use Committee in accordance with the National Institutes ofHealth Guide for Care and Use of Laboratory Animals.

Hematocrit and Hemodynamic Measurements

Hematocrit levels were determined in blood samples taken fromthe right orbita. Right ventricular systolic pressure was measured(MIDAC software, Radboud University, Nijmegen, The Nether-lands) with an ultaminiature catheter (model SPR-320, MillarInstruments, Houston, TX) attached to a pressure transducer intro-duced into the right external jugular vein and placed in the rightventricle by pressure tracing. The right ventricle was dissected fromthe heart, weighed, and the weight expressed as a percentage of thetotal body weight.

Myosin Heavy Chain Composition

To determine the myosin heavy chain (MHC) composition (type I,IIA, IIX, and IIB) of the plantaris muscle, we used a modification ofthe SDS-PAGE method used previously (13). In short, a 10-%msection was dissolved in 300 %l Laemmli buffer, and 11 %l wereloaded onto 6% acryl amide gels. Gels were run at 250 V for 25 h andsubsequently stained with a Silverstain Plus Kit (Bio-Rad, HemelHempstead, UK).

Staining of Histological Sections

Serial 10-%m cross sections from the middle of the plantaris musclewere cut on a cryostat at $20°C and mounted on polylysine-coatedslides. Sections were stored at $80°C until further analysis unlessotherwise stated. All sections were mounted in glycerin-gelatin afterbeing stained.

Myosin ATPase

Sections were stained for myosin ATPase to classify fibers as typeI, IIa, and IIx/b as previously described (8).

Succinate Dehydrogenase Activity

Succinate dehydrogenase (SDH) activity in individual muscle cellsin histological sections was determined immediately after sectioningas previously described (57). Sections were incubated at 37°C in thedark for 20 min in medium consisting of 37 mM sodium phosphatebuffer (pH 7.6), 74 mM sodium succinate, and 0.4 mM tetranitrobluetetrazolium. After incubation, the reaction was stopped in 0.01 MHCl. The staining intensity of SDH was determined by measuring theabsorbance of the final reaction product using an interference filter at

660 nm. Absorbances were converted to the rate of staining andexpressed as absorbance units (&A660) per micrometer section thick-ness per second of incubation time (&A660 !%m$1 !s$1). The stainingrate is proportional to the maximum rate of oxygen uptake by themuscle cell when oxygen is not rate limiting (76).

Mb Concentration

The Mb concentration in individual muscle cells was determined aspreviously described (74). In short, frozen sections were defrosted ina vacuum to avoid the condensation of water on the sections andhence the redistribution of Mb. Sections were then vapor fixed for 1 hin paraformaldehyde at 70°C. Subsequently, sections were fixed for10 min at room temperature in 2.5% glutaraldehyde in 0.07 M sodiumphosphate buffer (pH 7.4) and quickly rinsed with distilled water. Todetect Mb, sections were incubated for 1 h at room temperature inmedium containing 59 ml of 50 mM Tris and 80 mM KCl buffer (pH8.0), 25 mg ortho-toluidine dissolved in 2 ml of 96% ethanol (at#50°C), and 1.43 ml of 70% tertiary-butyl-hydroperoxide. Sectionswere then washed with distilled water and mounted. Calibratedimages were studied on a DMRB microscope (Leica, Wetzlar, Ger-many) at 436 nm.

Capillarization

Capillaries were depicted by staining sections for alkaline phos-phatase as previously described (18, 21, 22). The capillarization wasanalyzed using the method of capillary domains (18, 37). This methoddoes not only provide the overall indexes of capillary density (CD)and capillary-to-fiber ratio but also provides measures of the capillarysupply to individual fibers, even when they lack direct capillarycontacts, and allows the calculation of capillary supply to differentfiber types. Also, an index for the heterogeneity in capillary spacingcan be calculated, which is an important factor for tissue oxygenation(14). Since the deep and superficial regions of the muscle differ in theirfiber type composition and capillary supply, we analyzed these regionsseparately. In short, photomicrographs of cross sections of the deepand superficial regions of the plantaris muscle were taken. The fibertype distribution was assessed, and the outlines of the muscle fibers aswell as the location of each capillary were digitized (model MMII1201, Summagraphics) and fed into the computer program. Theoverall CD was defined as the number of capillaries per millimetersquared of tissue. Capillary domains, defined as the area surroundinga capillary delineated by equidistant boundaries from adjacent ones,were constructed, and their surface area was calculated. The SD of thelog-transformed domain area (logRSD) gives an indication of theheterogeneity of the capillary spacing. A decrease in this parameterindicates a more homogeneous distribution of capillaries. From theoverlap of the capillary domains with muscle fibers, the local capil-lary-to-fiber ratio (LCFR) and the capillary fiber density (CFD) wereobtained. The LCFR for a fiber was defined as the sum of the fractionsof each domain area overlapping the fiber. This variable has acontinuous distribution and takes into account remote capillaries, thusallowing the determination of the capillary supply to a fiber evenwhen it lacks direct capillary contacts. Such situations were observedoccasionally in the glycolytic region of the plantaris muscle. CFD,which is LCFR divided by FCSA, provides the CD of that fiber andis expressed as the number of capillaries per millimeter squared.

Modeling of Skeletal Muscle Tissue Oxygenation

We applied the Hill model and an extended Krogh model to obtainsome insight as to how the observed adaptations affected tissueoxygenation during hypoxia. Both models assume that oxygen dif-fuses over the cell membrane and through the cytoplasm into themitochondria and take into account Mb-facilitated oxygen diffusion.The Hill model assumes homogeneous PO2 in the interstitial spacearound the fiber, whereas the Krogh model assumes that oxygen

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diffuses from point sources of oxygen (capillaries). We assumed thatat the maximal oxygen consumption of the cell (VO2max), the flowthrough and PO2 in all capillaries are the same.

Hill model. The critical extracellular PO2 required to prevent thedevelopment of an anoxic core in a cylindrical cell (PO2 crit; inmmHg) at V'O2 max (in mM/s) was calculated with the following Hillmodel (32, 54):

PO2 crit ! (VO2max ! FCSA " 4) !DMb ! *MbO2+R,/4) !-M !DO2

where V'O2 max was calculated using SDH activity (76), FCSA wasmeasured in millimeters squared, DMb is the radial diffusion coeffi-cient of Mb [0.27 . 10$4 mm2/s (4)], [MbO2]R is the concentrationof oxygenated Mb at the sarcolemma (in mM) and was calculatedfrom the total Mb concentration in individual fibers using methodsdescribed in Des Tombe et al. (19), -M is the solubility of oxygen inskeletal muscle, and DO2 is the diffusion coefficient for oxygen inskeletal muscle. The product of the latter two (-M . DO2) is knownas Krogh’s diffusion coefficient (2 nM !mm2 !mmHg$1 !s$1) (75).

Krogh model. The critical PO2 just outside the capillary required toprevent the development of anoxic tissue areas when the muscle isworking at VO2max (PO2 cap; in mmHg) was calculated using auniversal Krogh’s tissue model (36). We used photomicrographs ofsections with a representative capillarization for each region andcondition. For the model calculations, the capillary radius was set at2.4 %m. The weighted average VO2max and Mb concentration were fedinto the model. Where the Hill model calculates a PO2 that should beuniformly present around a muscle fiber, Krogh’s tissue model cal-culates a minimal PO2 at the capillary that ensures the absence ofanoxic areas.

We also used the Krogh’s tissue model to estimate the distributionof oxygen pressure within the muscle tissue during normoxia (meancapillary PO2 of 55 mmHg) and acute hypoxia (mean capillary PO2 of25 mmHg, with both conditions using tissue characteristics of thenormoxic situation). We then fed into the model the observed skeletalmuscle adaptations to chronic hypoxia and assessed how these af-fected the PO2 distribution in the tissue during hypoxia (mean capil-lary PO2 of 25 mmHg). Finally, we fed into the model not only theobserved skeletal muscle adaptations to chronic hypoxia but also thereported increase in mean capillary PO2 [35 mmHg (9)] to see how thiswould affect the tissue PO2 distribution.

Statistical Analysis

Independent t-tests were used to compare means between the twogroups. Multilevel analysis (MLwiN, version 2.02, Centre for Multi-level Modeling, Bristol, UK) was used to test for significant differ-ences in capillarity, oxidative capacity, Mb concentration, and fibersize between region, fiber type, and hypoxia/normoxia. Multilevelanalysis can be considered as an extension to the commonly usedlinear regression analysis, which has the disadvantage that only acontinuous outcome variable can be analyzed (73). Moreover, itincorporates variance at the level of the cell and the individual.Differences were considered significant at P ! 0.05. Unless statedotherwise, values are means / SD.

RESULTS

Body weights of hypoxic (363 / 40 g) and control (383 /28 g) rats were not significantly different. In addition, weightsof control and hypoxic plantaris muscles (397 / 76 vs. 370 /31 mg, respectively) did not differ significantly. Ratios ofplantaris muscle mass to body mass were also similar in controland hypoxic rats: 1.04 / 0.17 and 1.02 / 0.05 mg/g, respec-tively. That the rats were really hypoxic was reflected by thehigher hematocrit than control rats (49.9 / 0.8 vs. 58.9 /0.9%, P ! 0.001) as well as elevated right ventricular systolicpressure (from 15 / 32 to 74 / 11 mmHg in controls vs.

hypoxic rats, P ! 0.001), and right ventricular hypertrophy(from 0.75 / 0.15 to 1.02 / 0.12 g/g body wt, P ! 0.001),similar to that reported previously in hypobaric hypoxia (80).

In total, 990 control and 1,130 hypoxic fibers were analyzed.For each fiber, all parameters were assessed in serial musclesections; a typical example is shown in Fig. 1.

Fiber Type Composition and FCSA

The overall MHC composition of the plantaris muscle did notchange significantly after exposure to hypoxia (Fig. 2). The deepregion had a higher proportion of type I fibers and a lowerproportion of type IIx/b fibers than the superficial region (P !0.001), whereas there were no significant regional differ-ences in the proportion of type IIa fibers. In line with theMHC data, hypoxia did not result in an altered fiber typecomposition (Fig. 3A).

In both regions, the size of the fibers was in the order of I !IIa ! IIx/b (P ! 0.05). Type I fibers in the deep region werelarger and type IIx/b fibers were smaller than those in thesuperficial region (P ! 0.05), whereas type IIa fibers had asimilar size in both regions. Hypoxia resulted in an overall10% reduction in FCSA (P ! 0.05; Fig. 3B). The absence of aninteraction between fiber type and/or region with hypoxiaindicates that this effect was similar for each fiber type andindependent of region.

SDH Activity

SDH activity in type IIx/b fibers was lower than that in theother fibers (P ! 0.001; Fig. 4A). Interestingly, in the deepregion only, the SDH activity of type IIa fibers was higher thanthat of type I fibers (P ! 0.001). Overall, the SDH activity was26% lower in the superficial region than in the deep region(P ! 0.001). This was explicable by the larger number of typeIIx/b fibers in the superficial region than in the deep region. Inaddition, the SDH activity in type IIx/b fibers in the superficialregion was lower than that in the deep region (P ! 0.001),whereas there were no significant differences in activity in typeI and IIa fibers between the two regions. Hypoxia induced anoverall 25% increase in the SDH activity in fibers in thesuperficial region (P ! 0.05), whereas no change in the SDHactivity occurred in the deep region.

Spatially integrated SDH activity is proportional to theVO2max per millimeter of fiber length (in nmol !mm$1 !s$1) ofthe cell (76). In control rats, type I fibers had a lower integratedSDH activity compared with type II fibers (IIa and IIx/b fibers,P ! 0.001), whereas there were no differences between IIa andIIx/b fibers (Fig. 4B). Integrated SDH activity was higher infibers from the deep region compared with those from thesuperficial region (P ! 0.001). This was due to a higherintegrated SDH activity of type II fibers (P ! 0.001) but not oftype I fibers in the deep region than in the superficial region ofthe muscle. After exposure to hypoxia, this difference betweenthe two regions disappeared (P " 1.00).

Mb Concentration

Overall, fibers in the superficial region of the plantaris musclehad a 30% lower Mb concentration than those in the deep region(P ! 0.001; Fig. 5). The Mb concentration of type IIa fibers inboth the deep and superficial regions was higher than that of typeI fibers (P ! 0.05), whereas type IIx/b fibers had the lowest Mb

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concentration (P ! 0.001). Hypoxia did not significantly affectthe Mb concentration in fibers of any type or region.

Capillarization

The LCFR for each fiber type and the overall capillary-to-fiber ratio (shown as “total”) are shown in Fig. 6A. In both

regions, the LCFR of type IIx/b fibers was higher than that ofthe other fibers (P ! 0.001). In the deep region, type I and IIafibers had a similar LCFR, whereas in the superficial region,the LCFR of type IIa fibers was higher compared with type Ifibers (P ! 0.001). The LCFR for each fiber type was higherin the deep region compared with the superficial region (P !0.001). There was an interaction between region and hypoxia(P ! 0.001), which was apparent as a 14% increase in theLCFR after hypoxia of fibers in the deep region (P ! 0.05) butnot in the superficial region.

The CFD of fibers of each type was lower in the superficialregion than in the deep region (P ! 0.001; Fig. 6B). The CFDof type IIx/b fibers was lower than those of type I and IIa fibers(P ! 0.001), which had a similar CFD. Hypoxia resulted in a24% higher CFD in the deep region (P ! 0.01) but not thesuperficial region of the muscle.

The heterogeneity of capillary spacing (logRSD) was largerin the superficial region compared with the deep region(0.097 / 0.013 vs. 0.112 / 0.011, means / SE, P ! 0.001)but was not significantly changed by hypoxia in either region(0.091 / 0.010 and 0.118 / 0.022 for the deep and superficialregions, respectively).

Relationships Among Capillarization, SDH Activity,and FCSA

To investigate how the adaptations in response to hypoxiaaffected oxygen supply and demand, we calculated the rela-tionships among LCFR, SDH activity, and FCSA for all fibertypes. Relations for type IIa and IIx/b fibers, as they are thepredominant fibers in the plantaris muscle, are shown in Fig. 7.

As expected, LCFR was linearly related to the maximaloxygen uptake of the cell (as reflected by the spatially inte-

Fig. 1. Typical examples of serial sections stained forATPase after preincubation at pH 4.55 (A), capillaries (B),succinate dehydrogenase (SDH) activity (C), and myoglo-bin (Mb) concentration (D; note the dark spots stained forhemoglobin). Type I cells (stained dark for ATPase) had ahigher capillary fiber density (CFD), higher SDH activity,and more Mb. *Same type IIx/b fiber in each section.Bars " 50 %m.

Fig. 2. A: myosin heavy chain (MHC) composition in the extensor digitorumlongus (lane 1), normoxic plantaris muscle (lanes 2–4), hypoxic plantarismuscle (lanes 5–7), and soleus muscle (lane 8) of rats using 6% gel electro-phoresis. B: no significant differences were observed between normoxia andhypoxia.




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grated SDH activity) in type I fibers (R2 ranged from 0.165 to0.640 for each region and condition, P ! 0.001), type IIa fibers(R2 ranged from 0.110 to 0.336, P ! 0.001; Fig. 7A), and typeIIx/b fibers (R2 ranged from 0.070 to 0.284, P ! 0.001; Fig.7B) except for type IIx/b fibers in the normoxic superficialregion (R2 " 0.012, P " 0.06). After hypoxia, the correlationcoefficient and slope of this relation increased for type IIx/bfibers in both regions (P ! 0.001).

There was no significant relationship between LCFR andSDH activity for type I fibers (R2 ranged from 0.000 to 0.144)and IIa fibers (R2 ranged from 0.008 to 0.052; Fig. 7C), and intype IIx/b fibers in the superficial region, a slight negativerelationship was observed (R2 " 0.018, P ! 0.01; Fig. 7D,dotted line), which disappeared (P ! 0.03) after exposure tohypoxia (R2 " 0.000).

Whereas no correlation was found between LCFR and SDHactivity, LCFR did not significantly correlate positively withFCSA in type I fibers (R2 ranged from 0.226 to 0.487), IIafibers (R2 ranged from 0.115 to 0.491; Fig. 7E) and IIx/b fibers(R2 ranged from 0.127 to 0.341; Fig. 7F). For all fiber types,the slope between FCSA and LCFR significantly increased inthe deep region (P ! 0.05) but not in the superficial regionafter hypoxia. Interestingly, whereas the region-specific differ-ence in the slope was not significant in normoxia, hypoxiaamplified the difference in the slope between the deep andsuperficial regions for all fiber types, which was entirely due toan increased slope in the deep region only. This indicated thatat a given FCSA, type I, IIa, and IIx/b fibers in the superficial

region had a similar LCFR, whereas those in the deep regionhad a higher LCFR after hypoxia (P ! 0.001). This wasreflected by the increased CFD for each fiber type in the deepregion but not in the superficial region during hypoxia (Fig.6B). Overall, the CFD of type I fibers was higher than that fortype IIa fibers. Type IIx/b fibers had the lowest CFD (P !0.001).

Fig. 3. A and B: effects of hypoxia on muscle fiber type distribution (A) andfiber cross-sectional area (FCSA; B) in the deep and superficial region of theplantaris muscle. The deep (oxidative) region had a significantly higherpercentage of type I fibers (P ! 0.001) compared with the superficial (glyco-lytic) region. No significant fiber type shift was observed after 4 wk ofhypoxia. FCSA decreased significantly after exposure to hypoxia (P ! 0.05) inall fiber types and both regions. See RESULTS for further statistically significantdifferences between types and regions.

Fig. 4. A and B: SDH activity (A) and spatially integrated SDH activity (B) indifferent fiber types and regions of the plantaris muscle after 4 wk of hypoxiacompared with normoxia. SDH activity in the superficial region was increasedby 25% in the hypoxic group (P ! 0.05), but this did not occur in the deepregion. The difference in spatially integrated SDH activity between the deepand superficial regions, which was apparent in control rats (P ! 0.001),disappeared after hypoxia (P " 1.00). SDH activity was measured as absor-bance units (&A660) per micrometer section thickness per second of incubationtime (&A660 !%m$1 ! s$1). See RESULTS for further statistically significant dif-ferences between types and regions.

Fig. 5. Mb concentration in different fiber types and regions of the plantarismuscle after 4 wk of hypoxia compared with normoxia. No significantdifferences were observed after the exposure to hypoxia. See RESULTS forstatistically significant differences between fiber types and regions.




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Model Calculations

Model calculations were used to asses the functionalconsequences of the observed adaptations. During nor-moxia, the PO2 crit for each individual muscle fiber (Hillmodel) was higher in the deep region compared with thesuperficial region (P ! 0.001; Fig. 8A). In response tochronic hypoxia, the regional difference in PO2 crit disap-peared, due to a decrease in the deep region and an increasein the superficial region (interaction between hypoxia andregion, P ! 0.001). The decrease in PO2 crit in the deepregion was caused by muscle fiber atrophy, whereas theincrease in the superficial region resulted from an increasein SDH activity.

Subsequently, we calculated the PO2 that is required in thecapillaries to prevent the occurrence of any anoxic area at VO2max

(as was calculated for PO2 crit), which is referred to as PO2 cap. Tothis end, we chose photographs with representative capillarydistributions for each region and condition. PO2 cap in the super-ficial region was higher compared with the deep region. PO2 cap inthe deep region did not differ between normoxia and hypoxia, buthypoxia resulted in a 25% increase in PO2 cap in the superficialregion of the muscle (Fig. 8B).

The PO2 distribution, shown as a cumulative curve (in %), inthe muscle tissue at VO2max was calculated using the Kroghmodel in normoxia [Fig. 9A; mean capillary PO2 set to 55mmHg (9)], acute hypoxia [Fig. 9B; mean capillary PO2 set to25 mmHg (9)], and after chronic exposure to hypoxia (mean

capillary PO2 set to 25 mmHg), taking into account skeletalmuscle adaptations (Fig. 9B). Figure 9C shows the distributionof tissue PO2 when not only the adaptations in the skeletalmuscle but also the reported changes in mean capillary PO2

(presumably due to increases in hematocrit and blood flow)were fed into the model (mean capillary PO2 " 35 mmHg).While during normoxia no anoxic regions were present ineither the deep or superficial regions of the muscle (Fig. 9A),acute hypoxia (25 mmHg) resulted in a marked leftwardshift in the PO2 distribution in the tissue; during acutehypoxia, #75% of the skeletal muscle tissue in the super-ficial region was anoxic and 40% of the skeletal muscletissue in the deep region was anoxic. The adaptations of theskeletal muscle tissue to chronic hypoxia resulted in amarked region-specific adaptation: whereas in the deepregion the adaptations in the skeletal muscle (higher capil-larization and muscle atrophy) resulted in a rightward shiftcompared with the acute hypoxic situation, indicating im-proved tissue oxygenation due to the increase in capillar-ization and the reduction in FCSA, a leftward shift wasobserved in the superficial region. However, the hypoxia-induced adaptations in oxygen delivery (9) resulted in aslightly improved tissue oxygenation in the superficial re-gion and a more improved tissue oxygenation in the deepregion (Fig. 9C). In the deep region, mean tissue PO2

decreased from 32.1 to 5.9 mmHg after acute exposure tohypoxia. After chronic exposure, this increased to 9.9 and18.6 mmHg (Fig. 9). In the superficial region, mean tissuePO2 decreased from 26.0 to 3.9 mmHg after acute exposureto hypoxia and decreased even further to 2.3 mmHg aftertaking into account the skeletal muscle adaptations tochronic exposure (Fig. 9). Use of the increased mean cap-illary PO2 reported by Calbet et al. (9), most likely as aconsequence of the increased hematocrit and possible in-crease in blood flow, resulted in an improved mean tissuePO2 of 5.9 mmHg.

DISCUSSION

Using an integrative approach to study the parameters de-termining oxygen delivery and utilization, we observed that themuscular adaptations to chronic hypoxia are region specificand not fiber type specific. Overall, hypoxia did cause an#10% muscle fiber atrophy, irrespective of fiber type andmuscle region. In the deep oxidative region of the plantarismuscle, this and angiogenesis resulted in improved capillariza-tion. In the superficial glycolytic region, there was no angio-genesis but, in contrast to our expectations, a 25% increase inthe oxidative capacity, indicating mitochondrial biogenesis.The absence of a change in the oxidative capacity in the deep(oxidative) region of the muscle indicates that the loss ofmitochondria was proportional to the degree of atrophy. Whilethe adaptations to chronic hypoxia in the deep region attenu-ated the reduction in tissue PO2 and prevented the occurrence ofanoxic areas during hypoxia, the increased oxidative capacityin the superficial region appeared to aggravate problems withtissue oxygenation, which was reversed to some extent by anincrease in mean capillary PO2 due to an alteration in theoxygen delivery during conditions of elevated oxygen demandby the muscle.

Fig. 6. A and B: local capillary-to-fiber ratio (LCFR; A) and CFD (B) indifferent fiber types and regions of the plantaris muscle after 4 wk of hypoxiacompared with control values. Note that the total value in both regions is theoverall capillary-to-fiber ratio (A) or overall capillary density (B). Hypoxiaincreased LCFR in the deep region (P ! 0.05) but not in the superficial region.CFD increased in the deep region (P ! 0.01) but not in the superficial region.See RESULTS for further statistically significant differences between types andregions.




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Relationship Between Oxidative Capacity and CapillarySupply in Normoxia and Hypoxia

The general idea is that in many species, including birds(52), humans (5), and rats (present study), the capillary supplyto a fiber is related to its oxidative capacity. This relationshipis modulated by the metabolic surroundings of a fiber, where afiber in an oxidative region has a larger capillary supply than asimilar fiber surrounded by glycolytic fibers (18). Here, weshowed, using quantitative histochemistry, that although thenumber of capillaries supplying a fiber (LCFR) was indeedrelated to its VO2max, as estimated from its integrated SDHactivity, LCFR correlated more with fiber size (Fig. 7) (53, 79).The lack of a correlation between SDH activity per millimetersquared of fiber and LCFR could be due, at least in part, to thefact that the mitochondrial density within a fiber decreasesfrom the periphery to the center of the fiber (70, 79). Weobserved that the capillary supply per unit fiber perimeter wasmore or less constant [data not shown and Ref. 14], suggestingthat diffusion distance or capillary contact per fiber perimeter,

rather than the total number of mitochondria in a cell, is a moreimportant determinant of the capillary supply to a fiber.

Capillaries do not only supply oxygen but also remove wasteproducts and heat from the exercising muscle cell. This mayexplain the absence of a positive relationship between theintegrated SDH activity and LCFR of the glycolytic fibers witha low oxidative capacity (type IIx/b fibers) in the glycolyticregion of the muscle (Fig. 7B).

During exposure to hypoxia, the integrated SDH activity inthese fibers increased and the relationship between spatiallyintegrated SDH activity and LCFR became stronger for typeIIx/b fibers only. This suggests that, during hypoxia, thefunction of the capillaries to deliver oxygen to the (previouslylow oxidative) muscle cells becomes more important.

Overall Adaptations to Chronic Hypoxia

Similar to previous studies, we observed no differences inthe fiber type composition (30, 55). Hypoxia did, however,cause an #10% muscle atrophy (6, 20, 22, 25) in fibers of each

Fig. 7. Relationship between LCFR and spa-tially integrated SDH activity (A and B), SDHactivity (C and D), and FCSA (E and F) for typeIIa fibers (A, C, and E) and IIx/b fibers (B, D, andF). The correlation between the spatially inte-grated SDH activity and capillary supply (A andB) was due to a significant correlation betweenFCSA and LCFR (E and F) rather than a corre-lation between LCFR and SDH activity (C andD). For R2 and P values, see RESULTS.




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type and region, which may serve to decrease the diffusiondistance from the capillaries to the interior of the muscle fibers.

A factor that is often overlooked, but may have a majorimpact on the tissue oxygenation, is the heterogeneity ofcapillary spacing (logRSD): a more homogeneous distributionof capillaries might enhance tissue oxygenation, whereas astrong heterogeneous distribution of capillaries may result inanoxic areas (14, 28, 56). In the deep oxidative region of themuscle, with a higher demand of oxygen, the capillaries weremore homogenously distributed than in the superficial glyco-lytic region. Yet, we did not observe a more homogeneousdistribution of capillaries after exposure to hypoxia, not even inthe glycolytic region that became more oxidative. Also, duringhypertrophy (16) and atrophy (15), the heterogeneity of capil-lary spacing remains unaltered, and it has been suggested thatthe absence of changes might be related to physical constraints,determined by the morphology of the muscle fibers, of placingnew capillaries (16).

Mb might act to facilitate the diffusion of oxygen (62). Anelevated Mb concentration during hypoxia (59) might thusserve to enhance intracellular oxygen transport and ensureadequate tissue oxygenation. In agreement with previous ob-servations (77), we observed that oxidative type I and IIa fiberscontain more Mb than glycolytic IIx/b fibers. Whereas somereports (26, 72) have suggested that the Mb content of themuscle is increased after exposure to hypoxia, others have notshown such changes in Mb protein content (41, 66), even whenendurance training was superimposed (50). In these studies, theMb content was determined in muscle homogenates, and re-

gion- and/or fiber type-specific adaptations might be missed.Here, we showed with quantitative histochemistry that al-though the oxidative capacity of fibers in the superficial regionwas elevated after hypoxia, there were no changes in the Mbconcentration in fibers of any type or region after exposure tohypoxia. Although iron deficiency during hypoxia may induceelevated Mb mRNA levels without the formation of functionalMb (61), it is unlikely to play a role in our study as irondeficiency also hampers mitochondrial synthesis (35), some-thing that did not appear to be limited in our study. Our modelcalculations show that the facilitation of oxygen diffusion byMb in our tissue was equivalent to a reduction of 5 mmHg inthe required PO2 cap (data not shown).

We observed that the deep oxidative and superficial glycolyticregions of the muscle differed in their adaptations in capillariza-tion and oxidative capacity to chronic hypoxia. It is interesting tonote that the adaptations were region specific and not fiber typespecific. In other words, no matter what type a fiber was, itsintegrated SDH activity was increased in the superficial regionand its capillary supply in the deep region.

Region-Specific Increase in Capillarity

Whereas capillary proliferation took place in the deep region, asreflected by the increased LCFR, no capillary proliferation oc-curred in the superficial region. Similar to the present results, otherinvestigators (2, 21, 22) have also observed region-specific adap-tations in capillarization. During submaximal exercise particu-larly, the deep oxidative region of the muscle is recruited (42). Todeliver the same amount of oxygen, the blood flow to the muscleat a given submaximal workload is increased, as is the extractionof oxygen (44), and it is expected that particularly in the deepregion and less so in the superficial glycolytic region, the endo-thelium is exposed to elevated shear stress, which is a potentstimulant for angiogenesis (40). Also in the superficial region anincreased blood flow and shear stress is to be expected, which,however, may have been be too low to induce angiogenesis.

Although our model calculations showed that the degree ofhypoxia is more severe in the superficial region than in the deepregion of the muscle during maximal exercise, it is possible thatmuscle fibers in the deep region, being recruited more also duringnormal activity, are exposed to much longer periods of lowoxygen pressures than the superficial region. Hypoxia-induciblefactor (HIF)-1- protein expression is elevated during hypoxia andis a master transcription regulator in the adaptations, such as thoseof the capillary network, to hypoxia (63). Indeed, elevated HIF-1-expression induces the increased expression of VEGF and itsreceptor (FLT-1). Thus, although speculative, it might also be thatparacrine factors, such as VEGF and/or other substances, areexcreted by or diffuse out of the cell in relatively larger quantitiesfrom cells that are recruited more, such as in the deep region (11),than elsewhere and hence affect the capillary proliferation indifferent regions differently.

Region-Specific Adaptation in Oxidative Capacity

The spatially integrated SDH activity, reflecting the total num-ber of mitochondria per fiber, decreased in the deep region andincreased in the superficial region. In the deep region, the de-creased integrated SDH activity per fiber is explicable by atrophy.This, together with an unaltered SDH activity per millimetersquare, reflecting the number of mitochondria per volume of

Fig. 8. Minimal PO2 around the fiber (Hill model; A) and in the capillary(Krogh model; B) that prevents the development of an anoxic core when thefibers work at their maximal oxygen consumption (VO2max). The region-specific difference in the critical extracellular PO2 required to prevent thedevelopment of an anoxic core in a cylindrical cell (PO2 crit) in A disappearedafter exposure to hypoxia. Using the Krogh model (incorporating the capillarysupply), the critical capillary PO2 did not change in the deep region, whereasit increased by 25% in the superficial region.




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tissue (71), indicates that atrophy is accompanied by a propor-tional loss of mitochondria in the deep region. The increasedintegrated SDH activity in the superficial region, on the otherhand, indicates that although atrophy also occurred in this region,mitochondrial biogenesis was elevated.

During hypoxia, even during submaximal exercise, an acceler-ated breakdown of phosphocreatine (PCr) and fall in pH occur(34), which may, independent from intracellular PO2 levels, causefatigue in single skeletal muscle fibers (68). The increased rate ofaccumulation of metabolites may require the earlier recruitment ofglycolytic fibers during submaximal activity. This more frequentrecruitment may be compared with an endurance training programinducing an increase in the oxidative capacity. An additional 5%duration of recruitment has been shown to be sufficient to increasethe oxidative capacity of a muscle cell, as seen with electricalstimulation (43), and also training during hypoxia induces anincrease in the oxidative capacity (1, 72). It is not clear why theoxidative capacity in the deep region is not affected, but ourobservations are analogous to the observation that the increasein oxidative capacity after electrical stimulation is inverselyrelated to the original oxidative capacity of the muscle ormuscle region (58).

The region-specific adaptation in mitochondrial biogenesismight be related to differential upregulation of HIF-1- in bothregions as HIF-1- is reported to inhibit mitochondrial biogenesis(49). Other potential mediators for the region-specific adaptationin oxidative capacity are differences in the expression of factorsthat are involved in the transcriptional regulation of mitochondrialgenes, such as peroxidase proliferator-actived receptor (PPAR)-0and its ligand PPAR-0 coactivator-1- (45).

Functional Implications of the Adaptations to Hypoxia

Both the Krogh and Hill model calculations gave a qualitativeindication that the improved capillarization and atrophy in thedeep region of the muscle served to improve tissue oxygenation.

In the superficial region, however, the adaptations did not appearto improve tissue oxygenation, as reflected by an increased pro-portion of anoxic tissue areas calculated with the Krogh model.The Hill model indicated that in the superficial region the PO2

allowing the muscle cell to work at VO2max was elevated afterchronic hypoxia, which corresponded with an increase in thecapillary PO2, calculated with the Krogh model, required for thetissue to work at VO2max without the development of anoxic tissueareas. The higher values for PO2 cap than PO2 crit (but similarqualitative changes after hypoxia) can be ascribed to the differentassumptions of the models: the Hill model assumes that the PO2

around a fiber is homogeneous and diffusion thus essentially takesplace from an infinite number of point sources, whereas in theKrogh model oxygen diffuses from capillaries (point sources). Inthe resting and isometric situations capillaries are running essen-tially in parallel with muscle fibers, approximating the Kroghtissue cylinders, whereas during shortening the capillaries becomemore tortuous, resulting in an increasing capillary-to-fiber perim-eter contact and hence resembling more the Hill model (31).Nonetheless, both models agree that the increased oxidative ca-pacity in the superficial region after chronic hypoxia does notappear to help but rather to aggravate the situation. Thus, in termsof tissue oxygenation, this seems to be an inappropriate adaptationto hypoxia.

During chronic hypoxia we did not only observed adaptationsin the tissue but also an increase in hematocrit. This increase inhematocrit, together with possible increases in blood flow throughthe active muscle during hypoxia compared with normoxia, mayexplain the increased mean capillary PO2 after chronic hypoxiacompared with acute hypoxia in the exercising muscle (9). Whenwe took this into account, our model calculations showed aconsiderable improvement in tissue oxygenation during hypoxia,with a significant reduction in the percentage of anoxic areas from74% to 55% in the superficial region and from 19% to 6% in thedeep region of the muscle (Fig. 9C).

Fig. 9. Cumulative PO2 distribution (in %) in the muscle tissue calculated at VO2max using the Krogh model in normoxia (A; mean capillary PO2 " 55 mmHg),during acute hypoxia, and after skeletal muscle adaptations to chronic hypoxia (B; mean capillary PO2 " 25 mmHg), and after adaptations in the skeletal musclein oxygen delivery (C; mean capillary PO2 " 35 mmHg). The values for mean capillary PO2 were derived from Calbet et al. (9). A marked differential responsein both regions can be observed.




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The respiration of the mitochondria is a hyperbolic function ofthe PO2 that the mitochondria experience, and when the PO2

becomes too low, the respiration of the mitochondria decreases(27). Yet, the demand for ATP by the fibers in the superficialregion may increase because of a more frequent recruitment, asdiscussed above. An increase in mitochondria may enhance theflux of oxygen and hence total respiration, even though eachindividual mitochondrion is not working maximally (33). Accord-ing to our model calculations, there are virtually no anoxic areasduring hypoxia in the deep region of the muscle, suggesting thatthe mitochondria may still be able to work near maximally. It isthus not necessary to increase their number to enhance flux in thisregion of the muscle.

Conclusions

In conclusion, using an integrative approach to study theparameters determining oxygen delivery and utilization, we ob-served that the adaptation of skeletal muscle to hypoxia is regionspecific rather than fiber type specific. Overall, hypoxia results inmuscle atrophy. In oxidative regions, hypoxia results in angiogen-esis, whereas fibers in a more glycolytic region of the musclesubstantially increase their oxidative capacity. Without improve-ments in the delivery of oxygen, such as those brought about byincreases in hematocrit and blood flow, the latter may causeproblems with tissue oxygenation. We hypothesize that the in-creased oxidative capacity may serve to ensure adequate aerobicATP generation during hypoxia by maintaining the flux of oxy-gen. It is currently unknown which signaling pathways underliethese region-specific adaptations to hypoxia. Finally, when con-sidering the effects of hypoxia during the later stages of chronicobstructive pulmonary disease and chronic heart failure, whereatrophy and tissue hypoxia are common observations (29), onehas to bear in mind that different muscles or muscle regions mightbe affected differently.

ACKNOWLEDGMENTS

The authors appreciate the help from Remco Haasdijk and Jos Evers for thecare of the animals in the hypoxic chamber and assistance with surgery. Theauthors also thank Brechje van Beek-Harmsen for technical help.

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